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S Kirischuk

Publications and source records attributed to S Kirischuk.

At least 19 recordsLinked to original sources
PubMed2005

Brain-derived neurotrophic factor modulates GABAergic synaptic transmission by enhancing presynaptic glutamic acid decarboxylase 65 levels, promoting asynchronous release and reducing the number of activated postsynaptic receptors.

Brain-derived neurotrophic factor is known to modulate the function of GABAergic synapses, but the site of brain-derived neurotrophic factor action is still a matter of controversy. This study was aimed at further dissecting the functional alterations produced by brain-derived neurotrophic factor treatment of GABAergic synaptic connections in cultures of the murine superior colliculus. The functional consequences of long-term brain-derived neurotrophic factor treatment were assessed by analysis of unitary evoked and delayed inhibitory postsynaptic currents in response to high frequency stimulation of single axons. It was found that brain-derived neurotrophic factor facilitated the asynchronous release, but had no effect on the probability of evoked release, the size of the readily releasable pool, and the paired-pulse behavior of evoked inhibitory postsynaptic currents. However, the amplitudes of evoked inhibitory postsynaptic currents, delayed inhibitory postsynaptic currents and miniature inhibitory postsynaptic currents were significantly reduced. Non-stationary fluctuation analysis revealed a decrease in the open channel number at the miniature/evoked inhibitory postsynaptic current peak, but no effect on the mean GABA(A) receptor single channel conductance. Quantitative immunocytochemistry uncovered a significant elevation of presynaptic levels of glutamic acid decarboxylase 65. Together, these findings indicate that brain-derived neurotrophic factor treatment induces pre- as well as postsynaptic changes. What effect predominates will depend on the presynaptic activity pattern: at low activation rates brain-derived neurotrophic factor-treated synapses display a pronounced postsynaptic depression, but at high frequencies this depression is fully compensated by an enhancement of asynchronous release.

Animals↗
PubMed2000

A readily releasable pool of single inhibitory boutons in culture.

The number of presynaptic vesicles that are immediately available for release, the readily releasable pool (RRP), is a strong determinant of synaptic strength and plasticity. The properties of the RRP in individual GABAergic synapses were examined in superior colliculus cultures. The RRP was depleted by high frequency trains and cumulative evoked IPSC amplitudes (CA) were calculated. The amplitude of monoquantal responses (q) was determined on the basis of mIPSC histograms. On average, the RRP, defined as CA/q, comprised about 10 vesicles. About 60% of the RRP could be released by a single stimulus. After depletion, the RRP was replenished with a time constant of about 14 s. These data provide information for further studies on the capacity of individual inhibitory synapses to modulate sensory information transfer.

Animals↗
PubMed1999

Relationship between presynaptic calcium transients and postsynaptic currents at single gamma-aminobutyric acid (GABA)ergic boutons.

Postsynaptic responses to stereotyped activation of single axons are known to fluctuate, but the origin of synaptic variability in the vertebrate central nervous system is still unclear. To test the hypothesis that fluctuations of inhibitory postsynaptic currents reflect variations in presynaptic Ca2+ concentration, we examined single GABAergic axodendritic contacts in low-density cultures. Collicular neurons from rat embryos were loaded with the Ca2+ indicator Oregon Green 488 BAPTA-1. Presynaptic axon terminals were visualized by staining with the styryl dye RH414. Under the condition of action potential block, RH414-labeled boutons were activated selectively by current pulses applied through a fine-tipped glass pipette. Short (1- to 3-ms) depolarization of isolated boutons resulted in stimulus-locked changes of presynaptic Ca2+ concentration ([Ca2+]pre) and in evoked inhibitory postsynaptic currents (eIPSCs). Varying the strength of the stimulating currents produced a wide amplitude range of both presynaptic fluorescence transients (up to 220% of the resting value) and postsynaptic conductance changes (up to 2-3 nS). It was found that average eIPSCs displayed an approximately third-power dependency on [Ca2+]pre. Transmitter release retained its probabilistic character throughout the range of observed [Ca2+]pre values. In any tested single bouton, maximal eIPSCs occurred in association with the largest [Ca2+]pre transients, but failures were present at any [Ca2+]pre. The increase of maximal eIPSC amplitudes in connection with higher [Ca2+]pre supports the hypothesis that GABAergic boutons have the capacity to regulate synaptic strength by changing the number of simultaneously released vesicles.

Animals↗
PubMed1999

Single-bouton-mediated synaptic transmission: postsynaptic conductance changes in their relationship with presynaptic calcium signals.

Most central neurons contact their dendritic targets at several sites. However, it is not known whether all synapses formed by a single parent axon make the same contribution to the postsynaptic response. In order to answer this question it is necessary to isolate the synaptic currents generated by individual axon terminals. This paper describes a method that was designed to activate transmitter release from solitary synaptic boutons in culture. Neurons from the embryonic rat superior colliculus were grown at low density and double-loaded with a fluorescent marker of synaptic vesicles (FM1-43 or RH414) and a fluorescent Ca2+ indicator (Fura-2, Mag-fura-2, Oregon Green BAPTA-1 or Oregon Green BAPTA-5N). Action potential generation was blocked by tetrodotoxin. Appropriate synaptic boutons were selected under phase-contrast and fluorescence illumination at a magnification of 1000. They were activated by short electrical pulses via a fine-tipped glass pipette filled with bath solution. Presynaptic Ca2+ transients were measured in a region delineated by the FM1-43/RH414 fluorescence. By simultaneous presynaptic Ca2+ imaging and whole-cell recording of postsynaptic responses to single depolarizing pulses, the quantitative relationships between pre- and postsynaptic parameters of synaptic strength in a small synapse of central origin could, for the first time, be analysed. The experiments showed that the average postsynaptic currents depend strongly on the size of the presynaptic Ca2+ transients. However, at any level of presynaptic Ca2+ concentration postsynaptic responses fluctuated in amplitude.

Animals↗
PubMed1999

Glutamate-triggered calcium signalling in mouse bergmann glial cells in situ: role of inositol-1,4,5-trisphosphate-mediated intracellular calcium release.

The mechanisms of glutamate-induced changes in intracellular free calcium concentration in Bergmann glial cells in mouse cerebellar slices were investigated by Fura-2-based microfluorimetry. Extracellular applications of glutamate, quisqualate and kainate triggered an increase in cytoplasmic calcium concentration, whereas N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate were ineffective. The calcium elevation triggered by kainate was completely blocked by removal of calcium ions from the external solutions or by slice incubation with 6-cyano-7-nitroquinoxaline-2,3-dione. Conversely, both glutamate- and quisqualate-induced intracellular calcium transients were only slightly attenuated by slice incubation with either 6-cyano-7-nitroquinoxaline-2,3-dione or calcium-free solution, suggesting the intracellular origin for calcium ions. The glutamate-triggered cytosolic calcium increases were inhibited by slice incubation with thapsigargin, the inhibitor of intracellular calcium pumps, or by intracellular perfusion of Bergmann glial cells with heparin, the antagonist of inositol-1,4,5-trisphosphate-gated calcium release channels. Therefore the calcium release from inositol-1,4,5-trisphosphate-sensitive intracellular stores plays the major role in glutamate-induced calcium signalling. We concluded that Bergmann glial cells express calcium permeable ionotropic glutamate receptors, which might be important for generation of fast calcium signals. However, slow glutamate-evoked calcium signals are mostly determined by inositol-1,4,5-trisphosphate-dependent intracellular signalling chain.

Amino Acids↗
PubMed1997

Bergmann glial cells in situ express endothelinB receptors linked to cytoplasmic calcium signals.

The endothelin (ET) isoforms ET-1, ET-2 and ET-3 applied at 100 nM triggered a transient increase in [Ca2+]i in Bergmann glial cells in cerebellar slices acutely isolated from 20-25 day-old mice. The intracellular calcium concentration ([Ca2+]i) was monitored using Fura-2-based [Ca2+]i microfluorimetry. The ET-triggered [Ca2+]i transients were mimicked by ETB receptor agonist BQ-3020 and were inhibited by ETB receptor antagonist BQ-788. ET elevated [Ca2+]i in Ca(2+)-free extracellular solution and the ET-triggered [Ca2+]i elevation was blocked by 500 nM thapsigargin indicating that the [Ca2+]i was released from InsP3-sensitive intracellular pools. The ET-triggered [Ca2+]i increase in Ca(2+)-free solution was shorter in duration. Restoration of normal extracellular [Ca2+] briefly after the ET application induced a second [Ca2+]i increase indicating the presence of a secondary Ca2+ influx which prolongs the Ca2+ signal. Pre-application of 100 microM ATP or 10 microM noradrenaline blocked the ET response suggesting the involvement of a common Ca2+ depot. The expression of ETB receptor mRNAs in Bergmann glial cells was revealed by single-cell RT-PCR. The mRNA was also found in Purkinje neurones, but no Ca2+ signalling was triggered by ET. We conclude that Bergmann glial cells are endowed with functional ETB receptors which induce the generation of intracellular [Ca2+]i signals by activation of Ca2+ release from InsP3-sensitive intracellular stores followed by a secondary Ca2+ influx.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1997

Na+/Ca2+ exchanger modulates kainate-triggered Ca2+ signaling in Bergmann glial cells in situ.

The role of sodium-calcium exchanger in calcium homeostasis in Bergmann glial cells in situ was investigated by monitoring cytoplasmic calcium ([Ca2+]i) and sodium ([Na+]i) concentrations. The [Ca2+]i and [Na+]i transients were measured either separately by using fluorescent indicators fura-2 and SBFI, respectively, or simultaneously using the indicators fluo-3 and SBFI. Since the removal of extracellular Na+ induced a relatively small (approximately 50 nM) elevation of [Ca2+]i, the Na+/Ca2+ exchanger seems to play a minor role in regulation of resting [Ca2+]i. In contrast, kainate-triggered [Ca2+]i increase was significantly suppressed by lowering of the extracellular Na+ concentration ([Na+]o). In addition, manipulations with [Na+]o dramatically affected the recovery of the kainate-induced [Ca2+]i transients. Simultaneous recordings of [Ca2+]i and [Na+]i revealed that kainate-evoked [Ca2+]i transients were accompanied with an increase in [Na+]i. Moreover, kainate induced significantly larger [Ca2+]i and smaller [Na+]i transients under current-clamp conditions as compared to those recorded when the membrane voltage was clamped at -70 mV. The above results demonstrate that the Na(+)-Ca2+ exchanger is operative in Bergmann glial cells in situ and is able to modulate dynamically the amplitude and kinetics of [Ca2+]i signals associated with an activation of ionotropic glutamate receptors.

Adenosine Triphosphate↗
PubMed1996

[Ca2+]i recordings from neural cells in acutely isolated cerebellar slices employing differential loading of the membrane-permeant form of the calcium indicator fura-2.

This paper contains a description of the procedure for monitoring the cytoplasmic free calcium concentration ([Ca2+]i) from intact neurones and glial cells in acutely isolated cerebellar slices. The loading of cells with the calcium indicator fura-2 was achieved by slice incubation in Tyrode solution containing 5 "mu"M fura-2 acetoxymethylester (fura-2/AM) and 0.02% (w/v) pluronic-F127 under a controlled (temperature, 35 degrees C; humidity, 98%; and gas, 5% O2 +95% CO2) environment. In such conditions, different cellular elements of the cerebellum (namely granule neurones, Bergmann glial cells and Purkinje neurones) acquired fura-2 at different rates. Ten minutes of slice incubation gave adequate staining of granule neurones only, 20 min of incubation allowed calcium-dependent changes of fluorescence signal measurements in Bergmann glial cells, whereas loading of Purkinje neurones required 40 min of slice exposure to fura-2/AM. In order to assure dye deesterification, slices were kept in continuously gassed bicarbonate-buffered solution for not less than 1 h thereafter. The fluorescence signals (excited at 360 and 380 nm) were collected from either a 25- "mu"m or 40- "mu"m area limited by fixed diaphragm inserted in front of the photomultiplier tube; an individual cell was positioned in approximately the centre of the fluorescence measurement area. These signals were comprised of [Ca2+]i-related changes in fura-2 fluorescence recorded from a cell of interest and background fluorescence. The latter resulted from the summation of slice autofluorescence, signals from the fura-2 acquired by neighbouring tissue and signals from fura-2 compartmentalized by intracellular organelles. After the end of fluorescence recordings, the cell was internally dialysed with dye-free intracellular solution in order to determine the actual levels of background fluorescence. In parallel, electrophysiological properties were determined, allowing identification of cell type and viability. The background fluorescence values were then used to correct fluorescence recordings by subtraction from original traces. Corrected records were used for [Ca2+]i calculation.

Animals↗
PubMed1996

Calcium homeostasis in aged neurones.

Mechanisms of cytoplasmic calcium homeostasis were investigated in peripheral and central neurones isolated from neonatal, adult and old Wistar rats and in granule neurones in acutely prepared cerebellar slices of adult and old CBA mice. The cytoplasmic calcium concentration ([Ca2+]i) was measured by either indo-1-or fura-2-based microfluorimetry. The resting [Ca2+]i was significantly higher in senile neurones. The depolarization-induced [Ca2+]i transients were markedly altered in old neurones when compared with adult ones: the age-associated changes in stimulus-evoked [Ca2+]i signalling comprised of (i) significant decrease of the amplitudes of [Ca2+]i transients; (ii) prolongation of the rising phase and (iii) prominent deceleration of the recovery of the [Ca2+]i elevation towards the resting level after the end of depolarization. The amplitudes of calcium release from caffeine/Ca(2+)-sensitive endoplasmic reticulum calcium stores became significantly smaller in old central neurones, whereas they remained unaffected in peripheral neurones. Based on our observations we can conclude that ageing of the nervous system is associated with significant changes in mechanisms of [Ca2+]i homeostasis in individual neurones. These changes lead to a stable increase in the resting [Ca2+]i and to a substantial prolongation of stimulus-evoked [Ca2+]i signals. We could suggest also that the ability of the old neurones to handle Ca2+ loads is diminished, which may determine higher vulnerability of aged neurones to excess of calcium ions.

Aging↗
PubMed1996

Activation of P2-purino-, alpha 1-adreno and H1-histamine receptors triggers cytoplasmic calcium signalling in cerebellar Purkinje neurons.

The cytoplasmic calcium concentration ([Ca2+]i) was measured from Purkinje neurons in acutely prepared cerebellar slices. Neurons were loaded with calcium indicator Fura-2 by 40-min slice incubation in Tyrode solution containing 5 microM Fura-2/AM and 0.02% pluronic-F127. Bath applications of ATP (100 microM), epinephrine (10 microM) and histamine (100 microM) triggered a transient increase of [Ca2+]i in Purkinje neurons. ATP-induced [Ca2+]i elevation in Purkinje neurons was mimicked by ADP, but not AMP or adenosine pointing to the involvement of P2Y metabotropic purinoreceptors. Epinephrine-triggered [Ca2+]i responses were blocked by the selective alpha 1-antagonist prazosin and were mimicked by the alpha 1-adrenoreceptor agonist phenylephrine, and were not affected by beta- and alpha 2-adrenoreceptor agonists (isoproterenol and clonidine) and antagonists (propranolol and yohimbine). Histamine-induced [Ca2+]i responses demonstrated specific sensitivity to selective H1 antagonist chlorpheniramine, and were not sensitive to H2 and H3 histamine receptors modulators. The [Ca2+]i responses to all three agonists persisted in Ca(2+)-free extracellular media and were blocked by slice preincubation with thapsigargin (500 nM). We conclude that cerebellar Purkinje neurons are endowed with metabotropic P2 gamma purinoreceptors, alpha 1-adrenoreceptors and H1 histamine receptors which mediate the generation of intracellular [Ca2+]i signals via activation of Ca2+ release from inositol-1,4,5-trisphosphate-sensitive intracellular stores.

Adenosine Triphosphate↗
PubMed1996

Calcium signalling in granule neurones studied in cerebellar slices.

The cytoplasmic free calcium concentration ([Ca2+]i) was studied in Fura-2/AM loaded granule neurones in acutely prepared cerebellar slices isolated from neonatal (6 days old) and adult (30 days old) mice. Bath application of elevated (10-50 mM) KCl-containing extracellular solutions evoked [Ca2+]i rise which was dependent on extracellular Ca2+. The K(+)-induced [Ca2+]i elevation was inhibited to different extends by verapamil, nickel and omega-conotoxin suggesting the coexpression of different subtypes of plasmalemmal voltage-gated Ca2+ channels. Bath application of caffeine (10-40 mM) elevated [Ca2+]i by release of Ca2+ from intracellular stores. Caffeine-induced [Ca2+]i elevation was inhibited by 100 microM ryanodine and 500 nM thapsigargin. Depletion of internal Ca2+ stores by caffeine, or blockade of Ca2+ release channels by ryanodine, did not affect depolarization-induced [Ca2+]i transients, suggesting negligible involvement of Ca(2+)-induced Ca2+ release in [Ca2+]i signal generation following cell depolarization. External application of 100 microM glutamate, but not acetylcholine (1-100 microM), carbachol (10-100 microM) or (1S,3R)-ACPD (100-500 microM) evoked [Ca2+]i elevation. Part of glutamate-triggered [Ca2+]i transients in neurones from neonatal mice was due to Ca2+ release (presumably via inositol-(1,4,5)-trisphosphate-sensitive mechanisms) from internal Ca2+ stores. In adult animals, glutamate-triggered [Ca2+]i elevation was exclusively associated with plasmalemmal Ca2+ influx via both voltage-gated and glutamate-gated channels.

Acetylcholine↗
PubMed1996

Calcium signalling in mouse Bergmann glial cells mediated by alpha1-adrenoreceptors and H1 histamine receptors.

The presence of adrenergic and histaminergic receptors in Bergmann glial cells from cerebellar slices from mice aged 20-25 days was determined using fura-2 Ca2+ microfluorimetry. To measure the cytoplasmic concentration of Ca2+ ([Ca2+]i), either individual cells were loaded with the Ca2+-sensitive probe fura-2 using the whole-cell patch-clamp technique or slices were incubated with a membrane permeable form of the dye (fura-2/AM) and the microfluorimetric system was focused on individual cells. The monoamines adrenalin and noradrenalin (0.1-10 microM) and histamine (10-100 microM) triggered a transient increase in [Ca2+]i. The involvement of the alpha1-adrenoreceptor was inferred from the observations that monoamine-triggered [Ca2+]i responses were locked by the selective alpha1-adreno-antagonist prazosin and were mimicked by the alpha1-adreno-agonist phenylephrine. The monoamine-induced [Ca2+]i signals were not affected by beta- and alpha2-adrenoreceptor antagonists (propranolol and yohimbine), and were not mimicked by beta- and alpha2-adrenoreceptor agonists (isoproterenol and clonidine). Histamine-induced [Ca2+]i responses demonstrated specific sensitivity to only H1 histamine receptor modulators. [Ca2+]i responses to monoamines and histamine did not require the presence of extracellular Ca2+ and they were blocked by preincubation of slices with thapsigargin (500 nM), indicating that the [Ca2+]i responses were recorded after application of aspartate, bradykinin, dopamine, GABA, glycine, oxytocin, serotonin, somatostatin, substance P, taurine or vasopressin. We conclude that cerebellar Bergmann glial cells are endowed with alpha1-adrenoreceptors and H1 histamine receptors which induce the generation of intracellular [Ca2+]i signals via activation of Ca2+ release from inositol-1,4,5-trisphosphate-sensitive intracellular stores.

Adrenergic Agonists↗
PubMed1995

Preferential localization of active mitochondria in process tips of immature retinal oligodendrocytes.

Active mitochondria were visualized with a laser scanning confocal microscope using the dye rhodamine 123, which is accumulated in mitochondria with a high respiratory activity. In glial precursor cells cultured from rabbit retina the fluorescence signal of active mitochondria was confined to the tips of processes, while in oligodendrocytes an even distribution was found. The developmental stage of cultured oligodendrocytes was determined by staining with O4 and O10 antibodies after the end of microfluorometric measurements. In glial precursor cells, the mitochondrial uncoupler CCCP generated an increase in the cytoplasmic calcium concentration ([Ca2+]i) in regions corresponding to those revealed by rhodamine 123 staining. In mature oligodendrocytes CCCP evoked [Ca2+]i elevation without significant spatial heterogeneity. However, electron microscopy did not reveal an uneven distribution of mitochondria in either immature or mature oligodendrocytes. We conclude that in the precursor cells, active mitochondria are confined to process tips and serve as a Ca2+ pool, while in oligodendrocytes such a spatial heterogeneity cannot be detected. This preferential localization of active mitochondria may play a role in the neurone-glia interaction during the formation of the contact between the myelinating glial process and the axonal membrane.

Aniline Compounds↗
PubMed1995

Activation of P2-purinoreceptors triggered Ca2+ release from InsP3-sensitive internal stores in mammalian oligodendrocytes.

1. The subcellular characteristics of an ATP-induced elevation of the cytoplasmic free calcium concentration ([Ca2+]i) were studied in cultured cells of the oligodendrocyte lineage obtained from mouse cortex and rabbit retina, as well as in oligodendrocytes from mouse corpus callosum slices, using laser scanning confocal microfluorometry. 2. With the stage- and lineage-specific antibodies O4 and O10, three developmental stages within the oligodendrocyte lineage were distinguished prior to Ca2+ recording. 3. Bath application of 1-100 microM ATP induced a transient increase of [Ca2+]i in late precursors and oligodendrocytes but not in early glial precursor cells from retinal and cortical cultures and from corpus callosum slices. This effect of ATP was observed in Ca(2+)-free extracellular solution, suggesting that the ATP-mediated elevation of [Ca2+]i is due to a Ca2+ liberation from intracellular stores. 4. In both late precursors and oligodendrocytes from retina, the amplitude of ATP-induced [Ca2+]i transients was significantly higher in processes as compared with the soma; in cortical cultures such an uneven response was only observed in oligodendrocytes, while in immature cells responses in soma and processes were of similar amplitude. 5. The rank order of potency for the purine and pyrimidine nucleotides was UTP > or = ATP > ADP >> AMP = adenosine = Me-ATP for retinal oligodendrocytes, and ADP > or = ATP >> UTP = AMP = adenosine = Me-ATP for cortical oligodendrocytes. The response to ATP and related nucleotides was blocked by suramin, indicating the involvement of a P2-purinoreceptor in the ATP-mediated [Ca2+]i response. 6. ATP-induced elevation of the cytosolic Ca2+ concentration was inhibited by incubating cells with thapsigargin (10 microM) and by intracellular administration of heparin (1 microM). These findings indicate that ATP triggers a release of Ca2+ ions from InsP3-sensitive internal stores. 7. The ATP receptors may play a role in neuron-glial signal transfer; ATP is released as neurotransmitter, but also under pathological conditions from damaged cells.

Adenosine Triphosphate↗
PubMed1995

Subcellular heterogeneity of voltage-gated Ca2+ channels in cells of the oligodendrocyte lineage.

We studied the distribution of voltage-gated Ca2+ channels in cells of the oligodendrocyte lineage from retinal and cortical cultures. Influx of Ca2+ via voltage-gated channels was activated by membrane depolarization with elevated extracellular K+ concentration ([K+]e) and local, subcellular increases in cytosolic free Ca2+ concentration ([Ca2+]in) could be monitored with a fluometric system connected to a laser scanning confocal microscope. In glial precursor cells from both retina and cortex, small depolarizations (with 10 or 20 mM K+) activated Ca2+ transients in processes indicating the presence of low-voltage-activated Ca2+ channels. Larger depolarizations (with 50 mM K+) additionally activated high-voltage-activated Ca2+ channels in the soma. An uneven distribution of Ca2+ channels was also observed in the mature oligodendrocytes; Ca2+ transients in processes were considerably larger. Recovery of Ca2+ levels after the voltage-induced influx was achieved by the activity of the plasmalemmal Ca2+ pump, while mitochondria played a minor role to restore Ca2+ levels after an influx through voltage-operated channels. During the development of white matter tracts, cells of the oligodendrocyte lineage contact axons to form myelin. Neuronal activity is accompanied by increases in [K+]e; this may lead to Ca2+ changes in the processes and the Ca2+ increases might be a signal for the glial precursor cell to start myelin formation.

Animals↗
PubMed1995

ATP-induced cytoplasmic calcium mobilization in Bergmann glial cells.

ATP receptor mediated Ca2+ signaling was recorded from Bergmann glial cells in cerebellar slices obtained from mice of different ages (postnatal days 6 to 45). To measure the cytoplasmic concentration of Ca2+ ([Ca2+]in), either individual cells were loaded with the Ca(2+)-sensitive probes using the whole cell patch clamp technique or slices were incubated with the dye and the microfluorimetric system was focused on individual cells. Signals were recorded either with single-detector microfluorimetry of the dye fura-2 or by confocal laser scanning microfluorimetry (fluo-3-based recordings). Extracellular application of 100 microns ATP caused a transient elevation of [Ca2+]in, which amplitude was significantly higher in Bergmann glial cell processes as compared with their soma. The rank order of potency for the purinoreceptor agonists was: ADP > or = ATP > UTP >> AMP = adenosine = alpha, beta-methylene-ATP. ATP-triggered Ca2+ transients were reversibly inhibited by the P2 purinoreceptor agonist suramin (100 microM). The involvement of P2 metabotropic receptors is inferred by the observation that ATP mediated cytoplasmic Ca2+ transients were not associated with a measurable change in membrane conductance. The [Ca2+]in increase was due to release from inositol-1,4,5-trisphosphate (InsP3)-sensitive intracellular stores since responses were still observed in Ca(2+)-free extracellular solutions and were irreversibly blocked by the inhibitor of the sarco(endo)plasmic reticulum Ca2+ ATPase, thapsigargin, and by the competitive inhibitor of the InsP3-gated intracellular Ca2+ channels heparin. Intracellular dialysis altered the refilling process of the InsP3-sensitive stores, suggesting that cytoplasmic factors control ATP mediated Ca2+ signalling.

Adenosine Triphosphate↗
PubMed1994

Different properties of caffeine-sensitive Ca2+ stores in peripheral and central mammalian neurones.

Using indo-1 based microfluorometry for measuring the cytoplasmic free calcium concentration ([Ca2+]i), the properties of caffeine-induced Ca2+ release from internal stores were studied in rat cultured central and peripheral neurones, including dorsal root ganglia (DRG) neurones, neurones from nucleus cuneatus, CA1 and CA3 hippocampal region and pyramidal neocortical neurones. Under resting conditions the Ca2+ content of internal stores in DRG neurones was high enough to produce caffeine-triggered [Ca2+]i transients. Caffeine-induced Ca2+ release depleted internal stores in DRG neurones, but they refilled themselves spontaneously up to 81.4 +/- 5.67% within 10 minutes. In contrast, in all types of central neurones the resting Ca2+ content of internal stores was low, but the stores could be charged by transmembrane Ca2+ influx through voltage-operated calcium channels. After charging, the stores in central neurones spontaneously lost releasable calcium content and within 10 minutes they emptied again. We suggest that in sensory neurones calcium stores are continuously filled by releasable calcium and after discharge they can refill themselves spontaneously, while in central neurones internal calcium stores can be charged by releasable calcium only transiently.

Animals↗