Sergej Kirischuk

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.

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 μM) and histamine (10‐100 μM) triggered a transient increase in [Ca2+]i. The involvement of the α1‐adrenoreceptor was inferred from the observations that monoamine‐triggered [Ca2+]i responses were blocked by the selective α1‐adreno‐antagonist prazosin and were mimicked by the α1‐adreno‐agonist phenylephrine. The monoamine‐induced [Ca2+]i signals were not affected by β‐ and α2‐adrenoreceptor antagonists (propranolol and yohimbine), and were not mimicked by β‐ and α2‐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 increase is due to release from intracellular pools. No [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 α1 ‐adrenoreceptors and H1 histamine receptors which induce the generation of intracellular [Ca2+]i signals via activation of Ca2+ release from inositol‐l,4,5‐trisphosphate‐sensitive intracellular stores.

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 (~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.—Kirischuk, S., Kettenmann, H., Verkhratsky, A. Na+/Ca2+ exchanger modulates kainate‐triggered Ca2+ signaling in Bergmann glial cells in situ. FASEB J. 11, 566–572 (1997)

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.

Activation of P2-purino-,α1-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.

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.

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.

Measurements of intracellular calcium in sensory neurons of adult and old rats

Cytosolic free calcium ([Ca2+]i) was measured using fluorescent digital imaging microscopy in rat dorsal root ganglion neurons isolated from animals of two age groups (adult: seven months; and old: 30 months). Neurons were enzymatically isolated and maintained in primary culture for 14 days. Cultured neurons were loaded with the fluorescent dye, Fura-2. The spatial distribution of resting [Ca2+]i was even in both adult and old rats, but the value of cytoplasmic free calcium in old neurons was significantly higher (207 +/- 37 nmol/l vs 96 +/- 23 nmol/l) in comparison with adult ones. Depolarization with 50 mmol/l K+ produced a rapid increase in [Ca2+]i in all neurons, but the values of depolarization-induced increase of [Ca2+]i in old neurons were significantly lower (423 +/- 54 nmol/l) compared with cells isolated from adult rats (1011 +/- 91 nmol/l). The time of the complete restoration of [Ca2+]i to the resting level was 10-times longer in old neurons. The caffeine-induced rise of intracellular calcium was somewhat higher in neurons from old animals, and its restoration to normal level was delayed. The findings indicate a substantial alteration of the mechanisms of regulation of intracellular calcium homeostasis with neuronal ageing.

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.

[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 µM fura-2 acetoxymethylester (fura-2/AM) and 0.02% (w/v) pluronic-F127 under a controlled (temperature, 35° 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-µm or 40-µm 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.