Alexej Verkhratsky

Calcium signalling in glial cells

Calcium signals are the universal way of glial responses to the various types of stimulation. Glial cells express numerous receptors and ion channels linked to the generation of complex cytoplasmic calcium responses. The glial calcium signals are able to propagate within glial cells and to create a spreading intercellular Ca2+ wave which allow information exchange within the glial networks. These propagating Ca2+ waves are primarily mediated by intracellular excitable media formed by intracellular calcium storage organelles. The glial calcium signals could be evoked by neuronal activity and vice versa they may initiate electrical and Ca2+ responses in adjacent neurones. Thus glial calcium signals could integrate glial and neuronal compartments being therefore involved in the information processing in the brain.

Activation of mouse microglial cells affects P2 receptor signaling.

Microglial cells are the immunocompetent cells of the CNS, which are known to exist in several activation states. Here we investigated the impact of microglial activation on the P2 receptor-mediated intracellular calcium ([Ca(2+)](i)) signaling by means of fluo-3 based Ca(2+)-imaging. Cultured mouse microglial cells were treated with either astrocyte-conditioned medium to induce a ramified morphology or LPS to shift the cells toward the fully activated stage. The extracellular application of ATP (100 microM) induced a [Ca(2+)](i) elevation in 85% of both untreated and ramified microglial cells, whereas only 50% of the LPS-activated cells responded to the stimulus. To characterise the pharmacological profile of microglial P2 receptors we investigated the effects of various P2 agonists on [Ca(2+)](i) in cultured microglial cells. Untreated and ramified microglial cells demonstrated a very similar sensitivity to the different P2 agonists. In contrast, in LPS-activated microglia, a sharp decrease of responses to P2 agonist stimulation was seen. This indicates that microglial activation influences the capability of microglial cells to generate [Ca(2+)](i) signals upon P2 receptor activation.

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.

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.

ATP induces Ca2+ release from IP3-sensitive Ca2+ stores exclusively in large DRG neurones.

PURINORECEPTOR-MEDIATED intracellular Ca2+ release was studied in freshly isolated adult mouse dorsal root ganglia (DRG) neurones. The cytoplasmic Ca2+ concentration ([Ca2+]i) was measured using indo-1-based microfluorimetry. The application of 100 μM ATP in Ca2+-free solution triggered an increase in [Ca2+]i in 93% of large DRG neurones but in no small ones. The ATP-induced [Ca2+]i transients in large neurones were inhibited by cells incubation with thapsigargin or by intracellular dialysis with heparin-containing solution. The ATP-triggered increase in [Ca2+]i was not mimicked by adenosine and was blocked by suramin, suggesting the involvement of metabotropic (P2Y) purinoreceptors. We conclude that large (proprioceptive) DRG neurones express P2Y purinoreceptors linked to the inositol 1,4,5-triphosphate-Ca2+ intracellular signal transduction cascade, whereas small (nociceptive) DRG neurones are devoid of such a mechanism.

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.

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.

Integrative aspects of calcium signalling

Introduction: Principles of Calcium Signaling E.C. Toescu, A. Verkhratsky. Calcium Regulation of Cellular Systems: The Fate of Calcium Ions Entering a Cell: Calcium Transport Through Organelles M. Hoth. Calcium Regulation of Cytosolic Enzymes H. Schulman. Function of Nuclear and Cytoplasmic Calcium in the Control of Gene Expression S. Chawla, H. Bading. Calcium Regulation of Ion Channels D. Swandulla, H.U. Zeilhofer. Modulation of NMDA Receptor Channels by Intracellular Calcium N. Burnashev. Regulation of InsP3R by Ca2+ and Cytosolic Ca2+ Dynamics V.S. Markin, I. Bezprozvanny. Modulation of the Cytoskeletal Architecture by Calcium: A Putative Role in Plastic Synapses D. van Rossum. Calcium Regulation of Cellular Functions: Calcium and Exocytosis J. Hartmann. Calcium and Neuronal Development and Growth F. Archer, et al. Calcium and Cellular Death R. Sattler, M. Tymianski. Calcium-Dependent Signaling in Apoptosis T. Jayaraman, A.R. Marks. The Spatial Range of Dendritic Signals for Cerebellar Long-Term Depression: Studies with Local Photolysis of Caged Compounds G.J. Augustine, et al. 5 Additional Chapters. Index.

Endothelin-induced calcium signaling in cultured mouse microglial cells is mediated through ETB receptors

MICROGLIAL cells are the intrinsic immunocompetent cells of the central nervous system, which are activated by brain tissue damage. In this paper we investigated the ability of endothelins (ETs), which are potent vasoconstrictors, to induce intracellular calcium signals in cultured microglia cells. Both endothelin-1 and endothelin-3 increased intracellular Ca2+ concentration ([Ca2+]i). These [Ca2+]i transients were mimicked by BQ3020, an ETB receptor agonist and blocked by BQ788, a selective ETB antagonist, respectively. The calcium signals induced by the endothelins persisted in Ca2+-free media. Transcripts encoding the ETB receptor were detected in purified microglial cultures and cDNA fragments derived from ETB receptor mRNA were amplified from 9% of electrophysiologically characterized microglial cells by the use of single-cell RT-PCR.