ATP-induced cytoplasmic calcium mobilization in Bergmann glial cells

Abstract

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.

Radially expanding transglial calcium waves in the intact cerebellum

“…BG express a variety of G protein-coupled receptors that couple to calcium release from intracellular stores (17, 31), including P2Y receptors, which are activated by ATP (16,18,19). To test whether spontaneous transglial waves rely on a purinergic receptor-dependent mechanism, we imaged transglial waves in rats before, during, and after surface application of the P2 receptor antagonists PPADS (500 M, Fig.…”

“…We used in vivo 2-photon microscopy in combination with either bolus loading of calcium indicators or expression of the genetically encoded calcium sensor protein G-CaMP2 targeted to Bergmann glia (BG), the astrocytes of the molecular layer (ML). In brain slices, BG mobilize calcium in response to a number of neurotransmitters (16,17) and can generate subcellular domains of calcium release in response to synaptic stimulation via activation of purinergic receptors (18,19).…”

P2X1and P2X5Subunits Form the Functional P2X Receptor in Mouse Cortical Astrocytes

“…ATP triggers cytosolic Ca 2ϩ signals or membrane current responses in many types of CNS glia, including astrocytes and radial glia (Salter and Hicks, 1994;Kirischuk et al, 1995b;King et al, 1996), oligodendrocytes (Kirischuk et al, 1995a;James and Butt, 2001), and microglial cells (Haas et al, 1996;Moller et al, 2000;Verderio and Matteoli, 2001;Farber and Kettenmann, 2006). Cellular effects of ATP are mediated through metabotropic P2Y (Erb et al, 2006;Hussl and Boehm, 2006) and ionotropic P2X (Egan et al, 2006) receptors.…”

“…Cellular effects of ATP are mediated through metabotropic P2Y (Erb et al, 2006;Hussl and Boehm, 2006) and ionotropic P2X (Egan et al, 2006) receptors. The importance of P2Y receptors in triggering Ca 2ϩ signaling within astroglial networks is well documented (Kirischuk et al, 1995b;Verkhratsky and Kettenmann, 1996;Verkhratsky et al, 1998;Fellin et al, 2006). The functional role and relevance of ionotropic P2X receptors in astroglial signaling remains much less explored, primarily attributable to the absence of functional evidence on P2X receptors types operating in astrocytes in situ.…”

Brief Bursts of Parallel Fiber Activity Trigger Calcium Signals in Bergmann Glia

“…BGs express high levels of glutamate transporters (Chaudhry et al, 1995;Ottersen et al, 1997) and are therefore particularly effective in confining glutamate to activated synapses (Bergles et al, 1997;Clark and Barbour, 1997;Huang and Bordey, 2004). In addition, exogenous agonist application (Muller et al, 1992;Kirischuk et al, 1995;Kirischuk et al, 1996) and synaptic activation (Grosche et al, 1999) via the release of nitric oxide (NO) (Matyash et al, 2001) or noradrenaline (Kulik et al, 1999) can lead to calcium increases in BGs. Although activation of metabotropic glutamate receptors (mGluRs) and purinergic receptors (P2Rs) via synaptically released glutamate and ATP are mediators of calcium signaling in other types of glia (Porter and McCarthy, 1996;Pasti et al, 1997;Newman, 2005), no such role has been has been described in BGs.…”