Müller cell Ca2+ waves evoked by purinergic receptor agonists in slices of rat retina.

We have measured agonist evoked Ca2+ waves in Müller cells in situ within freshly isolated retinal slices. Using an eye cup dye loading procedure we were able to preferentially fill Müller glial cells in retinal slices with calcium green. Fluorescence microscopy revealed that bath perfusion of slices with purinergic agonists elicits Ca2+ waves in Müller cells, which propagate along their processes. These Ca2+ signals were insensitive to tetrodotoxin (TTX, 1.0 microM) pretreatment. Cells were readily identified as Müller cells by their unique morphology and by subsequent immunocytochemical labeling with glial fibrillary acidic protein antibodies. While cells never exhibited spontaneous Ca2+ oscillations, purinoreceptor agonists, ATP, 2 MeSATP, ADP, 2 MeSADP, and adenosine readily elicited Ca2+ waves. These waves persisted in the absence of [Ca2+]o but were abolished by thapsigargin pretreatment, suggesting that the purinergic agonists tested act by releasing Ca2+ from intracellular Ca2+ stores. The rank order of potency of different purines and pyrimidines for inducing Ca2+ signals was 2 MeSATP = 2MeSADP > ADP > ATP >> alphabetameATP = uridine triphosphate (UTP) > uridine diphosphate (UDP). The Ca2+ signals evoked by ATP, ADP, and 2 MeSATP were inhibited by reactive blue (100 microM) and suramin (200 microM), and the adenosine induced signals were abolished only by 3,7-dimethyl-1-propargylxanthine (200 microM) and not by 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine) or 8-cyclopentyl-1,3-dipropylxanthine at the same concentration. Based on these pharmacological characteristics and the dose-response relationships for ATP, 2 MeSATP, 2 MeSADP, ADP, and adenosine, we concluded that Müller cells express the P1A2 and P2Y1 subtypes of purinoceptors. Analysis of Ca2+ responses showed that, similar to glial cells in culture, wave propagation occurred by regenerative amplification at specialized Ca2+ release sites (wave amplification sites), where the rate of Ca2+ release was significantly enhanced. These data suggest that Müller cells in the retina may participate in signaling, and this may serve as an extra-neuronal signaling pathway.

Characterization of ryanodine receptors in oligodendrocytes, type 2 astrocytes, and O-2A progenitors.

In this study we have investigated the expression of ryanodine receptors (RyRs), and the ability of caffeine to evoke RyR-mediated elevation of intracellular Ca2+ levels ([Ca2+]i) in glial cells of the oligodendrocyte/type 2 astrocyte lineage. Immunocytochemistry with specific antibodies identified ryanodine receptors in cultured oligodendrocytes, type 2 astrocytes, and O-2A progenitor cells, at high levels in the perinuclear region and in a variegated pattern along processes. Glia acutely isolated from rat brain and in aldehydefixed sections of cortex were similarly found to express RyRs. Caffeine (5-50 mM) caused an increase in [Ca2+]i in most cultured type 2 astrocytes and in 50% of oligodendrocytes. Responses elicited by caffeine were inhibited by pretreatment with ryanodine (10 microM) or thapsigargin (1 microM), and the peak response was unaffected by removal of [Ca2+]o. O-2A progenitor cells, in contrast, were largely unresponsive to caffeine treatment. Pretreatment with kainate (200 microM) to activate Ca2+ entry increased the magnitude of caffeine-evoked [Ca2+]i elevations in type 2 astrocytes and oligodendrocytes, and caused caffeine to activate responses in a significant proportion of previously non-responding O-2A progenitors. In both type 2 astrocytes and oligodendrocytes, caffeine evoked Ca2+ changes which propagated as wavefronts from several initiation sites. These wave amplification sites were characterized by significantly higher local Ca2+ release kinetics. Our results indicate that several glial cell types express RyRs, and that their functionality differs within different cell types of the oligodendrocyte lineage. In addition, ionotropic glutamate receptor activation fills the caffeine-sensitive Ca2+ stores in these cells.

AMPA receptors shape Ca2+ responses in cortical oligodendrocyte progenitors and CG-4 cells.

Intracellular calcium signals triggered by glutamate receptor activation were studied in primary cortical oligodendrocyte lineage cells and in the oligodendrocyte cell line CG-4. Glutamate, kainate, and AMPA (30-300 microM) increased [Ca2+]i in both types of cells at the stage of oligodendrocyte progenitors (O-2A; GD3+) or pro-oligodendroblasts (O4+). The peak amplitude of Ca2+ responses to glutamate receptor agonists was significantly larger in cortical cells. In CG-4 and in cortical cells, the majority (more than 90%) of bipolar GD3+ or multipolar O4+ cells responded to kainate. In all the cells analyzed, kainate was more efficacious than AMPA and glutamate. The percentage of bipolar or multipolar cells responding to glutamate was significantly lower in the CG-4 cell line than in primary cultures. Cellular responses typical of metabotropic glutamate receptor activation were observed in 20% of the cortical O-2A progenitors, but in none of the CG-4 cells. The AMPA-selective antagonist GYKI 52466 blocked kainate-induced Ca2+ responses in cortical O-2A cells. The selective AMPA receptor modulator cyclothiazide (30 microM) greatly potentiated the effects of AMPA (30-100 microM) on [Ca2+]i in cortical and CG-4 cells. Our findings indicate that Ca2+ responses in cells of the oligodendrocyte lineage are primarily shaped by functional AMPA receptors.

Subcellular calcium oscillators and calcium influx support agonist-induced calcium waves in cultured astrocytes.

We have analysed Ca2+ waves induced by norepinephrine in rat cortical astrocytes in primary culture using fluorescent indicators fura-2 or fluo-3. The temporal pattern of the average [Ca2+]i responses were heterogeneous from cell to cell and most cells showed an oscillatory response at concentrations of agonist around EC50 (200 nM). Upon receptor activation, [Ca2+]i signals originated from a single cellular locus and propagated throughout the cell as a wave. Wave propagation was supported by specialized regenerative calcium release loci along the length of the cell. The periods of oscillations, amplitudes, and the rates of [Ca2+]i rise of these subcellular oscillators differ from each other. These intrinsic kinetic properties of the regenerative loci support local waves when stimulation is continued over long periods of time. The presence of local waves at specific, invariant cellular sites and their inherent kinetic properties provide for the unique and reproducible pattern of response seen in a given cell. We hypothesize that these loci are local specializations in the endoplasmic reticulum where the magnitude of the regenerative Ca2+ release is higher than other regions of the cell. Removal of extracellular Ca2+ or blockade of Ca2+ channels by inorganic cations (Cd2+ and Ni2+) during stimulation of adrenergic receptors alter the sustained plateau component of the [Ca2+]i response. In the absence of Ca2+ release, due to store depletion with thapsigargin, agonist occupation alone does not induce Ca2+ influx in astrocytes. This finding suggests that, under these conditions, receptor-operated Ca2+ entry is not operative. Furthermore, our experiments provide evidence for local Ca2+ oscillations in cells which can support both wave propagation as well as spatially discrete Ca2+ signalling.

Glutamate regulates intracellular calcium and gene expression in oligodendrocyte progenitors through the activation of DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors.

Oligodendrocytes and their progenitors (O-2A) express functional kainate- and DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-preferring glutamate receptors. The physiological consequences of activation of these receptors were studied in purified rat cortical O-2A progenitors and in the primary oligodendrocyte cell line CG-4. Changes in the mRNA levels of a set of immediate early genes were studied and were correlated to intracellular Ca2+ concentration, as measured by fura-2 Ca2+ imaging. Both in CG-4 and in cortical O-2A progenitors, basal mRNA levels of NGFI-A were much higher than c-fos, c-jun, or jun-b. Glutamate, kainate, and AMPA greatly increased NGFI-A mRNA and protein by activation of membrane receptors in a Ca(2+)-dependent fashion. Agonists at non-N-methyl-D-aspartate receptors promoted transmembrane Ca2+ influx through voltage-dependent channels as well as kainate and/or AMPA channels. The influx of Ca2+ ions occurring through glutamate-gated channels was sufficient by itself to increase the expression of NGFI-A mRNA. AMPA receptors were found to be directly involved in intracellular Ca2+ and NGFI-A mRNA regulation, because the effects of kainate were greatly enhanced by cyclothiazide, an allosteric modulator that selectively suppresses desensitization of AMPA but not kainate receptors. Our results indicate that glutamate acting at AMPA receptors regulates immediate early gene expression in cells of the oligodendrocyte lineage by increasing intracellular calcium. Consequently, modulation of these receptor channels may have immediate effects at the genomic level and regulate oligodendrocyte development at critical stages.

Vasoactive intestinal peptide increases intracellular calcium in astroglia: synergism with alpha-adrenergic receptors.

In type I astrocytes from rat cerebral cortex, vasoactive intestinal peptide (VIP) at concentrations below 1 nM evoked an increase in intracellular calcium ion concentration. This response, however, was observed in only 18% of the astrocytes examined. alpha-Adrenergic stimulation with phenylephrine or norepinephrine also resulted in an intracellular calcium response in these cells and the threshold sensitivity of astrocytes to phenylephrine was vastly different from cell to cell. Treatment of these astrocytes with VIP (0.1 nM) together with phenylephrine at subthreshold concentrations produced large increases in intracellular Ca2+ concentration ([Ca2+]i) and oscillations. The continued occupation of the alpha-adrenergic receptor was required for sustained synergism. Both alpha-receptor stimulation and stimulation with the mixture of agonists induced the cellular calcium response by triggering release of calcium from cellular stores, since the response persisted in the absence of extracellular calcium. Furthermore, thapsigargin pretreatment, which depletes intracellular stores, abolished the agonist-induced [Ca2+]i response. VIP (0.1 nM) and phenylephrine were found to increase cellular levels of inositol phosphates; however, there was no apparent additivity in this response when the agonists were added together. These observations suggest a calcium-mediated second messenger system for the high-affinity VIP receptor in astrocytes and that alpha-adrenergic receptors act synergistically with the VIP receptor to augment an intracellular calcium signal. The synergism between diverse receptor types may constitute an important mode of cellular signaling in astroglia.