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.

InsP3-induced Ca2+ release in dorsal root ganglion neurones.

The intracellular calcium signalling was studied on subpopulation of freshly isolated adult mouse dorsal root ganglia (DRG) neurones with large somatas (30-45 microns in diameter). The cytoplasmic Ca2+ concentration ([Ca2+]i) was measured using indo-1 based microfluorimetry. The extracellular application of ATP (100 microM) triggered both inward current and [Ca2+]i elevation.

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 microM ATP in Ca(2+)-free solution triggered an increase in [Ca2+]i in 93% of large DRG neurones but in no small ones.

Mechanisms of C5a and C3a complement fragment-induced [Ca2+]i signaling in mouse microglia.

Microglial cells are activated in response to brain insults; the mechanisms of this process are not yet understood. One of the important signaling mechanisms that might be involved in microglia activation is related to changes in the intracellular calcium concentration ([Ca2+]i). Using fluo-3 microfluorimetry, we have found that external application of the complement fragment C5a (4-10 nM) induced [Ca2+]i elevation in microglial cells in situ in corpus callosum slices.

ATP-induced membrane currents in ameboid microglia acutely isolated from mouse brain slices.

Microglial cells were harvested from the surface of corpus callosum slices acutely isolated from the brain of neonatal (five- to seven-day-old) mice. Transmembrane ionic currents were measured employing a standard whole-cell voltage-clamp technique. The extracellular application of 1 mM ATP triggered the generation of a complex membrane current comprising three components: (i) an initial fast inward current which had a reversal potential at about -20 to -15 mV; (ii) this initial component was followed by a steady-state inward current with reversal potential about -50 to -40 mV; and (iii) a delayed inward current with a reversal potential close to 0 mV.

Calcium signalling in glial cells.

Glial cells respond to a variety of external stimuli such as neurotransmitters, hormones or even mechanical stress by generating complex changes in the cytoplasmic Ca2+ concentration. This Ca2+ signal is controlled by an interplay of different mechanisms including plasmalemmal and intracellular Ca2+ channels, Ca2+ transporters and cytoplasmic Ca2+ buffers. In astrocytes, the Ca2+ signal can travel as waves within the syncytium spreading via gap junctions which might be regarded as a possible means for interglial communication.

Gradual caffeine-induced Ca2+ release in mouse dorsal root ganglion neurons is controlled by cytoplasmic and luminal Ca2+.

Cytosolic free calcium concentration ([Ca2+]c) was recorded from acutely isolated mouse dorsal root ganglion neurons loaded with Ca(2+)-indicator indo-1. The initiation of intracellular Ca2+ release by low (1-5 mM) caffeine concentrations failed to completely empty the caffeine-sensitive stores; subsequent challenge with higher doses of caffeine produced an additional [Ca2+]c elevation. This indicates a gradual Ca2+ release from caffeine-sensitive stores.

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.

Age-associated changes of cytoplasmic calcium homeostasis in cerebellar granule neurons in situ: investigation on thin cerebellar slices.

Mechanisms of cytoplasmic calcium homeostasis were investigated in adult and old CBA mice. The cytoplasmic calcium concentration ([Ca2+]i) was measured on fura-2/AM loaded granule neurons in acutely isolated cerebellar slices. The resting [Ca2+]i was significantly higher in senile cerebellar granule neurons, being on average 60 +/- 15 nM (n = 163) in adult and 107 +/- 12 nM (n = 129) in old neurons.

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.

[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.

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.

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.

Calcium-induced calcium release in neurones.

Neurones express several subtypes of intracellular Ca2+ channels, which are regulated by cytoplasmic calcium concentration ([Ca2+]c) and provide the pathway for Ca(2+)-induced Ca2+ release (CICR) from endoplasmic reticulum Ca2+ stores. The initial studies of CICR which employed several pharmacological tools (and in particular caffeine and ryanodine) demonstrated that: (i) caffeine induces intracellular calcium release in various peripheral and central neurones; and (ii) inhibition of CICR affects the parameters of depolarization-triggered [Ca2+]c responses. Experiments with caffeine demonstrated also that Ca2+ release from internal pools was incremental, suggesting the coexistence of several subpopulations of Ca2+ release channels with different sensitivity to caffeine.

Calcium-induced calcium release in rat sensory neurons.

1. In isolated dorsal root ganglion cells (DRG neurons), changes in the concentration of global cytosolic Ca2+ (delta [Ca2+]c) were measured by the fluorescence of K5-indo-1. Depolarizations from -60 to 0 mV (500 ms) and Ca2+ influx through Ca2+ channels (ICa) increased [Ca2+]c by 480 +/- 113 nM, the peak occurring 542 +/- 76 ms (mean +/- S.

Depolarization-induced calcium signals in the somata of cerebellar Purkinje neurons.

Cerebellar Purkinje neurons express voltage-gated Ca2+ channels that are located on their somata and dendrites. Previous reports, based on microelectrode recordings and fura-2 Ca2+ imaging, suggested that depolarization-mediated intracellular Ca2+ signaling is confined almost completely to the dendrites. We investigated the spatial distribution of depolarization-induced Ca2+ signals in Purkinje neurons by applying whole-cell patch-clamp recordings combined with fluorometric Ca2+ imaging to cerebellar slices.

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).

Ryanodine receptor-mediated intracellular calcium release in rat cerebellar Purkinje neurones.

1. Ryanodine receptor-mediated Ca2+ release was investigated in Purkinje neurones of rat cerebellar slices by using whole-cell patch-clamp recordings combined with fluorometric digital imaging of cytoplasmic Ca2+ concentration ([Ca2+]i). 2.

3-Isobutyl-1-methylxanthine (IBMX) affects potassium permeability in rat sensory neurones via pathways that are sensitive and insensitive to [Ca2+]in.

The effects of externally applied 3-isobutyl-1-methylxanthine (IBMX), in millimolar concentrations, on the membrane currents in dorsal root ganglia (DRG) neurones isolated from newborn rats were investigated using the amphotericin-based "perforated" patch-clamp technique. In some experiments, simultaneous measurements of intracellular Ca2+ concentration ([Ca2+]in) were performed using fura-2 microfluorimetry. Applications of IBMX induced elevation of [Ca2+]in resulting from Ca2+ release from caffeine-ryanodine-sensitive internal stores.

Dual action of thapsigargin on calcium mobilization in sensory neurons: inhibition of Ca2+ uptake by caffeine-sensitive pools and blockade of plasmalemmal Ca2+ channels.

The action of thapsigargin on intracellular calcium homeostasis and voltage-activated calcium currents was studied on freshly isolated adult mouse dorsal root ganglia neurons. The cytoplasmic Ca2+ concentration ([Ca2+]i) was measured using indo-1-based microfluorimetry; transmembrane Ca2+ currents were recorded under voltage-clamp in the whole-cell configuration of the patch-clamp technique. Extracellular applications of thapsigargin at concentrations of 20-2000 nM did not cause substantial changes of basal [Ca2+]i level in the majority of neurons studied.

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.

IBMX induces calcium release from intracellular stores in rat sensory neurones.

The intracellular free calcium concentration ([Ca2+]i) was recorded from freshly isolated rat dorsal root ganglia (DRG) neurones by means of Fura-2 or Indo-1-based microfluorimetry. Extracellular application of IBMX at millimolar concentrations evoked transient elevations in [Ca2+]i. The amplitude and rate of rise of the [Ca2+]i transient increased with increasing IBMX concentration.

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.

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.