Lucia Pasti

The spatial-temporal characteristics of intracellular calcium ([Ca2+]i) changes elicited in neurons and astrocytes by various types of stimuli were investigated by means of confocal fluorescent microscopy in acute rat brain slices loaded with the Ca2+ indicator indo-1. Neurons and astrocytes from the visual cortex and CA1 hippocampal region were identified in situ on the basis of their morphological, electrophysiological, and pharmacological features. We show here that stimulation of neuronal afferents triggered periodic [Ca2+]i oscillations in astrocytes. The frequency of these oscillations was under a dynamic control by neuronal activity as it changed according to the pattern of stimulation. After repetitive episodes of neuronal stimulation as well as repetitive stimulation with a metabotropic glutamate receptor agonist, astrocytes displayed a long-lasting increase in [Ca2+]i oscillation frequency. Oscillating astrocytes were accompanied by repetitive [Ca2+]i elevations in adjacent neurons, most likely because of the release of glutamate via a tetanus toxin-resistant process. These results reveal that [Ca2+]i oscillations in astrocytes represent a highly plastic signaling system that underlies the reciprocal communication between neurons and astrocytes.

The spatial–temporal characteristics of intracellular calcium ([Ca 2+ ] i ) changes elicited in neurons and astrocytes by various types of stimuli were investigated by means of confocal fluorescent microscopy in acute rat brain slices loaded with the Ca 2+ indicator indo-1. Neurons and astrocytes from the visual cortex and CA1 hippocampal region were identified in situ on the basis of their morphological, electrophysiological, and pharmacological features. We show here that stimulation of neuronal afferents triggered periodic [Ca 2+ ] i oscillations in astrocytes. The frequency of these oscillations was under a dynamic control by neuronal activity as it changed according to the pattern of stimulation. After repetitive episodes of neuronal stimulation as well as repetitive stimulation with a metabotropic glutamate receptor agonist, astrocytes displayed a long-lasting increase in [Ca 2+ ] i oscillation frequency. Oscillating astrocytes were accompanied by repetitive [Ca 2+ ] i elevations in adjacent neurons, most likely because of the release of glutamate via a tetanus toxin-resistant process. These results reveal that [Ca 2+ ] i oscillations in astrocytes represent a highly plastic signaling system that underlies the reciprocal communication between neurons and astrocytes.

To obtain insights into the spatiotemporal characteristics and mechanism of Ca(2+)-dependent glutamate release from astrocytes, we developed a new experimental approach using human embryonic kidney (HEK) 293 cells transfected with the NMDA receptor (NMDAR), which act as glutamate biosensors, plated on cultured astrocytes. We here show that oscillations of intracellular Ca(2+) concentration ([Ca(2+)](i)) in astrocytes trigger synchronous and repetitive [Ca(2+)](i) elevations in sensor HEK cells, and that these elevations are sensitive to NMDAR inhibition. By whole-cell patch-clamp recordings, we demonstrate that the activation of NMDARs in HEK cells results in inward currents that often have extremely fast kinetics, comparable with those of glutamate-mediated NMDAR currents in postsynaptic neurons. We also show that the release of glutamate from stimulated astrocytes is drastically reduced by agents that are known to reduce neuronal exocytosis, i.e., tetanus toxin and bafilomycin A(1). We conclude that [Ca(2+)](i) oscillations represent a frequency-encoded signaling system that controls a pulsatile release of glutamate from astrocytes. The fast activation of NMDARs in the sensor cells and the dependence of glutamate release on the functional integrity of both synaptobrevin and vacuolar H(+) ATPase suggest that astrocytes are endowed with an exocytotic mechanism of glutamate release that resembles that of neurons.

To obtain insights into the spatiotemporal characteristics and mechanism of Ca 2+ -dependent glutamate release from astrocytes, we developed a new experimental approach using human embryonic kidney (HEK) 293 cells transfected with the NMDA receptor (NMDAR), which act as glutamate biosensors, plated on cultured astrocytes. We here show that oscillations of intracellular Ca 2+ concentration ([Ca 2+ ] i ) in astrocytes trigger synchronous and repetitive [Ca 2+ ] i elevations in sensor HEK cells, and that these elevations are sensitive to NMDAR inhibition. By whole-cell patch-clamp recordings, we demonstrate that the activation of NMDARs in HEK cells results in inward currents that often have extremely fast kinetics, comparable with those of glutamate-mediated NMDAR currents in postsynaptic neurons. We also show that the release of glutamate from stimulated astrocytes is drastically reduced by agents that are known to reduce neuronal exocytosis, i.e., tetanus toxin and bafilomycin A 1 . We conclude that [Ca 2+ ] i oscillations represent a frequency-encoded signaling system that controls a pulsatile release of glutamate from astrocytes. The fast activation of NMDARs in the sensor cells and the dependence of glutamate release on the functional integrity of both synaptobrevin and vacuolar H + ATPase suggest that astrocytes are endowed with an exocytotic mechanism of glutamate release that resembles that of neurons.