Astrocytes in the brain form an intimately associated network with neurons. They respond to neuronal activity and synaptically released glutamate by raising intracellular calcium concentration ([Ca2+]i), which could represent the start of back-signalling to neurons. Here we show that coactivation of the AMPA/kainate and metabotropic glutamate receptors (mGluRs) on astrocytes stimulates these cells to release glutamate through a Ca2+-dependent process mediated by prostaglandins. Pharmacological inhibition of prostaglandin synthesis prevents glutamate release, whereas application of prostaglandins (in particular PGE2) mimics and occludes the releasing action of GluR agonists. PGE2 promotes Ca2+-dependent glutamate release from cultured astrocytes and also from acute brain slices under conditions that suppress neuronal exocytotic release. When applied to the CA1 hippocampal region, PGE2 induces increases in [Ca2+]i both in astrocytes and in neurons. The [Ca2+]i increase in neurons is mediated by glutamate released from astrocytes, because it is abolished by GluR antagonists. Our results reveal a new pathway of regulated transmitter release from astrocytes and outline the existence of an integrated glutamatergic cross-talk between neurons and astrocytes in situ that may play critical roles in synaptic plasticity and in neurotoxicity.

The spatial-temporal characteristics of intracellular calcium ([Ca 2ϩ ] 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 2ϩ 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 2ϩ ] 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 2ϩ ] i oscillation frequency. Oscillating astrocytes were accompanied by repetitive [Ca 2ϩ ] i elevations in adjacent neurons, most likely because of the release of glutamate via a tetanus toxin-resistant process. These results reveal that [Ca 2ϩ ] 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 2ϩ -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 2ϩ concentration ([Ca 2ϩ ] i ) in astrocytes trigger synchronous and repetitive [Ca 2ϩ ] 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 2ϩ ] 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 ϩ ATPase suggest that astrocytes are endowed with an exocytotic mechanism of glutamate release that resembles that of neurons.

Activation of nuclear transcription factors, breakdown of nuclear envelope and apoptosis represent a group of nuclear events thought to be modulated by changes in nucleoplasmic Ca2+ concentration, [Ca2+]n. Direct evidence for, or against, this possibility has been, however, difficult to obtain because measurements of [Ca2+]n are hampered by major technical problems. Here we describe a new approach for selectively monitoring Ca2+ concentrations inside the nucleus of living cells, which is based on the construction of a chimeric cDNA encoding a fusion protein composed of the photoprotein aequorin and a nuclear translocation signal derived from the rat glucocorticoid receptor. This modified aequorin (nuAEQ), stably expressed in HeLa cells, was largely confined to the nucleoplasm and thus utilized for monitoring [Ca2+]n in intact cells. No significant differences were observed between [Ca2+]n and cytosolic Ca2+ concentration ([Ca2+]i) under resting conditions. Upon stimulation of surface receptors linked to inositol‐1,4,5‐trisphosphate (InsP3) generation, and thus to intracellular Ca2+ signalling, the kinetics of [Ca2+]i and [Ca2+]n increases were indistinguishable. However, for the same rise in [Ca2+]i, the amplitude of [Ca2+]n increase was larger when evoked by Ca2+ mobilization from internal stores than when induced by Ca2+ influx across the plasma membrane. The functional significance of these transient nucleus‐cytosol Ca2+ gradients is discussed.

Calcium ions play crucial roles in a large variety of cell functions. The recent proposal that changes in the intracellular calcium concentration ([Ca 2ϩ

Long-term changes of synaptic strength in the central nervous system are mediated by an increase of cytosolic calcium concentration ([Ca2+]i) following activation of excitatory neurotransmitter receptors. These phenomena, which represent a possible cellular basis for learning and memory processes in eukaryotes, are believed to be restricted to neurons. Here we provide evidence for a long-term change of the response elicited by the excitatory neurotransmitter glutamate in a non-neuronal cell population of the central nervous system, i.e. visual cortical astrocytes in culture. Stimulation with glutamate induces in astrocytes a regular pattern of [Ca2+]i oscillations. A second stimulation, after an interval ranging from 2 to 60 min, induces an oscillatory response characterized by an increased frequency. Induction of this change in the astrocyte response is abolished by a specific inhibitor of the nitric oxide synthase and recovers upon exogenous nitric oxide generation or addition of a permeant cGMP analogue. Local brief pulses of glutamate to individual astrocytes, at a rate of 1 Hz, also elicit [Ca2+]i oscillations whose frequency increases following a second series of pulses. The long-lasting modification in the [Ca2+]i oscillatory response induced by glutamate in astrocytes demonstrates that in the central nervous system cellular memory is not a unique feature of neurons.

The substantia gelatinosa of the spinal cord (lamina II) is the major site of integration for nociceptive information. Activation of NMDA glutamate receptor, production of nitric oxide (NO), and enhanced release of substance P and calcitonin gene-related peptide (CGRP) from primary afferents are key events in pain perception and central hyperexcitability. By combining reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry for NO-producing neurons with immunogold labeling for substance P, CGRP, and glutamate, we show that (1) NO-producing neurons in lamina IIi are islet cells; (2) these neurons rarely form synapses onto peptide-immunoreactive profiles; and (3) NADPH diaphorase-positive dendrites are often in close spatial relationship with peptide-containing terminals and are observed at the periphery of type II glomeruli showing glutamate-immunoreactive central endings. By means of confocal fluorescent microscopy in acute spinal cord slices loaded with the Ca2+ indicator Indo-1, we also demonstrate that (1) NMDA evokes a substantial [Ca2+]i increase in a subpopulation of neurons in laminae I-II, with morphological features similar to those of islet cells; (2) a different neuronal population in laminae I-IIo, unresponsive to NMDA, displays a significant [Ca2+]i increase after slice perfusion with either substance P and the NO donor 3morpholinosydnonimine (SIN-1); and (3) the responses to both substance P and SIN-1 are either abolished or significantly inhibited by the NK1 receptor antagonist sendide. These results provide compelling evidence that glutamate released at type II glomeruli triggers the production of NO in islet cells within lamina IIi after NMDA receptor activation. The release of substance P from primary afferents triggered by newly synthesized NO may play a crucial role in the cellular mechanism leading to spinal hyperexcitability and increased pain perception.

Cellular calcium handling was examined in brain slices from transgenic antisense mice with a regional deficiency in the neuronal calcium binding protein calbindin D28k and from their non transgenic wild type litter mate controls. Depolarization of brain slices with NMDA or potassium produced a prolonged elevation of neuronal calcium signal in neurons in brain slices from calbindin D28k-deficient transgenic mice. This effect was selective and was seen only in brain areas where the antisense construct produced a significant depletion of calbindin D28k protein. In other regions where calbindin D28k protein was not modified by the construct and in all glial cells whether from wild type or transgenic mice, cellular calcium handling was normal.