Astrocytes are a key conduit for upstream signaling of vasodilation during cerebral cortical neuronal activation in vivo

Astrocytes play an important role in the coupling between neuronal activity and brain blood flow via their capacity to "sense" neuronal activity and transmit that information to parenchymal arterioles. Here we show another role for astrocytes in neurovascular coupling: the ability to act as a signaling conduit for the vitally important process of upstream vasodilation (represented by pial arterioles) during both excessive (seizure) and physiological (sciatic nerve stimulation) increases in cerebral cortical neuronal activity. The predominance of an astrocytic rather than a vascular route was indicated by data showing that pial arteriolar-dilating responses to neuronal activation were completely blocked following selective disruption of the superficial glia limitans, whereas interference with interendothelial signaling was without effect. Results also revealed contributions from connexin 43, implying a role for gap junctions and/or hemichannels in the signaling process and that signaling from the glia limitans to pial arterioles may involve a diffusible mediator.     

Role of astrocytes in cerebrovascular regulation.

Astrocytes send processes to synapses and blood vessels, communicate with other astrocytes through gap junctions and by release of ATP, and thus are an integral component of the neurovascular unit. Electrical field stimulations in brain slices demonstrate an increase in intracellular calcium in astrocyte cell bodies transmitted to perivascular end-feet, followed by a decrease in vascular smooth muscle calcium oscillations and arteriolar dilation. The increase in astrocyte calcium after neuronal activation is mediated, in part, by activation of metabotropic glutamate receptors. Calcium signaling in vitro can also be influenced by adenosine acting on A2B receptors and by epoxyeicosatrienoic acids (EETs) shown to be synthesized in astrocytes. Prostaglandins, EETs, arachidonic acid, and potassium ions are candidate mediators of communication between astrocyte end-feet and vascular smooth muscle. In vivo evidence supports a role for cyclooxygenase-2 metabolites, EETs, adenosine, and neuronally derived nitric oxide in the coupling of increased blood flow to increased neuronal activity. Combined inhibition of the EETs, nitric oxide, and adenosine pathways indicates that signaling is not by parallel, independent pathways. Indirect pharmacological results are consistent with astrocytes acting as intermediaries in neurovascular signaling within the neurovascular unit. For specific stimuli, astrocytes are also capable of transmitting signals to pial arterioles on the brain surface for ensuring adequate inflow pressure to parenchymal feeding arterioles. Therefore, evidence from brain slices and indirect evidence in vivo with pharmacological approaches suggest that astrocytes play a pivotal role in regulating the fundamental physiological response coupling dynamic changes in cerebral blood flow to neuronal synaptic activity. Future work using in vivo imaging and genetic manipulation will be required to provide more direct evidence for a role of astrocytes in neurovascular coupling.     

Regulation of cell-to-cell communication mediated by astrocytic ATP in the CNS

It has become apparent that glial cells, especially astrocytes, not merely supportive but are integrative, being able to receive inputs, assimilate information and send instructive chemical signals to other neighboring cells including neurons. At first, the excitatory neurotransmitter glutamate was found to be a major extracellular messenger that mediates these communications because it can be released from astrocytes in a Ca(2+)-dependent manner, diffused, and can stimulate extra-synaptic glutamate receptors in adjacent neurons, leading to a dynamic modification of synaptic transmission. However, recently extracellular ATP has come into the limelight as an important extracellular messenger for these communications. Astrocytes express various neurotransmitter receptors including P2 receptors, release ATP in response to various stimuli and respond to extracellular ATP to cause various physiological responses. The intercellular communication "Ca(2+) wave" in astrocytes was found to be mainly mediated by the release of ATP and the activation of P2 receptors, suggesting that ATP is a dominant "gliotransmitter" between astrocytes. Because neurons also express various P2 receptors and synapses are surrounded by astrocytes, astrocytic ATP could affect neuronal activities and even dynamically regulate synaptic transmission in adjacent neurons as if forming a "tripartite synapse". In this review, we summarize the role of astrocytic ATP, as compared with glutamate, in gliotransmission and synaptic transmission in neighboring cells, mainly focusing on the hippocampus. Dynamic communication between astrocytes and neurons mediated by ATP would be a key event in the processing or integration of information in the CNS.     

Dynamic inositol trisphosphate-mediated calcium signals within astrocytic endfeet underlie vasodilation of cerebral arterioles

Active neurons communicate to intracerebral arterioles in part through an elevation of cytosolic Ca(2+) concentration ([Ca(2+)](i)) in astrocytes, leading to the generation of vasoactive signals involved in neurovascular coupling. In particular, [Ca(2+)](i) increases in astrocytic processes ("endfeet"), which encase cerebral arterioles, have been shown to result in vasodilation of arterioles in vivo. However, the spatial and temporal properties of endfoot [Ca(2+)](i) signals have not been characterized, and information regarding the mechanism by which these signals arise is lacking. [Ca(2+)](i) signaling in astrocytic endfeet was measured with high spatiotemporal resolution in cortical brain slices, using a fluorescent Ca(2+) indicator and confocal microscopy. Increases in endfoot [Ca(2+)](i) preceded vasodilation of arterioles within cortical slices, as detected by simultaneous measurement of endfoot [Ca(2+)](i) and vascular diameter. Neuronal activity-evoked elevation of endfoot [Ca(2+)](i) was reduced by inhibition of inositol 1,4,5-trisphosphate (InsP(3)) receptor Ca(2+) release channels and almost completely abolished by inhibition of endoplasmic reticulum Ca(2+) uptake. To probe the Ca(2+) release mechanisms present within endfeet, spatially restricted flash photolysis of caged InsP(3) was utilized to liberate InsP(3) directly within endfeet. This maneuver generated large amplitude [Ca(2+)](i) increases within endfeet that were spatially restricted to this region of the astrocyte. These InsP(3)-induced [Ca(2+)](i) increases were sensitive to depletion of the intracellular Ca(2+) store, but not to ryanodine, suggesting that Ca(2+)-induced Ca(2+) release from ryanodine receptors does not contribute to the generation of endfoot [Ca(2+)](i) signals. Neuronally evoked increases in astrocytic [Ca(2+)](i) propagated through perivascular astrocytic processes and endfeet as multiple, distinct [Ca(2+)](i) waves and exhibited a high degree of spatial heterogeneity. Regenerative Ca(2+) release processes within the endfeet were evident, as were localized regions of Ca(2+) release, and treatment of slices with the vasoactive neuropeptides somatostatin and vasoactive intestinal peptide was capable of inducing endfoot [Ca(2+)](i) increases, suggesting the potential for signaling between local interneurons and astrocytic endfeet in the cortex. Furthermore, photorelease of InsP(3) within individual endfeet resulted in a local vasodilation of adjacent arterioles, supporting the concept that astrocytic endfeet function as local "vasoregulatory units" by translating information from active neurons into complex InsP(3)-mediated Ca(2+) release signals that modulate arteriolar diameter.     

Long-term facilitation of spontaneous calcium oscillations in astrocytes with endogenous adenosine in hippocampal slice cultures

It is well established that astrocytes release gliotransmitters and moderate neuronal activity in the central nervous system via intracellular Ca(2+) dynamics. Astrocytic Ca(2+) oscillations are one type of spontaneous Ca(2+) mobilization that occurs in astrocytes. However, the modulation of spontaneous astrocytic Ca(2+) oscillations, especially in pathophysiological conditions, is not yet fully understood. Here, we demonstrate that activation of adenosine receptors induces a long-lasting increase in the frequency of astrocytic Ca(2+) oscillations in rat hippocampal slice cultures. The long-term facilitation of the frequency of Ca(2+) oscillations was mediated by endogenous adenosine generated via breakdown of extracellular ATP by ecto-ATPase. We also demonstrate that local tissue injury with ultraviolet irradiation can cause this long-term facilitation of Ca(2+) oscillations via endogenous adenosine. Our data suggest that endogenous adenosine is one of the modulators of spontaneous astrocytic Ca(2+) oscillations in the rat hippocampus, and may play a significant role in altered Ca(2+) dynamics in astrocytes observed during pathophysiological conditions.     

Intercellular calcium signaling in astrocytes via ATP release through connexin hemichannels.

Astrocytes are capable of widespread intercellular communication via propagated increases in intracellular Ca(2+) concentration. We have used patch clamp, dye flux, ATP assay, and Ca(2+) imaging techniques to show that one mechanism for this intercellular Ca(2+) signaling in astrocytes is the release of ATP through connexin channels ("hemichannels") in individual cells. Astrocytes showed low Ca(2+)-activated whole-cell currents consistent with connexin hemichannel currents that were inhibited by the connexin channel inhibitor flufenamic acid (FFA). Astrocytes also showed molecular weight-specific influx and release of dyes, consistent with flux through connexin hemichannels. Transmembrane dye flux evoked by mechanical stimulation was potentiated by low Ca(2+) and was inhibited by FFA and Gd(3+). Mechanical stimulation also evoked release of ATP that was potentiated by low Ca(2+) and inhibited by FFA and Gd(3+). Similar whole-cell currents, transmembrane dye flux, and ATP release were observed in C6 glioma cells expressing connexin43 but were not observed in parent C6 cells. The connexin hemichannel activator quinine evoked ATP release and Ca(2+) signaling in astrocytes and in C6 cells expressing connexin43. The propagation of intercellular Ca(2+) waves in astrocytes was also potentiated by quinine and inhibited by FFA and Gd(3+). Release of ATP through connexin hemichannels represents a novel signaling pathway for intercellular communication in astrocytes and other non-excitable cells.     

Comparison of the effect of ATP on intracellular calcium ion dynamics between rat testicular and cerebral arteriole smooth muscle cells

Adenosine 5'-triphosphate (ATP), when released from neuronal and non-neuronal tissues, interacts with cell surface receptors produces a broad range of physiological responses. The goal of the present study was to examine the issue of whether vascular smooth muscle cells respond to ATP. To this end, the dynamics of the intracellular concentration of calcium ions ([Ca(2+)](i)) in smooth muscle cells in testicular and cerebral arterioles was examined by laser scanning confocal microscopy. ATP produced an increase in [Ca(2+)](i) in arteriole smooth muscle cells. While P1 purinoceptor agonists had no effect on this process, P2 purinoceptor agonists induced a [Ca(2+)](i) increase and a P2 purinoceptor antagonist, suramin, completely inhibited ATP-induced [Ca(2+)](i) dynamics in both arteriole smooth muscle cells.In testicular arterioles, Ca(2+) channel blockers and the removal of extracellular Ca(2+), but not thapsigargin pretreatment, abolished the ATP-induced [Ca(2+)](i) dynamics. In contrast, Ca(2+) channel blockers and the removal of extracellular Ca(2+) did not completely inhibit ATP-induced [Ca(2+)](i) dynamics in cerebral arterioles. Uridine 5'-triphosphate caused an increase in [Ca(2+)](i) only in cerebral arterioles and alpha,beta-methylene ATP caused an increase in [Ca(2+)](i) in both testicular and cerebral arterioles.We conclude that testicular arteriole smooth muscle cells respond to extracellular ATP via P2X purinoceptors and that cerebral arteriole smooth muscle cells respond via P2X and P2Y purinoceptors.     

Functional characterization of P2Y1 versus P2X receptors in RBA-2 astrocytes: elucidate the roles of ATP release and protein kinase C

A physiological concentration of extracellular ATP stimulated biphasic Ca(2+) signal, and the Ca(2+) transient was decreased and the Ca(2+) sustain was eliminated immediately after removal of ATP and Ca(2+) in RBA-2 astrocytes. Reintroduction of Ca(2+) induced Ca(2+) sustain. Stimulation of P2Y(1) receptors with 2-methylthioadenosine 5'-diphosphate (2MeSADP) also induced a biphasic Ca(2+) signaling and the Ca(2+) sustains were eliminated using Ca(2+)-free buffer. The 2MeSADP-mediated biphasic Ca(2+) signals were inhibited by phospholipase C (PLC) inhibitor U73122, and completely blocked by P2Y(1) selective antagonist MRS2179 and protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) whereas enhanced by PKC inhibitors GF109203X and Go6979. Inhibition of capacitative Ca(2+) entry (CCE) decreased the Ca(2+)-induced Ca(2+) entry; nevertheless, ATP further enhanced the Ca(2+)-induced Ca(2+) entry in the intracellular Ca(2+) store-emptied and CCE-inhibited cells indicating that ATP stimulated Ca(2+) entry via CCE and ionotropic P2X receptors. Furthermore, the 2MeSADP-induced Ca(2+) sustain was eliminated by apyrase but potentiated by P2X(4) allosteric effector ivermectin (IVM). The agonist ADPbetaS stimulated a lesser P2Y(1)-mediated Ca(2+) signal and caused a two-fold increase in ATP release but that were not affected by IVM whereas inhibited by PMA, PLC inhibitor ET-18-OCH(3) and phospholipase D (PLD) inhibitor D609, and enhanced by removal of intra- or extracellular Ca(2+). Taken together, the P2Y(1)-mediated Ca(2+) sustain was at least in part via P2X receptors activated by the P2Y(1)-induced ATP release, and PKC played a pivotal role in desensitization of P2Y(1) receptors in RBA-2 astrocytes.     

Prostaglandin E(2) stimulates glutamate receptor-dependent astrocyte neuromodulation in cultured hippocampal cells.

Recent Ca(2+) imaging studies in cell culture and in situ have shown that Ca(2+) elevations in astrocytes stimulate glutamate release and increase neuronal Ca(2+) levels, and that this astrocyte-neuron signaling can be stimulated by prostaglandin E(2) (PGE(2)). We investigated the electrophysiological consequences of the PGE(2)-mediated astrocyte-neuron signaling using whole-cell recordings on cultured rat hippocampal cells. Focal application of PGE(2) to astrocytes evoked a Ca(2+) elevation in the stimulated cell by mobilizing internal Ca(2+) stores, which further propagated as a Ca(2+) wave to neighboring astrocytes. Whole-cell recordings from neurons revealed that PGE(2) evoked a slow inward current in neurons adjacent to astrocytes. This neuronal response required the presence of an astrocyte Ca(2+) wave and was mediated through both N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors. Taken together with previous studies, these data demonstrate that PGE(2)-evoked Ca(2+) elevations in astrocyte cause the release of glutamate which activates neuronal ionotropic receptors.     

Oxygen-glucose deprivation and interleukin-1α trigger the release of perlecan LG3 by cells of neurovascular unit

Two of the main stresses faced by cells at the neurovascular unit (NVU) as an immediate result of cerebral ischemia are oxygen-glucose deprivation (OGD)/reperfusion and inflammatory stress caused by up regulation of IL-1. As a result of these stresses, perlecan, an important component of the NVU extracellular matrix, is highly proteolyzed. In this study, we describe that focal cerebral ischemia in rats results in increased generation of laminin globular domain 3 (LG3), the c-terminal bioactive fragment of perlecan. Further, in vitro study of the cells of the NVU was performed to locate the source of this increased perlecan-LG3. Neurons, astrocytes, brain endothelial cells and pericytes were exposed to OGD/reperfusion and IL-1α/β. It was observed that neurons and pericytes showed increased levels of LG3 during OGD in their culture media. During in vitro reperfusion, neurons, astrocytes and pericytes showed elevated levels of LG3, but only after exposure to brief durations of OGD. IL-1α and IL-1β treatment tended to have opposite effects on NVU cells. While IL-1α increased or had minimal to no effect on LG3 generation, high concentrations of IL-1β decreased it in most cells studied. Finally, LG3 was determined to be neuroprotective and anti-proliferative in brain endothelial cells, suggesting a possible role for the generation of LG3 in the ischemic brain.     

Neurovascular pathways to neurodegeneration in Alzheimer's disease and other disorders

The neurovascular unit (NVU) comprises brain endothelial cells, pericytes or vascular smooth muscle cells, glia and neurons. The NVU controls blood-brain barrier (BBB) permeability and cerebral blood flow, and maintains the chemical composition of the neuronal 'milieu', which is required for proper functioning of neuronal circuits. Recent evidence indicates that BBB dysfunction is associated with the accumulation of several vasculotoxic and neurotoxic molecules within brain parenchyma, a reduction in cerebral blood flow, and hypoxia. Together, these vascular-derived insults might initiate and/or contribute to neuronal degeneration. This article examines mechanisms of BBB dysfunction in neurodegenerative disorders, notably Alzheimer's disease, and highlights therapeutic opportunities relating to these neurovascular deficits.     

New players in the neurovascular unit: Insights from experimental and clinical epilepsy

The conventional notion that neurons are exclusively responsible for brain signaling is increasingly challenged by the idea that brain function in fact depends on a complex interplay between neurons, glial cells, vascular endothelium, and immune-related blood cells. Recent data demonstrates that neuronal activity is profoundly affected by an entire cellular and extracellular ‘orchestra’, the so-called neurovascular unit (NVU). Among the ‘musical instruments’ of this orchestra, there may be molecules long-known in biomedicine as important mediators of inflammatory and immune responses in the organism, as well as non-neuronal cells, e.g., leukocytes. We here review recent evidence on the structure and function of the NVU, both in the healthy brain and in pathological conditions, such as the abnormal NVU activation observed in epilepsy. We will argue that a better understanding of NVU function will require the addition of new players to the ‘orchestra’.     

Neurovascular abnormalities in brain disorders: highlights with angiogenesis and magnetic resonance imaging studies

The coupling between neuronal activity and vascular responses is controlled by the neurovascular unit (NVU), which comprises multiple cell types. Many different types of dysfunction in these cells may impair the proper control of vascular responses by the NVU. Magnetic resonance imaging, which is the most powerful tool available to investigate neurovascular structures or functions, will be discussed in the present article in relation to its applications and discoveries. Because aberrant angiogenesis and vascular remodeling have been increasingly reported as being implicated in brain pathogenesis, this review article will refer to this hallmark event when suitable.     

Proinflammatory treatment of astrocytes with lipopolysaccharide results in augmented Ca2+ signaling through increased expression of via phospholipase A2 (iPLA2)

Many Ca(2+)-regulated intracellular processes are involved in the development of neuroinflammation. However, the changes of Ca(2+) signaling in the brain under inflammatory conditions were hardly studied. ATP-induced Ca(2+) signaling is a central event of signal transmission in astrocytic networks. We investigated primary astrocytes after proinflammatory stimulation with lipopolysaccharide (LPS; 100 ng/ml) for 6-24 h. We reveal that Ca(2+) responses to purinergic ATP stimulation are significantly increased in amplitude and duration after stimulation with LPS. We detected that increased amplitudes of Ca(2+) responses to ATP in LPS-treated astrocytes can be explained by substantial increase of Ca(2+) load in stores in endoplasmic reticulum. The mechanism implies enhanced Ca(2+) store refilling due to the amplification of capacitative Ca(2+) entry. The reason for the increased duration of Ca(2+) responses in LPS-treated cells is also the amplified capacitative Ca(2+) entry. Next, we established that the molecular mechanism for the LPS-induced amplification of Ca(2+) responses in astrocytes is increased expression and activity of VIA phospholipase A(2) (VIA iPLA(2)). Indeed, both gene silencing with specific small interfering RNA and pharmacological inhibition of VIA iPLA(2) with S-bromoenol lactone reduced the load of the Ca(2+) stores and caused a decrease in the amplitudes of Ca(2+) responses in LPS-treated astrocytes to values, which were comparable with those in untreated cells. Our findings highlight a novel regulatory role of VIA iPLA(2) in development of inflammation in brain. We suggest that this enzyme might be a possible target for treatment of pathologies related to brain inflammation.     

Store-operated Ca2+ entry in astrocytes: different spatial arrangement of endoplasmic reticulum explains functional diversity in vitro and in situ

Ca(2+) signaling is the astrocyte form of excitability and the endoplasmic reticulum (ER) plays an important role as an intracellular Ca(2+) store. Since the subcellular distribution of the ER influences Ca(2+) signaling, we compared the arrangement of ER in astrocytes of hippocampus tissue and astrocytes in cell culture by electron microscopy. While the ER was usually located in close apposition to the plasma membrane in astrocytes in situ, the ER in cultured astrocytes was close to the nuclear membrane. Activation of metabotropic receptors linked to release of Ca(2+) from ER stores triggered distinct responses in cultured and in situ astrocytes. In culture, Ca(2+) signals were commonly first recorded close to the nucleus and with a delay at peripheral regions of the cells. Store-operated Ca(2+) entry (SOC) as a route to refill the Ca(2+) stores could be easily identified in cultured astrocytes as the Zn(2+)-sensitive component of the Ca(2+) signal. In contrast, such a Zn(2+)-sensitive component was not recorded in astrocytes from hippocampal slices despite of evidence for SOC. Our data indicate that both, astrocytes in situ and in vitro express SOC necessary to refill stores, but that a SOC-related signal is not recorded in the cytoplasm of astrocytes in situ since the stores are close to the plasma membrane and the refill does not affect cytoplasmic Ca(2+) levels.     

Astrocyte inositol triphosphate receptor type 2 and cytosolic phospholipase a(2) alpha regulate arteriole responses in mouse neocortical brain slices

Functional hyperemia of the cerebral vascular system matches regional blood flow to the metabolic demands of the brain. One current model of neurovascular control holds that glutamate released by neurons activates group I metabotropic glutamate receptors (mGluRs) on astrocytes, resulting in the production of diffusible messengers that act to regulate smooth muscle cells surrounding cerebral arterioles. The acute mouse brain slice is an experimental system in which changes in arteriole diameter can precisely measured with light microscopy. Stimulation of the brain slice triggers specific cellular responses that can be correlated to changes in arteriole diameter. Here we used inositol trisphosphate receptor type 2 (IP(3)R2) and cytosolic phospholipase A(2) alpha (cPLA(2)α) deficient mice to determine if astrocyte mGluR activation coupled to IP(3)R2-mediated Ca(2+) release and subsequent cPLA(2)α activation is required for arteriole regulation. We measured changes in astrocyte cytosolic free Ca(2+) and arteriole diameters in response to mGluR agonist or electrical field stimulation in acute neocortical mouse brain slices maintained in 95% or 20% O(2). Astrocyte Ca(2+) and arteriole responses to mGluR activation were absent in IP(3)R2(-) (/-) slices. Astrocyte Ca(2+) responses to mGluR activation were unchanged by deletion of cPLA(2)α but arteriole responses to either mGluR agonist or electrical stimulation were ablated. The valence of changes in arteriole diameter (dilation/constriction) was dependent upon both stimulus and O(2) concentration. Neuron-derived NO and activation of the group I mGluRs are required for responses to electrical stimulation. These findings indicate that an mGluR/IP(3)R2/cPLA(2)α signaling cascade in astrocytes is required to transduce neuronal glutamate release into arteriole responses.     

Modeling of ATP-mediated signal transduction and wave propagation in astrocytic cellular networks

Astrocytes, a special type of glial cells, were considered to have supporting role in information processing in the brain. However, several recent studies have shown that they can be chemically stimulated by neurotransmitters and use a form of signaling, in which ATP acts as an extracellular messenger. Pathological conditions, such as spreading depression, have been linked to abnormal range of wave propagation in astrocytic cellular networks. Nevertheless, the underlying intra- and inter-cellular signaling mechanisms remain unclear. Motivated by the above, we constructed a model to understand the relationship between single-cell signal transduction mechanisms and wave propagation and blocking in astrocytic networks. The model incorporates ATP-mediated IP3 production, the subsequent Ca2+ release from the ER through IP3R channels and ATP release into the extracellular space. For the latter, two hypotheses were tested: Ca2+- or IP3-dependent ATP release. In the first case, single astrocytes can exhibit excitable behavior and frequency-encoded oscillations. Homogeneous, one-dimensional astrocytic networks can propagate waves with infinite range, while in two dimensions, spiral waves can be generated. However, in the IP3-dependent ATP release case, the specific coupling of the driver ATP-IP3 system with the driven Ca2+ subsystem leads to one- and two-dimensional wave patterns with finite range of propagation.     

Cortical layer 1 and layer 2/3 astrocytes exhibit distinct calcium dynamics in vivo

Cumulative evidence supports bidirectional interactions between astrocytes and neurons, suggesting glial involvement of neuronal information processing in the brain. Cytosolic calcium (Ca(2+)) concentration is important for astrocytes as Ca(2+) surges co-occur with gliotransmission and neurotransmitter reception. Cerebral cortex is organized in layers which are characterized by distinct cytoarchitecture. We asked if astrocyte-dominant layer 1 (L1) of the somatosensory cortex was different from layer 2/3 (L2/3) in spontaneous astrocytic Ca(2+) activity and if it was influenced by background neural activity. Using a two-photon laser scanning microscope, we compared spontaneous Ca(2+) activity of astrocytic somata and processes in L1 and L2/3 of anesthetized mature rat somatosensory cortex. We also assessed the contribution of background neural activity to the spontaneous astrocytic Ca(2+) dynamics by investigating two distinct EEG states ("synchronized" vs. "de-synchronized" states). We found that astrocytes in L1 had nearly twice higher Ca(2+) activity than L2/3. Furthermore, Ca(2+) fluctuations of processes within an astrocyte were independent in L1 while those in L2/3 were synchronous. Pharmacological blockades of metabotropic receptors for glutamate, ATP, and acetylcholine, as well as suppression of action potentials did not have a significant effect on the spontaneous somatic Ca(2+) activity. These results suggest that spontaneous astrocytic Ca(2+) surges occurred in large part intrinsically, rather than neural activity-driven. Our findings propose a new functional segregation of layer 1 and 2/3 that is defined by autonomous astrocytic activity.     

ATP stimulates calcium-dependent glutamate release from cultured astrocytes

ATP caused a dose-dependent, receptor-mediated increase in the release of glutamate and aspartate from cultured astrocytes. Using calcium imaging in combination HPLC we found that the increase in intracellular calcium coincided with an increase in glutamate and aspartate release. Competitive antagonists of P(2) receptors blocked the response to ATP. The increase in intracellular calcium and release of glutamate evoked by ATP were not abolished in low Ca(2+)-EGTA saline, suggesting the involvement of intracellular calcium stores. Pre-treatment of glial cultures with an intracellular Ca(2+) chelator abolished the stimulatory effects of ATP. Thapsigargin (1 microM), an inhibitor of Ca(2+)-ATPase from the Ca(2+) pump of internal stores, significantly reduced the calcium transients and the release of aspartate and glutamate evoked by ATP. U73122 (10 microM, a phospholipase C inhibitor, attenuated the ATP-stimulatory effect on calcium transients and blocked ATP-evoked glutamate release in astrocytes. Replacement of extracellular sodium with choline failed to influence ATP-induced glutamate release. Furthermore, inhibition of the glutamate transporters p-chloromercuri-phenylsulfonic acid and Ltrans-pyrolidine-2,4-dicarboxylate failed to impair the ability of ATP to stimulate glutamate release from astrocytes. However, an anion transport inhibitor, furosemide, and a potent Cl(-) channel blocker, 5-nitro-2(3-phenylpropylamino)-benzoate, reduced ATP-induced glutamate release. These results suggest that ATP stimulates excitatory amino acid release from astrocytes via a calcium-dependent anion-transport sensitive mechanism.