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Astrocyte Control of Synaptic Transmission and Neurovascular Coupling

Silvio Conte Center for Integration at the Tripartite Synapse, Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania; and Istituto di Neuroscienze, Centro Nazionale Ricerche and Dipartimento di Scienze Biomediche Sperimentali, University of Padua, Padua, Italy

ABSTRACT

I. INTRODUCTION

II. ASTROCYTIC CALCIUM SIGNALING: THE BIOCHEMICAL BASIS OF GLIAL EXCITABILITY

NEURON-TO-ASTROCYTE SIGNALING IN THE CONTROL OF CEREBRAL CIRCULATION

IV. NEURONAL ACTIVITY-DEPENDENT CALCIUM ELEVATIONS IN ASTROCYTE ENDFEET

V. PROPAGATING CALCIUM WAVE IN ASTROCYTES MAY CONTRIBUTE TO CONTROL MICROCIRCULATION

VI. ACTIVATION OF ASTROCYTES CAN ALSO TRIGGER ARTERIOLE CONSTRICTION

VII. DISCOVERY OF GLIOTRANSMISSION: ASTROCYTES TALK TO NEURONS

VIII. MECHANISMS OF GLUTAMATE RELEASE FROM ASTROCYTES

IX. KISS-AND-RUN RELEASE OF GLUTAMATE FROM ASTROCYTES

X. THE TRIPARTITE SYNAPSE: ASTROCYTES MODULATE NEURONAL EXCITABILITY AND SYNAPTIC TRANSMISSION

XI. RELEASE OF GLUTAMATE FROM ASTROCYTES

XII. ASTROCYTES ACTIVATE EXTRASYNAPTIC NMDA RECEPTORS

XIII. WHY ARE ASTROCYTE-EVOKED NMDA CURRENTS SO LARGE IN AMPLITUDE?

XIV. ASTROCYTES SYNCHRONOUSLY ACTIVATE GROUPS OF PYRAMIDAL NEURONS

XV. D-SERINE: SELECTIVE SYNTHESIS IN AND RELEASE FROM ASTROCYTES

XVI. RELEASE OF ATP FROM ASTROCYTES

XVII. GLIAL-DERIVED ATP MODULATES NEURONAL EXCITABILITY

XVIII. PURINERGIC MODULATION OF SYNAPTIC TRANSMISSION

XIX. INTRODUCTION OF MOLECULAR GENETICS TO ADDRESS THE ROLES OF THE ASTROCYTE IN NEURONAL FUNCTION

XX. GLIOTRANSMISSION REGULATES SYNAPTIC CROSS-TALK

XXI. SUMMARY AND THE FUTURE

GRANTS

ACKNOWLEDGMENTS

REFERENCES

	ABSTRACT

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	I. INTRODUCTION

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The nervous system consists of two classes of cell, the neuron and glia. Although it is without doubt that neurons are essential for nervous system function, studies over the past decade are raising our awareness about the diversity of roles played by glial cells in nervous system function. In this review we focus on one of the subtypes of glial cells, the astrocyte, and discuss our current understanding of how these cells operate hand in hand with neurons to regulate integration in the central nervous system. Necessarily, we restrict our focus to the roles of astrocytes served by the release of three transmitters, glutamate, D-serine, and ATP; how these gliotransmitters regulate neuronal function; and how neuronal activity can, through astrocytic signaling cascades, locally regulate vascular tone. We do not attempt to discuss the roles of chemokines released from these glial cells, and instead alert the reader to an excellent recent review on this topic (219).

The discovery that chemical transmitters evoke Ca²⁺ elevations in cultured astrocytes (37) sparked the imagination of a small group of neuroscientists who diverted their attention to the investigation of this class of glial cell. Although these cells play critical roles in supporting neuronal function, astrocytic Ca²⁺ excitability and the consequent induced release of chemical transmitters, which we now term gliotransmitters, has led to an emerging new understanding of the functional roles played by these glial cells; we now appreciate that astrocytes listen and talk to synapses and play roles in synaptic modulation and in mediating synaptic cross-talk (9, 13, 30, 82, 148, 153, 205). In this review we have three goals: to provide a view of nervous system activity from the perspective of the astrocyte, to discuss how as an integrative hub the astrocyte exerts control over cerebrovascular as well as neuronal functions, and to discuss how the astrocyte and gliotransmission play a fundamental role in shaping and dynamically regulating the relative strengths of neighboring synaptic connections.

	II. ASTROCYTIC CALCIUM SIGNALING: THE BIOCHEMICAL BASIS OF GLIAL EXCITABILITY

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Anyone who has recorded, often by accident, from an astrocyte in a brain slice preparation or in vivo knows that these cells are electrically inexcitable, and offer little to study with electrophysiological approaches. Generally, astrocytes have a high resting K⁺ conductance, respond to depolarization with a linear current-voltage relationship, and are coupled by gap junctions. Recent studies have suggested that there may be many types of astrocytes. For example, Steinhauser's group has identified two classes of glial cell with distinct functional properties: one has the linear current-voltage relationship, high resting K⁺ conductance, and glutamate transporters expected of astrocytes, while the other expresses voltage-gated conductances together with AMPA receptors (93, 132, 220). We await the results of future studies to determine whether this second class of cell type is a subtype of astrocyte or a distinct glial cell type, and will therefore focus the remainder of our discussion to the more traditional gap junction-coupled astrocyte with a linear current-voltage relationship.

Because of the high resting K⁺ conductance and gap junction coupling, the first major function assigned to these cells was in the clearance of extracellular K⁺ following elevated periods of neuronal activity (158). Although early studies showed that the application of neurotransmitters can depolarize astrocytes (22, 100), perhaps the most significant discovery that initiated renewed vigor in studies of astrocytes was the discovery that the application of the chemical transmitter glutamate induces Ca²⁺ oscillations and Ca²⁺ waves between cultured hippocampal astrocytes (32, 37, 61).

Glutamate-induced Ca²⁺ oscillations in astrocytes result from the activation of class I metabotropic receptors that induce the phospholipase-dependent accumulation of inositol trisphosphate (IP₃) that stimulates the release of Ca²⁺ from IP₃-sensitive internal stores (99). Consequently, by measuring Ca²⁺ signaling, rather than membrane potential, it was discovered that astrocytes are an excitable system.

Since these initial observations it has been realized that astrocytes express a plethora of metabotropic receptors that can couple to second messenger systems (216, 217). For example, norepinephrine (48, 109), glutamate (48, 165, 174, 175, 190), GABA (96), acetylcholine (11, 190), histamine (190), adenosine (173), and ATP (24, 171, 173) have all been shown to induce Ca²⁺ elevations in glial cells in brain slice preparations. In culture, the list of metabotropic receptors is extensive. However, because culturing astrocytes can lead to the misexpression of proteins, it is not yet clear whether all of these receptors are normally expressed in astrocytes in vivo.

The presence of Ca²⁺ waves that propagate between cultured astrocytes has intrigued several groups who have attempted to identify the mechanism of signal propagation. Although these waves occur in cell culture, emerging evidence suggests that they do not occur under physiological conditions in vivo (85). Nonetheless, understanding the mechanism of wave propagation has provided important insights into signals that can be released from astrocytes. Two prominent hypotheses guided this work: 1) IP₃ could diffuse through gap junctions to evoke Ca²⁺ signals in neighboring unstimulated astrocytes (87, 115, 183, 193, 214), and 2) a message, ATP, is released from an astrocyte which, by activating P2Y receptors on adjacent astrocytes, stimulates additional Ca²⁺ signals (38, 51, 72). Although it is likely that both pathways contribute to wave propagation, that the wave can propagate between physically disconnected cells (81) provides compelling evidence for a role for the induced release of ATP signaling to neighboring cells and mediating the propagation of the wave.

Although in cell culture waves of Ca²⁺ elevation are the norm, it is appropriate to ask whether long-range Ca²⁺ waves could provide meaningful information if they were to occur within a nervous system. Several studies have now been performed using brain slice preparations, and these studies indicate that Ca²⁺ signals, under physiological conditions, are less extensive. For example, photorelease of glutamate in hippocampal slices to stimulate individual astrocytes demonstrated that activation of a single cell was able to evoke Ca²⁺ elevations in neighboring astrocytes (200). However, the range of this signal was extremely small compared with those observed in cultures. As will be discussed in section XVII, the extracellular concentration of ATP is tightly regulated by ectonucleotidases. It is likely that the difference in range over which Ca²⁺ signals propagate is in part regulated by the impact of ectonucleotidases that more effectively hydrolyze ATP to adenosine within the confined extracellular space of a brain slice and in vivo (49).

Studies in brain slice preparations have led to the proposal that astrocytes are functionally compartmentalized and Ca²⁺ oscillations are predominantly restricted to local microdomains. Imaging studies performed in hippocampal slices showed that astrocytic Ca²⁺ oscillations occur in portions of a process of individual astrocytes (165). Stimulation of the parallel fibers, which innervate Purkinje cells and Bergmann glia, evoke Ca²⁺ signals restricted to microdomains of the Bergmann glial cell processes (71). Three-dimensional reconstruction of the processes of Bergmann glia shows that microdomains are connected by extremely fine processes, providing a structural basis to support biochemical compartmentalization. Because one hippocampal astrocyte has been calculated to make contact with 100,000 synapses (29), this local Ca²⁺ signaling provides the opportunity for astrocytes to influence synaptic transmission in response to the glial Ca²⁺signal while retaining synaptic specificity. The idea of localized Ca²⁺ signaling is supported by in vivo imaging of astrocytes where synchronized Ca²⁺ waves are not generally detected (85).

What is the stimulus for the astrocytic Ca²⁺ signal? Since chemical transmitters can induce Ca²⁺ oscillations in these glial cells, the ability of neuronal activity to stimulate astrocytes was initially tested in studies in brain slice preparations. Trains of activity in the Schaffer collateral pathway of the hippocampus evoke Ca²⁺ signals in area CA1 astrocytes (165, 175). These signals result from synaptically released glutamate acting on subtype 5 of metabotropic glutamate receptors (mGluR5s), as well as a contribution from ATP acting through P2Y receptors (24). More recently, Newman (150) has shown that activation of the retina by light, to stimulate photoreceptors and the associated circuitry, does indeed stimulate Ca²⁺ signals in Müller glial cells. In support of local signaling, Ca²⁺ waves were not detected, but instead Ca²⁺ oscillations were detected in endfeet of these glia. An intriguing observation by McCarthy's group (149) suggests that astrocytes do not rely solely on instructive cues from neurons, but instead can intrinsically oscillate. With the use of a pharmacological cocktail of antagonists and despite blocking activity-dependent synaptic transmission, Ca²⁺ oscillations persisted in hippocampal astrocytes. Thus, although it is clear that neurons can activate astrocytic Ca²⁺ signals, and that this can occur in vivo, it is also possible that astrocytes have intrinsic capabilities of initiating Ca²⁺ signals. However, at this point we are only at the beginning of understanding the regulation of Ca²⁺ signals within astrocytes in vivo. Two-photon imaging in vivo shows that long-range Ca²⁺ signals are not the norm (85). However, we have little idea about how neuronal activity influences the spatial scale of glial Ca²⁺ signals nor how neuronal activity influences frequency encoding of glial Ca²⁺ signals. This is an extremely important area of investigation if we are to develop realistic models of the role of the astrocyte in the control of synaptic transmission, neuronal excitability, as well as the control of the cerebrovasculature.

These studies provide a different picture of Ca²⁺ signaling in the astrocyte than was first described in cultures. Oscillations are restricted to portions of the processes of individual cells, the so-called microdomains. They do not necessarily propagate over large distances, even within one astrocyte. Since, as we will discuss later, such Ca²⁺ signals cause gliotransmitters to be released that have feedback actions on neurons, localized responses to neuronal activity are likely to be of importance in maintaining synaptic specificity, while permitting gliotransmission to modulate neuronal function.

	NEURON-TO-ASTROCYTE SIGNALING IN THE CONTROL OF CEREBRAL CIRCULATION

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From a purely structural perspective, the astrocyte is situated much like a hub in which it receives inputs from thousands of synapses and at the same time can make contact with the local vasculature (Fig. 1). The combination of this structural relationship together with our new-found appreciation of the presence of dynamic, activity-dependent biochemical signaling between neurons and astrocytes suggests that the astrocyte is an important integrator of neuronal activity and consequently the local control of cerebrovasculature.

View larger version (59K): [in this window] [in a new window]   FIG. 1. Neuronal synaptic activity can act through the astrocyte network to regulate the cerebrovasculature. The activity of glutamatergic synapses can regulate astrocytic biochemical signaling through the coactivation of metabotropic glutamate and purinergic receptors to cause a phospholipase C-dependent increase in astrocytic Ca2+, which can propagate to the astrocytic endfoot to exert local actions on the vasculature. Through the activity of Ca2+-sensitive phospholipase A2, accumulated arachidonic acid can cause vasodilatory and vasoconstrictive actions through at least two of its metabolic pathways. Cyclooxygenase-2 (COX)-dependent accumulation of PGE2 leads to a vasodilation, while the diffusion of arachidonic acid to the smooth muscle, which contains high levels of CPY4A, leads to the accumulation of 20-HETE that causes vasoconstriction. While these two opposing actions seem in conflict, since both have been seen to occur in vivo, the challenge is to identify the conditions that select for the respective actions.

The increased energy demand of active neurons is also met by local increases in blood flow in the area of elevated neuronal activity. This phenomenon, which was first described by A. Mosso in the late 1800s (142) and later confirmed by Roy and Sherrington (180), is a fundamental event in brain function. Local increases in blood flow result from the rapid dilation of arterioles and capillaries of a restricted area in response to an episode of high neuronal activity. As a consequence, blood flow increases in that region within a few seconds, thereby ensuring that most active neurons receive an adequate supply of oxygen and metabolic substrates for energy consumption. Local accumulation of metabolic products has been initially proposed to directly control blood flow. Although under particular circumstances, such as brain hypoxia or ischemia, this process may indeed affect blood vessels, the time course of the neurovascular coupling argues against this hypothesis (122). Results obtained over the last few years provide conclusive support for the view that blood flow is directly coupled to neuronal activity rather than to local energy needs (16, 184). The present knowledge on the multiple signaling pathways that during activation lead to the production of vasoactive factors suggests that the molecular mechanism at the basis of functional hyperemia is highly complex and may not necessarily be the same in all brain regions. Although various aspects remain to be elucidated, most recent studies highlight a central role of neuron-to-astrocyte signaling in the local control of microcirculation (6, 60, 123, 145, 241, 242).

Because of their polarized anatomical structure and of the vicinity of their endfeet with contractile elements of blood vessels, such as smooth muscle cells in arterioles and pericytes in capillaries, astrocytes have been long proposed to contribute to the regulation of the blood flow during neuronal activity. The ability of astrocytes to remove from the extracellular space around active synapses potassium ions increasingly concentrated there following high neuronal activity and to redistribute them, through the syncytium, to distal regions, was originally considered a plausible mechanism to couple neuronal activity with dilation of vessels (207). This hypothesis was substantiated in the retina where a high potassium conductance was found in astrocyte endfeet and Müller cell processes in contact with blood vessels (152, 168).

The demonstration that astrocytes produce a plethora of vasoactive substances, such as nitric oxide (NO) (116, 146, 226), cyclooxygenase and epoxygenase activity-derived products (2, 4, 157, 169), and ATP (15, 36, 176), hints at the possibility that the control of microcirculation by astrocytes could not be based simply on the "spatial buffering" of K⁺ hypothesis, but rather involves a more complex mechanism and a number of different molecules. Among these, are epoxyeicosatrienoic acids (EETs) that cytochrome P-450 epoxygenase forms from arachidonic acid (AA) (179). Support for a distinct role of EETs in neurovascular coupling derives the observations that 1) by acting on K⁺ channels, EETs hyperpolarized smooth muscle cells and trigger dilation of cerebral vessels (5, 65, 88); 2) pharmacological inhibition of P-450 epoxygenase results in reduction of the basal blood flow in the cerebral cortex as measured by laser Doppler flowmetry (2); and 3) stimulation of astrocytes in culture with glutamate receptor agonists triggers formation of AA that is converted to various AA metabolites including EETs (1, 19). These observations led David Harper to propose a functional role of astrocyte EETs in neuronal activity-dependent regulation of blood flow (74, 75).

	IV. NEURONAL ACTIVITY-DEPENDENT CALCIUM ELEVATIONS IN ASTROCYTE ENDFEET

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Significant evidence in support of a distinct role of astrocytes in neurovascular coupling was then obtained in a series of experiments performed mainly in brain slice preparations. In hippocampal and cortical slices it was first observed that glutamate released at active synapses triggered Ca²⁺ oscillations in astrocytes that increased in frequency according to increasing levels of neuronal activity (165). These oscillations may represent a digital signal in the control of cell activity as it was originally proposed by Woods et al. (230) and Jacob et al. (94). While this observation demonstrates that astrocytes are sophisticated sensors of neuronal activity (243), it also represents a clue to the possibility that astrocytes transfer to blood vessels information on the level of neuronal activity. Indeed, neuronal activity-dependent Ca²⁺ elevations in astrocytes were observed to propagate to perivascular endfeet (242). Such a signal provides a mechanistic basis for the graded response of the blood flow to different levels of neuronal activity, thereby strengthening the idea of a distinct astrocytic role in neurovascular coupling. Importantly, high-frequency stimulation of neuronal afferents was found to trigger both Ca²⁺ elevations in astrocyte endfeet and dilation of cerebral arterioles. Furthermore, Ca²⁺ elevations triggered in astrocytes by either t-ACPD, a mGluR agonist, or direct mechanical stimulation of individual astrocytes by a patch pipette, also evoked dilation of cortical arterioles, while inhibition by mGluR antagonists of Ca²⁺ oscillations evoked in astrocytes by synaptic glutamate, or the incubation with cyclooxygenase (COX) inhibitors that block prostaglandin synthesis, reduced neuronal activity-dependent dilation of cerebral arterioles. Vasodilation appears to be mediated, at least in part, by prostaglandin E₂, since astrocytes in culture were observed to release this powerful dilating agent in a pulsatile manner according to the pattern of t-ACPD-mediated Ca²⁺ oscillations (244).

Activation of Ca²⁺ elevations in astrocyte endfeet has been also reported to suppress vasomotion (60), a rhythmic fluctuation in the diameter of cerebral arterioles that accompanies Ca²⁺ oscillations in smooth muscle cells (73, 224). Vasomotion, which is a natural property of cerebral microcirculation in the intact brain, requires some degree of tone that in arterioles from acute brain slice preparations is seriously compromised. Vasomotion could, however, recover upon treatment with agents that induce arteriole constriction (123). Although its precise functional significance and underlying mechanism remain undefined, vasomotion is believed to contribute to microvasculature hemodynamics by enhancing tissue oxygenation especially when perfusion is compromised (209). Interestingly, in arterioles from brain slice preparations, vasomotion and the accompanied Ca²⁺ oscillations in smooth muscle cells are suppressed by stimulation of neuronal afferents (27), suggesting that its inhibition or reduction contributes to neuronal activity-dependent blood flow changes. All together, these observations raise the possibility that the suppression of vasomotion, which accompanies Ca²⁺ elevations in astrocyte endfeet, contributes to the dilating action of astrocytes. In this action of astrocytes, EET release may be involved since blocking EET production with the epoxygenase inhibitor miconazole results in an increase in the frequency of vasomotion (123).

Results from in vivo experiments that used the same mGluR antagonists that in brain slices inhibited astrocyte-mediated vasodilation corroborated the role of astrocytes in functional hyperemia (242). By measuring the blood flow in the somatosensory cortex by laser Doppler flowmetry, the hyperemic response evoked by forepaw stimulation was found to be markedly reduced after the systemic application of mGluR antagonists. The action of the mGluR antagonists was unrelated to unspecific effects on the intensity of neuronal stimulation since the evoked somatosensory potential was unchanged. This is in agreement with the unchanged amplitude of the Ca²⁺ increase triggered by neuronal afferent stimulation in neurons from brain slices in the presence of the mGluR antagonists.

According to these results, a model is proposed in which astrocytes can encode different levels of neuronal activity into defined Ca²⁺ oscillation frequencies that, at the level of perivascular endfeet, mediate the release of dilating agents, such as EETs (2, 19, 123) and PGE₂ (242, 244) as well as constrictive agents such as 20-HETE (145; see also below). Neuronal activity-dependent Ca²⁺oscillations may ultimately represent the signaling system that allows blood flow to vary in a manner proportional to the intensity of neuronal activity.

	V. PROPAGATING CALCIUM WAVE IN ASTROCYTES MAY CONTRIBUTE TO CONTROL MICROCIRCULATION

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During functional hyperemia, the dilation of arterioles in the area of activation will not increase blood flow in that region effectively unless upstream vessels also dilate. How vasodilator and vasoconstrictor responses are conveyed from the initial site of activation to distant locations is unclear. Coordinated vasoactive responses may rely on coupling and communication between cells within the vessel wall. Endothelial cells are indeed extensively coupled (86, 118), and evidence has been also provided for an electronic coupling existing between endothelial and smooth muscle cells (118, 185, 231). Hyperpolarization of an individual smooth muscle cell can thus spread through the endothelial cells to other smooth muscle cells via myoendothelial coupling and evoke a coordinated dilating response along the length of an arteriole (70).

A Ca²⁺ wave propagating between perivascular astrocytes may also be involved. The Ca²⁺ response in an astrocyte in contact with a blood vessel, initially evoked either by neuronal activity (242) or by direct electrical stimulation (192), has been indeed observed to spread to other perivascular astrocytes. Furthermore, connexin43 and purinergic receptors, i.e., the basic elements which mediated the propagation of the Ca²⁺ wave in cultured astrocytes, are highly expressed at astrocyte endfeet (192), and filling single astrocytes that are in the proximity of a blood vessel with Lucifer yellow results in the diffusion of the dye to other astrocyte endfeet. Through the release of vasoactive factors, activation of perivascular astrocytes by the Ca²⁺ wave may affect the tone of upstream and/or downstream blood vessels, thereby regulating the overall conductance of the vascular network in a defined region.

	VI. ACTIVATION OF ASTROCYTES CAN ALSO TRIGGER ARTERIOLE CONSTRICTION

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In hippocampal slices, Ca²⁺ elevations in astrocyte endfeet triggered by either photolysis of a Ca²⁺caged compound or t-ACPD have been observed to evoke also arteriole constriction (145). Studies in cultured cells show that astrocytes can indeed produce, in addition to various dilating agents, constrictive agents such as the COX products PGF₂ (19, 195) and thromboxane A₂ (92, 169), endothelins (126), and 20-hydroxyeicosatetraenoic acid (20-HETE) (154). This latter compound, that derives from -hydroxylation of AA by CYP4A, a cytochrome P-450 enzyme subtype (179), depolarizes smooth muscle cells by inhibiting the opening of K⁺ channels (112), and also enhances Ca²⁺ influx through voltage-dependent Ca²⁺ channels (65). The formation of 20-HETE from AA in smooth muscle cells is proposed to mediate the constrictive action of astrocytes in the hippocampus (145). This action can also account for the constriction of cerebral blood vessels associated with spreading depression and ischemia (45), since Ca²⁺ elevations and Ca²⁺ waves are known to occur in the astrocytes during these pathological brain conditions (17, 110, 113, 114). The release of 20-HETE from astrocytes may, however, have a role also under normal physiological conditions. For example, 20-HETE has been proposed to play a crucial role in the maintenance of myogenic tone in cerebral blood vessels (64). The constrictive action of 20-HETE may also control, together with that of dilating agents, the extent of neuronal activity-dependent increases in blood flow (see also below).

The results reported by Mulligan and MacVicar's study (145) are in conflict with those reported by Zonta et al. (242) in which Ca²⁺ elevations in astrocyte endfeet were observed to trigger dilation of cerebral arterioles. How can these conflicting results be reconciled in a unifying hypothesis? In the latter study, most, although not all, experiments were performed in cortical slices incubated with N^G-nitro-L-arginine methyl ester (L-NAME), a NO synthase inhibitor that blocks the tonic action of NO on arterioles and thus results in a long-lasting constriction of arterioles. In contrast, this procedure was not applied in most of the experiments described in Mulligan and MacVicar's study in hippocampal slices. Therefore, the different initial state of contraction of cerebral arterioles in the two studies may account, at least in part, for the different results.

The central point here concerns the resting state of pressurized arterioles in vivo. Under normal physiological conditions in the intact brain, cerebral arteries are typically in a state of partial contraction (52). Although the exact mechanisms at the basis of myogenic tone remain uncertain, it is clear that this phenomenon is generated by an interplay of pressure-mediated stretching of the smooth muscle cell membrane, intraluminal blood flow, and various factors released by neurons, astrocytes, and endothelial cells (43, 44, 84). A change in ion gating in smooth muscle cell membrane, either directly or indirectly via membrane depolarization, results in increased levels of intracellular Ca²⁺ concentration and activation of the contractile process (84). The myogenic tone underlies cerebrovascular autoregulation, i.e., the ability of vessels to respond to changes in transmural pressure with either constriction when pressure increases or dilation when pressure decreases (43, 188, 224). This property has been also proposed to reflect a "regional blood reserve" that various control mechanisms use to produce vasodilation or vasoconstriction (62).

It is the importance of myogenic tone that likely accounts for the discrepancy between the results of these two studies. Accordingly, when the vascular tone is lost, arterioles tend to be in a dilated state, and dilating agents may be ineffective or less effective. Similarly, when smooth muscles are excessively contracted, the inner diameter of arterioles is reduced to such an extent that constrictive agents can hardly induce a further constriction. Therefore, given that in slice preparations the myogenic tone is lost, to detect a dilating effect of vasoactive agents, a certain degree of constriction that mimics the natural occurring myogenic tone of blood vessels in vivo is pharmacologically induced (56, 57, 77, 78, 124, 182). In agreement with this view, constriction of arterioles induced by t-ACPD in hippocampal slices changed to dilation when t-ACPD was applied after arterioles were preconstricted with L-NAME (145).

All together, these observations hint at the possibility that astrocytes release both dilating and constrictive agents. This ability of astrocytes seems, however, difficult to reconcile with a distinct role of the astrocyte in neurovascular coupling. Indeed, how can a Ca²⁺ signal in astrocyte endfeet lead to diametrically opposite changes in arteriole diameter? The hypothesis can be advanced that the ultimate effect of astrocyte activation may depend on the balance between the action of dilating and constrictive agents, on the one hand, and the resting state of arterioles, on the other. The release of 20-HETE may serve to generate a constrictive action that opposes the powerful action of other dilating agents, released by neurons and/or astrocytes themselves, ultimately modulating the amplitude of the neuronal activity-dependent increase in blood flow.

Certainly, to define the astrocyte's role in the control of cerebral blood flow, first of all, it will be important to provide conclusive evidence for the release from astrocytes of both dilating and constrictive agents. In such a case, are dilating and constrictive factors released simultaneously? If this corelease event does not occur, could it be possible that a dilating, or a constrictive, agent might be preferentially released according to a distinct pattern, or amplitude or compartmentalization, of the Ca²⁺ rise in the astrocyte? An additional interesting issue would be that of clarifying whether the various signaling pathways that rely on diverse enzymes to metabolize AA, i.e., COXs, lipoxygenases, epoxygenases, and -hydroxylases, can be differently regulated by modulatory factors. For example, NO that is produced by neurons as well as glia has been reported to downregulate the formation of the cytochrome P-450 subtypes CYP2 that produce EETs (121, 211) as well as to inhibit 20-HETE formation (3).

A recent in vivo study provides further support for the distinct role of astrocytes in neurovascular coupling (204). After loading of astrocytes from the somatosensory cortex of adult rats with both the Ca²⁺ indicator rhod 2 and the Ca²⁺caged compound 1-(4,5-dimethoxy-2-nitrophenyl)-EDTA (DMNP-EDTA), and after labeling of blood vessels with dextran-conjugated fluorescein, a Ca²⁺ elevation evoked in astrocyte endfeet by either photolysis of DMNP-EDTA or stimulation of neuronal activity was followed by a rapid (1 s delay) and marked dilation of the arteriole. Vasodilation and increase in blood flow, as measured by laser Doppler flowmetry, were sensitive to COX inhibitors (204), thereby confirming the previous finding that COX products are likely released from activated astrocyte endfeet to trigger arteriole dilation (242). When the activation of astrocytes, which accompanies neuronal activity, was blocked with the mGluR5 antagonist MPEP, the vascular response induced by stimulation of neuronal activity was significantly reduced, providing further evidence for the central role of neuron-to-astrocyte signaling pathway in the neurovascular coupling.

It should be noted in this in vivo study (204), as well as in Mulligan and MacVicar's brain slice investigation (145), that a membrane-permeant EDTA-based caged compound was used for the photolytic control of internal Ca²⁺. As discussed by Ellis-Davies (50), this will result in a compound that is 97% loaded with Mg²⁺ rather than Ca²⁺. Thus, at this time, it is not clear how photolysis raised internal Ca²⁺. Nonetheless, Mulligan and MacVicar (145) did perform an important control in which they loaded DMNP-EDTA with Ca²⁺, then, after dialysis into a single astrocyte, they showed that photolytic release of Ca²⁺ did replicate their results obtained using DMNP-EDTA acetoxymethyl ester.

It is important to note that in these in vivo experiments, Ca²⁺ elevations in astrocyte endfeet occasionally resulted in a significant arteriole constriction (204). Although it was detected only in a few arterioles, this response validates the hypothesis advanced above that activation of astrocytes can release both dilating and constrictive agents.

The ability of astrocytes to trigger also arteriole constriction has been further confirmed in whole-mounted retina preparations (135). Light stimulation as well as photolysis of caged Ca²⁺ that triggered a Ca²⁺ rise in the stimulated glial cell, i.e., an astrocyte or a Müller cell, were indeed found to evoke both dilation and constriction of retinal arterioles, mediated by different AA metabolites, EETs, and 20-HETE, respectively. Additional evidence for a role of glial cells as mediators of light-induced response of arterioles was the observation that the interruption of neuron-to-glia signaling also blocked the response of arterioles to light stimulation. Based on a series of experimental observations, the authors suggest that NO may play a modulatory role on arteriole responsiveness, favoring a vasodilating and vasoconstrictive response to light when its level is low and high, respectively (135).

While these observations confirm that the mechanism that governs the blood flow response to neuronal activity is complex and relies probably on different vasoactive agents in different brain regions, they underline the central role of astrocytes in functional hyperemia.

The important action of astrocytes in the control of microvasculature raises also the possibility that an astrocyte dysfunction could be implicated in the dysregulation of cerebral circulation in brain pathologies, for example, in the defective neurovascular coupling that is associated with Alzheimer's disease (90, 155), as well as in the vascular abnormal responses during stroke, trauma, and spreading depression (89). The full characterization of the molecular mechanisms that are at the basis of the astrocyte control on cerebral blood vessels in the normal and pathological brain certainly represents one of the most interesting challenges in neurobiological research in years to come.

	VII. DISCOVERY OF GLIOTRANSMISSION: ASTROCYTES TALK TO NEURONS

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In 1994 two studies (147, 160) provided the first suggestion that the astrocytic Ca²⁺ signals described by Stephen Smith's group do have functional consequences on integration in the nervous system. In these studies it was demonstrated that experimentally evoked Ca²⁺ elevations in astrocytes evoked elevations in the internal Ca²⁺ of adjacent neurons. These breakthrough discoveries, which were later reproduced in independent studies performed by Andrew Charles' (31) and Stan Kater's laboratories (80), provided a new insight into glial-neuron interactions in the nervous system. Although one study suggested an involvement of the gap junction-mediated communication between the astrocyte and neuron (147), the others offered a more compelling mechanistic insight in which the Ca²⁺-dependent release of the excitatory transmitter glutamate from the astrocyte evoked the depolarization of the neuron through the activation of ionotropic glutamate receptors (160). Indeed, ligands that elevated astrocytic Ca²⁺ led to the release of glutamate from pure cultures of astrocytes (95, 162), and optical assays for glutamate demonstrated external waves of glutamate elevation that followed the internal Ca²⁺ waves in culture (91).

After the demonstration of a glutamate-mediated astrocyte-neuron signaling pathway, it was several years until it was feasible to document a similar pathway in a more intact system to alleviate worries about potential culture artifacts. In 1997, Pasti et al. (165) demonstrated bidirectional signaling between astrocytes and neurons and showed that the activation of astrocytic metabotropic glutamate receptors to evoke glial Ca²⁺ elevations caused delayed neuronal Ca²⁺ signals mediated by ionotropic glutamate receptors. Given the previous culture studies, this result was readily interpreted as being due to the Ca²⁺-dependent release of glutamate from hippocampal astrocytes, an observation that was later confirmed in slice preparations by Bezzi et al. (19).

	VIII. MECHANISMS OF GLUTAMATE RELEASE FROM ASTROCYTES

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Several mechanisms of glutamate release have been proposed, and it is likely that more than one does operate within an astrocyte. Evidence has been provided to support roles for the four pathways: exocytosis, hemi-channels, anion transporters, and P2X receptors. However, under the condition of physiological Ca²⁺ elevations, there is a groundswell of support for an exocytotic mechanism. Although release through hemi-channels (234) and P2X₇ receptors (46) has been proposed, effective release requires the presence of low divalent saline to promote opening of these channels (67, 101). Since under resting conditions at the membrane potential of an astrocyte and with normal divalent cation concentrations the open probability of hemi-channels and of P2X₇ receptors is so low, these pathways are unlikely to be utilized in physiological conditions. However, it should be noted that during neuronal activity, external Ca²⁺ can fall substantially, opening the possibility for these pathways of gliotransmission to become activated under conditions of elevated neuronal activity.

Further doubt has been cast on the potential role of hemi-channel-mediated gliotransmission by the clear demonstration that several antagonists that have been used to block these channels are nonselective and also block P2X₇ receptors. Additionally, using genetically modified astrocytoma cells, as well as astrocytes from connexin43^–/– and P2X₇R^–/– mice, the release of ATP under low divalent cation conditions has been clearly demonstrated to be mediated by P2X₇R, not by hemi-channels (199).

Pharmacological evidence has supported a P2X₇ mechanism of release (46); however, debate about whether this receptor is expressed in the nervous system (108, 191), and because it is unlikely to be regulated by elevations of internal Ca²⁺, limits enthusiasm for this pathway. However, in hippocampal slices, sustained activation by BzATP of a receptor that has features similar to the P2X₇ receptor has been recently reported to mediate a sustained glutamate efflux from astrocytes (55). Under pathological conditions, such as ischemia and brain trauma, this P2X₇-like receptor in astrocytes might be activated by increasing concentrations of extracellular ATP (47, 59, 125, 130, 134). The consequent glutamate release may contribute to increasing the extracellular concentration of glutamate to the abnormal levels that cause excitotoxic cell death (35). Consistent with this hypothesis are the observations that following an acute spinal cord injury in rats, the functional recovery was enhanced and the death of motoneurons decreased when P2X₇ receptors were pharmacologically inhibited (222).

The role of anion transporters/channels is unclear at this time. One of the problems with studying this pathway is the poor selectivity of antagonists. For example, NPPB, which inhibits transporters, can inhibit the filling of vesicles with transmitters (212). Nonetheless, under conditions that promote swelling, it is likely that this pathway could contribute to the release of glutamate from the astrocyte (103, 104). The expression of dominant negative vesicle proteins in astrocytes to inhibit Ca²⁺-regulated exocytosis leaves a swelling-induced pathway of transmitter release from astrocytes unaffected (236). Thus a volumetric release pathway likely exists in parallel to an exocytotic pathway (203). A future challenge is to identify the conditions that select for each of these two pathways.

There is now compelling evidence supporting an exocytic mechanism of glutamate release from astrocytes. These glial cells express a variety of vesicle proteins that are essential for exocytosis (39, 83, 128, 161, 227, 236). Clostridial toxins, when introduced into the astrocyte to cleave target SNARE proteins, prevent glutamate release (10). Ca²⁺ elevations lead to an increase in membrane surface area synchronous with the release of glutamate (237). Bafilomycin A₁, which inhibits the V-ATPase that pumps protons into the vesicle (23) that are required to drive the transport of glutamate, inhibits the uptake of L-[³H]glutamate in astrocyte vesicles (39) and reduces the release of glutamate (10, 166). Rose Bengal, an inhibitor of vesicular glutamate transporters (VGLUT), similarly blocks Ca²⁺-dependent glutamate release (140). Finally, immunoelectron microscopy has revealed VGLUT expressing vesicles within the process of astrocytes in vivo (20).

Volterra and colleagues (20) have used total internal reflection fluorescence microscopy to reveal expressed VGLUT-EGFP fusion proteins as well as acridine orange (AO), a dye that accumulates in acidic organelles, and can be used as a marker for exocytosis. Because of an absorbance shift in AO when located within vesicles compared with physiological saline, fusion of an AO-filled vesicle with the plasma membrane leads to a rapid change in AO fluorescence emission, when excited at 490 nm, as this dye mixes with the extracellular saline. Stimuli that induce Ca²⁺ elevations were shown to cause a brief burst of AO fluorescence events together with a reduction in the numbers of VGLUT-EGFP fluorescent puncta, observations consistent with regulated exocytosis in astrocytes. Furthermore, cocultured glutamate receptor expressing reporter cells simultaneously detected the release of glutamate providing strong evidence supporting exocytotic release of this gliotransmitter from astrocytes. These results have recently been supported by an independent study which has shown Ca²⁺-dependent fusion of vesicles with the plasma membrane (39).

	IX. KISS-AND-RUN RELEASE OF GLUTAMATE FROM ASTROCYTES

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Although this evidence is overwhelmingly in support of a exocytic mechanism, there are still detractors who argue that it is possible that the vesicle is inserted into the membrane to provide a channel or transporter to mediate the release of glutamate. Although we feel that the evidence does not support their contention, there is one troubling aspect to the vesicular hypothesis of glutamate release from the astrocyte, since there are few vesicles within astrocytic profiles in vivo. However, one recent observation suggests that the mechanism of transmitter release from the vesicle may be biased towards a kiss-and-run fusion mechanism in which the vesicle does not fully fuse with the plasma membrane but instead forms an ephemeral pore that permits transmitter to be released (33). This possibility is based on the observation that synaptotagmin IV is essential for Ca²⁺-dependent glutamate release from astrocytes (236). The synaptotagmin gene family is known to play essential roles in the regulation of vesicle fusion in different cell types (105). In nerve terminals, synaptotagmin I plays a dominant role. However, when synaptotagmin IV is expressed in place of synaptotagmin I, fusion events are biased toward kiss-and-run release rather than full fusion (221). An important consequence is that the vesicle reacidifies, a critical step for refilling of the vesicle with transmitter, up to 20 times faster after kiss-and-run release compared with full fusion events (63). Consequently, if synaptotagmin IV does indeed promote kiss-and-run release of glutamate from astrocytes, one can envision that one astrocytic vesicle is equivalent to 20 nerve terminal vesicles.

Studies of exocytosis have been significantly advanced by the electrochemical detection of released transmitters. This approach has now been turned to investigate the Ca²⁺-dependent release of transmitter from astrocytes (33). Because glutamate is not directly detected by carbon fiber amperometry, astrocytes were preloaded with dopamine, which, if released, is readily detected electrochemically. Stimuli that elevate astrocytic Ca²⁺ were shown to cause rapid amperometric spikes. Mechanical stimuli, which cause large prolonged Ca²⁺ elevations in astrocytes, caused very large amperometric signals. However, physiological stimuli only led to the release of 1/10th of the vesicle content. During the brief opening of a fusion pore (2 ms), they showed quantal transmitter release and suggest that relatively large vesicles (300 nm diameter) normally serve to mediate glutamate release through a kiss-and-run mechanism that only depletes a portion of the vesicular transmitter store.

Such a kiss-and-run mechanism could account for transmission in vivo where there is a paucity of astrocytic vesicles. Though they did demonstrate similar results with acutely isolated astrocytes to answer concerns about culture artifacts, and an independent study supports the potential for large vesicular structures mediating transmitter release from astrocytes in brain slices (97), it is not yet clear where these large vesicles reside within an astrocyte in vivo. Indeed, immunoelectron microscopy has shown the presence of 30-nm vesicles in an independent study (20).

	X. THE TRIPARTITE SYNAPSE: ASTROCYTES MODULATE NEURONAL EXCITABILITY AND SYNAPTIC TRANSMISSION

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Following the discovery of the regulated release of glutamate from astrocytes, many studies have gone on to demonstrate that this gliotransmitter can modulate synaptic transmission and neuronal excitability. In addition to glutamate, glial-released D-serine and ATP have been discovered to mediate powerful synaptic actions. To maintain some coherence in our discussion, we present information related to each gliotransmitter in independent sections.

	XI. RELEASE OF GLUTAMATE FROM ASTROCYTES

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Initially cell culture studies showed that astrocytes can use glutamate to modulate neuronal excitability and synaptic transmission. Whole cell recordings from a neuron that was cocultured with astrocytes demonstrated slow glutamate-mediated inward currents (SICs) when a glial Ca²⁺ elevation confronted the recorded neuron (12). These results were later substantiated in thalamus and hippocampus by showing pure N-methyl-D-aspartate (NMDA) receptor-dependent neuronal currents following astrocytic Ca²⁺ elevations (54, 55, 163). In addition to direct activation of neuronal currents, glial-released glutamate was also shown to modulate synaptic transmission. Again in culture, astrocytic Ca²⁺ elevations augmented the frequency of miniature excitatory postsynaptic currents (EPSCs) and inhibitory postsynaptic currents (IPSCs), an action that was judged to be due to the gliotransmitter glutamate because effects were blocked by the NMDA receptor antagonist D-AP5 (14). In brain slice preparations, similar actions were observed because GABAergic activation of Ca²⁺ signals in astrocytes caused an NMDA receptor-dependent increase in inhibitory mIPSC frequency detected in pyramidal neurons and a strengthening of certain inhibitory synapses (96). Subsequently, other forms of synaptic modulation have been identified in which metabotropic glutamate receptors and kainate receptors mediate the actions of glial-released glutamate. Single astrocyte photolysis of caged IP₃ increases the frequency of excitatory mEPSCs, an action that is blocked by the metabotropic receptor antagonists LY367385 and 2-methyl-6-(phenylethynyl)-pyridine, suggesting that glutamate release from astrocytes can act through neuronal class I metabotropic glutamate receptors to augment the release of transmitter from nerve terminals (58). Flash photolysis of caged Ca²⁺ also increases spontaneous action potential-driven IPSCs, an action that is mediated by kainate receptors containing the GluR5 subunit (120).

These studies clearly show the potential for astrocytes to integrate neuronal activity and to provide feedback modulatory signals. The roles for these feedback pathways are not yet known in the hippocampus. However, the first critical demonstration of the integrated action of synaptic activity and synaptically associated glial signals was provided by studies performed at the frog neuromuscular junction. Associated with the neuromuscular junction are several perisynaptic Schwann cells that are molecularly and functionally distinct cells from the myelinating Schwann cell. Richard Robitaille (178) performed elegant experiments in which he directly manipulated GTP-binding protein signaling within these glia. Direct microinjection of guanosine 5'-O-(3-thiotriphosphate) (GTPS) into the perisynaptic Schwann cell to activate G protein signaling caused a reduction in the strength of the neuromuscular junction. To ask whether this glial-modulation pathway is recruited during physiological conditions, he studied activity-dependent depression of this synapse. High-frequency stimulation of the motoneuron axon causes a reversible depression of the neuromuscular connection. However, after injection of guanosine 5'-O-(2-thiodiphosphate) (GDPS) into the Schwann cell, to prevent G protein activation, stimulation of the nerve trunk led to a diminished depression. Taken together, these studies led to the proposal of the "tripartite synapse" in which the astrocyte listens to synaptic activity and provides feedback modulation of the strength of the synaptic connection (13).

	XII. ASTROCYTES ACTIVATE EXTRASYNAPTIC NMDA RECEPTORS

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As discussed above, cell culture studies initially demonstrated that astrocytes, by releasing glutamate, can activate neuronal NMDA receptors. Substantiation of this observation was recently provided in brain slice studies in which glial glutamate was shown to selectively access a specific class of NMDA receptor that contains the NR2B subunit (54). In these studies a variety of stimuli, each of which leads to astrocytic Ca²⁺ oscillations, all caused D-AP5-sensitive, NMDA receptor-mediated SICs in area CA1 pyramidal neurons. Moreover, blockade of synaptic transmission by brief incubation in tetanus toxin did not prevent the detection of these SICs showing that they were from a nonneuronal origin. (It should be noted that while tetanus toxin can prevent glutamate release from astrocytes when applied from the extracellular space, it does so with such a slow time course, due to a paucity of toxin receptors, that it is possible to selectively inactivate nerve terminals with short-term treatment.) Finally, single-cell stimuli such as flash photolysis of caged Ca²⁺, specifically in the astrocyte, or single astrocyte depolarization all evoked neuronally detected NMDA receptor (NMDAR)-dependent SICs.

Where are the NMDARs located that mediate the SIC? Using MK