PHYSIOLOGICAL REVIEWS   Vol. 78 No. 1 January 1998, pp. 99-141
Copyright ©1998 by the American Physiological Society

Glial Calcium: Homeostasis and Signaling Function

ALEXEJ VERKHRATSKY, RICHARD K. ORKAND, AND HELMUT KETTENMANN

Department of Cellular Neurosciences, Max-Delbrück Center for Molecular Medicine, Berlin-Buch, Germany; and Institute of Neurobiology, University of Puerto Rico, San Juan, Puerto Rico

I. INTRODUCTION
II. METHODOLOGICAL CONSIDERATIONS
    A. Types of Glial Cells
    B. Experimental Approaches to Measure Intracellular Ca²⁺
III. AN OVERVIEW OF CALCIUM HOMEOSTASIS IN GLIAL CELLS
    A. Resting Intracellular Ca²⁺ in Glial Cells
    B. Ca²⁺-Permeable Channels
    C. Ca²⁺ Storage Organelles, Intracellular Ca²⁺ Release, and Store-Operated Channels
    D. Ca²⁺ Transporters
    E. Intracellular Ca²⁺ Sensors and Effectors
IV. VOLTAGE-GATED CHANNELS AND DEPOLARIZATION-INDUCED CALCIUM SIGNALS
    A. Schwann Cells
    B. Astrocytes
    C. Oligodendrocytes
    D. Mechanisms of Glial Cell Depolarization
V. NEUROTRANSMITTER-INDUCED CALCIUM SIGNALING IN GLIAL CELLS
    A. Glutamate
    B. Purines and Pyrimidines
    C. Monoamines
    D. -Aminobutyric Acid and Glycine
    E. Acetylcholine
    F. Histamine
    G. Substance P
    H. Bradykinin
    I. Endothelins
    J. Other Agonists Linked to Intracellular Ca²⁺ Regulation in Glial Cells
    K. Heterogeneity of Neurotransmitter Receptor Expression in Glial Cells
VI. SPATIOTEMPORAL ORGANIZATION OF CALCIUM SIGNALS
    A. Intracellular Ca²⁺ Oscillations
    B. Intercellular Ca²⁺ Waves
VII. GLIAL CALCIUM SIGNALING AND NEURON-GLIAL INTERACTIONS
VIII. GLIAL CALCIUM AND BRAIN PATHOLOGY
IX. CALCIUM SIGNALS AND GLIAL FUNCTION
X. CONCLUDING REMARKS: CALCIUM SIGNALS ARE A CONSEQUENCE OF GLIAL EXCITABILITY
REFERENCES

	ABSTRACT

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Verkhratsky, Alexej, Richard K. Orkand, and Helmut Kettenmann. Glial Calcium: Homeostasis and Signaling Function. Physiol. Rev. 78: 99-141, 1998.  Glial cells respond to various electrical, mechanical, and chemical stimuli, including neurotransmitters, neuromodulators, and hormones, with an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i). The increases exhibit a variety of temporal and spatial patterns. These [Ca²⁺]_i responses result from the coordinated activity of a number of molecular cascades responsible for Ca²⁺ movement into or out of the cytoplasm either by way of the extracellular space or intracellular stores. Transplasmalemmal Ca²⁺ movements may be controlled by several types of voltage- and ligand-gated Ca²⁺-permeable channels as well as Ca²⁺ pumps and a Na⁺/Ca²⁺ exchanger. In addition, glial cells express various metabotropic receptors coupled to intracellular Ca²⁺ stores through the intracellular messenger inositol 1,4,5-trisphosphate. The interplay of different molecular cascades enables the development of agonist-specific patterns of Ca²⁺ responses. Such agonist specificity may provide a means for intracellular and intercellular information coding. Calcium signals can traverse gap junctions between glial cells without decrement. These waves can serve as a substrate for integration of glial activity. By controlling gap junction conductance, Ca²⁺ waves may define the limits of functional glial networks. Neuronal activity can trigger [Ca²⁺]_i signals in apposed glial cells, and moreover, there is some evidence that glial [Ca²⁺]_i waves can affect neurons. Glial Ca²⁺ signaling can be regarded as a form of glial excitability.

	I. INTRODUCTION

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Glial intracellular calcium ([Ca²⁺]_i), like that of other eukaryotic cells, is highly regulated; the free [Ca²⁺]_i is four to five orders of magnitude less than that in the narrow system of clefts that constitutes the functional extracellular environment of the nervous system. There is, therefore, a steep electrochemical gradient favoring Ca²⁺ entry; transient cellular activation increasing Ca²⁺ permeability will lead to a transient increase in [Ca²⁺]_i . In addition, there are Ca²⁺ stores within the cell that may release Ca²⁺ in response to specific intracellular chemical messengers, also increasing [Ca²⁺]_i . These transient rises of [Ca²⁺]_i in turn trigger or regulate various intracellular events, including metabolic processes, gene expression, and ion transport systems. Therefore, changes in [Ca²⁺]_i act as an eclectic second messenger system coordinating changes in the external environment with intracellular processes. These observations, in a wide variety of cells, have led to a general appreciation of the specific role of [Ca²⁺]_i in cell signaling (for general references, see Refs. 42, 73, 147, 245, 345). Cells have developed specialized machinery to control the spatial and temporal characteristics of these Ca²⁺ signals. These include transmembrane Ca²⁺ transporters and Ca²⁺-permeable channels, cytoplasmic buffers, and intracellular organelles that are able to accumulate, store, and release Ca²⁺.

View larger version (110K): [in this window] [in a new window]   FIG. 1.   Earliest illustration of all 3 glial elements in central nervous system by Del Rio-Hortega (95). A: protoplasmic astrocytes from gray matter. B: fibrous astrocytes from white matter. C: microglia. D: interfascicular glia, or oligodendrocytes, from white matter. [From Del Rio-Hortega (95).]

In the nervous system, Ca²⁺ regulation has been extensively investigated and well characterized in a variety of neurons (17, 134, 290, 387, 430). These investigations demonstrate that neuronal [Ca²⁺]_i participates in the control of important neuronal functions, like electrical excitability, neurotransmitter release, and long-term changes in synaptic efficacy. In parallel, but to a much lesser extent, knowledge has accumulated on the homeostasis and role of Ca²⁺ signaling in glial cells. The perception of the role of glia in brain function has changed dramatically over the last 10 years from that of a supporting glue (Greek glia is glue) with mainly trophic functions to that of a cell with dynamic interactions with neurons actively participating in nervous system function. This change occurred after the development of new techniques, like patch-clamp recording and Ca²⁺ imaging, that revealed that glial cells express a wide variety of ion channels and neurotransmitter receptors that make them able to detect and respond to neuronal activity (429, 432). Changes in glial [Ca²⁺]_i have been measured under a variety of conditions where glial cells are responding to electrical, mechanical, and chemical stimuli. These fluctuations in [Ca²⁺]_i appear to be a consistent response of glial cells to changes in the environment that lead to a change in glial function; they are not passive responses and, therefore, can be considered a form of glial excitability mediated by calcium. This review primarily includes recent insights into the major mechanisms involved in the control of [Ca²⁺]_i and the role of changes in [Ca²⁺]_i in glial signal transduction in response to neuronal activity. In addition, we consider what is known of changes in [Ca²⁺]_i that affect glial function and accompany pathological processes.

	II. METHODOLOGICAL CONSIDERATIONS

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Glial cells (Fig. 1) are found throughout the vertebrate central nervous system (CNS) (for overview on glial cell biology, see Ref. 223). The macroglial cells, astrocytes, and oligodendrocytes are of ectodermal origin, whereas the microglial cells are thought to stem from the mesoderm. Astrocytes are probably the most diverse population of glial cells. One of their hallmarks is the expression of intermediate filament proteins, glial fibrillary acidic protein (GFAP) or S100. There is a battery of commercially available antibodies that can be used as markers to identify astrocytes. However, the expression of GFAP can vary among astrocytes and can change during development, particularly in pathological conditions. The astrocytic response to injury is marked by an increase in GFAP expression, and these cells are termed reactive astrocytes. Astrocytes in culture probably represent reactive astrocytes, since they obviously sense the strange culture environment and prominently express GFAP. Expression of GFAP is, however, not a marker for all astrocytes: Müller cells in the retina do not express GFAP under normal conditions, but only under pathological conditions. What then is the definition of an astrocyte? The answer may be that they characteristically have two contact sites, the neuronal membrane (synaptic regions in the gray matter and axons in the white matter) and the border of the CNS, either the blood system or the ventricular walls. Astrocytes can be subdivided into three major populations: radial astrocytes, fibrous astrocytes, and protoplasmic astrocytes with transition forms between these populations. Bergmann glial cells of the cerebellum are a prominent example of a radial (astrocytic glial cell). Fibrous astrocytes send a large number of processes into all directions, whereas protoplasmic astrocytes, mainly located in the gray matter, have short ramified and crimped processes (for review of astrocyte morphology, see Ref. 354).

Oligodendrocytes are the myelin-producing cells of the CNS (406). They produce myelin proteins such as myelin basic protein, proteolipid protein, myelin-associated glycoprotein, and cyclic nucleotide phosphodiesterase. Antibodies against these proteins can be used as oligodendrocyte markers. In white matter, their function seems to be well defined: they enwrap axons and form the myelin sheath. Oligodendrocytes are prominently found in white matter but can also be found in gray matter. There are also oligodendrocytes that do not myelinate, namely, the perineuronal oligodendrocytes. At present, their functions are not known.

Microglial cells are thought to invade the brain during embryonic and early postnatal period. They stem from the monocytic lineage and thus have many common features with cells of the monocytic lineage. After invasion, they distribute equally in the brain parenchyma, and each cell seems to have a defined territory. Microglial cells are the immunocompetent cells of the CNS and can express the relevant molecules such as the major histocompatibility complex II. Under normal physiological conditions, microglial cells are in a resting state and have a small soma and fine ramified processes. After any disturbance of the nervous system, they can be activated and respond in a defined manner, converting from the resting form ultimately to a cytotoxic, phagocytic cell. This transition is graded and probably, in part, controlled by factors of the immune system, such as complement factors or cytokins (see Refs. 145, 246 for review).

All types of glial cells have been studied under a variety of conditions. There is increasing evidence that the expression of glial properties depends both on the origin of the cells and the precise experimental conditions for study. The variables are numerous and need to be precisely defined in terms of the following: 1) type of cell (including subtype where applicable), e.g., astrocyte, oligodendrocyte, Schwann cell, or microglia; 2) cellular origin, including not only the species and age of the animal but also the brain region; 3) type of preparation, in vivo, acutely prepared slice, or slice and dissociated cells in tissue culture (including time in culture and presence of other cell types), chemical environment during preparation, preservation, and experiment; and 4) experimental approaches, e.g., anatomy, electrophysiology, histochemistry, ion imaging techniques (ion-sensitive dyes, ion probe microscopy), and molecular biology.

Given the multitude of variables, it is hardly surprising that there is little unanimity of opinion or even consistent results regarding the properties of neuroglia. The diversity of results is tending to focus on a few central problems. The control of [Ca²⁺]_i and its variation during glial responses is one of those problems.

Attempts to measure [Ca²⁺]_i in glial cells have paralleled those in other tissues and include the use of radioisotope tracers, Ca²⁺ ion-selective electrodes, electron-probe microscopy, and in more recent time Ca²⁺-sensitive fluorescent dyes (see Refs. 159, 385, 417 for review). Each of the methods has serious limitations that dictate the choice of glial preparation for study. The initial measurements of [Ca²⁺]_i were carried out in steadily growing cultures of glial cell lines (47) and primary glial cultures (283). As indicated above, glial cells change during development. Therefore, it was important to correlate the [Ca²⁺]_i measurement with the developmental stage. Thus Ca²⁺ fluxes and [Ca²⁺]_i recordings were carried out along with immunostaining with stage-specific antibodies (e.g., Refs. 182, 230, 436).

Experiments on cultured cells raised the question of whether the [Ca²⁺]_i handling mechanisms remain unaltered after the cells were removed from their natural environment and maintained under artificial conditions in the absence of neurons. To solve this problem, [Ca²⁺]_i recording techniques were applied first to freshly isolated cells (e.g. Refs. 103, 131) and then to cells in acutely prepared brain slices. Initially, the technique combining patch-clamp electrophysiological recordings with Ca²⁺-sensitive fluorescent dyes was applied to neurons (13, 265); later, it was used in glial cells (235, 237, 301). In these experiments, the patch-clamp whole cell configuration was employed to inject Ca²⁺-sensitive probes into glial cells. This technique confines the [Ca²⁺]_i recording to a single, morphologically identified cell and allows simultaneous electrophysiological recording. However, prolonged intracellular dialysis can significantly disturb [Ca²⁺]_i regulation (235). As an alternative, Ca²⁺-sensitive dye can be injected via microelectrodes into the cell of interest (104), or the slices can be incubated with membrane-permeant forms of fluorescent Ca²⁺ probes (33, 239, 240, 349, 351). A major difficulty with this technique is that the background fluorescence is unknown; this may lead to a miscalculation of [Ca²⁺]_i . This problem can be resolved by combining [Ca²⁺]_i measurements from cells loaded with a permeable form of the dye with subsequent intracellular dialysis. The latter helps to wash the dye from the cell of interest so that an actual background fluorescence value can be determined (240).

	III. AN OVERVIEW OF CALCIUM HOMEOSTASIS IN GLIAL CELLS

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The general mechanisms of [Ca²⁺]_i homeostasis are common to all eukaryotic cells (see Refs. 73, 245, 345 for review). Intracellular Ca²⁺ is determined by the interaction of membrane Ca²⁺ transporters and cytoplasmic calcium buffers (Fig. 2). The Ca²⁺ transporters are represented by several superfamilies of transmembrane Ca²⁺-permeable channels, ATP-driven calcium pumps, and electrochemically driven Ca²⁺ exchangers. The resulting Ca²⁺ fluxes may either deliver or remove Ca²⁺ from the cytoplasm. Upon entering the cytoplasm, most Ca²⁺ is trapped by Ca²⁺-binding proteins; this determines the Ca²⁺-buffering capacity of the cell. Calcium transporters are localized in the cell membrane (providing Ca²⁺ exchange between the cell interior and exterior) and in the membrane of intracellular organelles [e.g., endoplasmic reticulum (ER), mitochondria, Golgi complex, and nucleus]. The latter forms the intracellular Ca²⁺ storage system (352), which actively accumulates Ca²⁺. Accumulated Ca²⁺ is bound to intraluminal proteins, and it can be rapidly released via intracellular Ca²⁺ channels. This general scheme is applicable to all types of glial cells (429). A peculiar feature of glial cells is their high degree of heterogeneity with respect to the expression of various molecular cascades involved in [Ca²⁺]_i regulation.

View larger version (40K): [in this window] [in a new window]   FIG. 2.   General scheme of molecular cascades involved in intracellular calcium signaling (see discussion in text). VGCC, voltage-gated Ca2+ channels; SOCC, stores-operated Ca2+ channels; PMCA, plasmalemmal Ca2+-ATPase; Ca2+-BP, Ca2+ binding proteins; InsP3R, inositol 1,4,5-trisphosphate receptor/inositol 1,4,5-trisphosphate-gated Ca2+ channel; RyR, ryanodine receptors/Ca2+-gated Ca2+ channel; SERCA, sarco(endo)plasmic reticulum Ca2+-ATPase. Intracellular Ca2+ sensors are as follows: CaM, calmodulin; CaM kinase, Ca2+/calmodulin-dependent protein kinase; CaM AC, Ca2+/calmodulin-dependent-adenylate cyclase; CaM-phosphatase, Ca2+/calmodulin-dependent-protein phosphatase; Ras, p21ras guanine nucleotide-binding proteins; Raf, raf protein kinase; MEK, mitogen-activated/extracellular regulated kinase; MAPK, mitogen-activated protein kinase; IEG, immediate early genes.

Free cytoplasmic Ca²⁺ is a minor part (<0.001%) of total calcium in glial cells. Most is associated with intracellular organelles (e.g., ER, mitochondria, and Golgi apparatus). Resting [Ca²⁺]_i in glial cells varies from 30-40 to 200-400 nM (see Table 1). This variation is not only among subtypes of glia, but also within the same population of cells. It may reflect method-induced artifacts or indicate the flexibility of [Ca²⁺]_i homeostasis. Most measurements were made using membrane-permeable forms of calcium indicators; thus all the problems associated with this method (uncertain calibration, dye Ca²⁺ buffering, compartmentalization, and photobleaching) may contribute to the variability. Nevertheless, even in experiments performed on Bergmann glial cells in cerebellar slices (235) with careful intracellular calibration procedures, the resting [Ca²⁺]_i ranged from 30 to 200 nM. This variability did not appear to reflect cell damage, because in all cases the resting potential determined by whole cell recordings remained about normal (75 to 60 mV).

The initial electrophysiological surveys of glial cells of various origin (248, 249, 361; see Ref. 392 for review) did not reveal voltage-sensitive channels. With improved techniques, e.g., voltage clamping and patch clamping, the surprising finding was made that some glial cells exhibit a variety of voltage-gated ion channels that were previously believed to be present only in electrically excitable cells (23, 37, 393). Several populations of both peripheral and central macroglia were shown to express voltage-gated Ca²⁺ channels similar to those found in neurons (392). Later, it was found that Ca²⁺ influx through voltage-gated channels significantly increases [Ca²⁺]_i in astro- and oligodendrocytes as well as in Schwann cells. However, not all glia express Ca²⁺ channels. For example, Bergmann glial cells, microglia, and certain populations of astrogliomas seem to lack voltage-dependent Ca²⁺ channels (192, 239, 429). Nevertheless, Ca²⁺ may enter glial cytoplasm via ligand-gated channels that are abundantly expressed in almost all glial cell subtypes (397). Finally, certain types of glial cells (e.g., retinal Müller cells or cultured astrocytes) express nonspecific cation channels that may also pass Ca²⁺ (227, 356).

Little is known about Ca²⁺ storage organelles in glial cells. Many contain an elaborate ER (143, 354) that presumably serves as a major substrate for rapidly exchanging Ca²⁺ stores. Calcium accumulation by glial ER involves ER pumps that, like other cells, are inhibited by thapsigargin (60, 235, 237) and cyclopiazonic acid (158). Aplysia glial cells were found to have an unusual analog of Ca²⁺ stores, so called "gliagrana" (217, 218), which may retain an enormously high (up to 50-100 mM) Ca²⁺ concentration. The density of these gliagrana varies with fluctuations in extracellular Ca²⁺ ([Ca²⁺]_o). Increases or decreases in glial calcium depending on [Ca²⁺]_o suggest the possible involvement of glia in the regulation of [Ca²⁺]_o . Similar stores have been described in frog ependymal glia and human astrocytes (143).

The major mechanism for Ca²⁺ release from internal stores involves activation of inositol 1,4,5-trisphosphate (InsP₃)-gated Ca²⁺ release channels (InsP₃ receptors, Refs. 34, 139). The production of InsP₃ , in turn, is achieved by the activation of phospholipase C (PLC) coupled via G proteins with numerous "metabotropic" plasmalemmal receptors. The nature of the InsP₃ receptors subtypes in different glial cells is not known in detail. In rat cortical astrocytes and cerebellar Bergmann glial cells, only type 3 but not type 1 and 2 InsP₃ receptors have been immunolocalized (453). Oligodendrocytes were reported to transiently express type 1 InsP₃ receptors in a short period during the onset of myelination (98). The direct activation of InsP₃ receptors by photorelease of InsP₃ from caged compound was shown in cultured astrocytes (224, 382). Astrocytic InsP₃ receptors appear to be substantially more sensitive to InsP₃ than InsP₃ receptors in Purkinje neurons; the threshold InsP₃ concentration for activation of the InsP₃-gated channel in astrocytes was 0.2-0.5 µM, whereas in Purkinje neurons, it was 9 µM (224). Inositol 1,4,5-trisphosphate-induced Ca²⁺ release is involved in the majority of glial responses to neurotransmitters and neurohormones (see sect. V). The glial expression of another type of intracellular Ca²⁺ release channel, the Ca²⁺-gated channel [or ryanodine receptor (RyR); Refs. 138, 287] is still debatable. Functional Ca²⁺-induced Ca²⁺ release (CICR), sensitive to the classical modulators ryanodine and caffeine and activated under physiological conditions, has been demonstrated only for periaxonal Schwann cells (258) and for freshly isolated Müller glial cells from salamander retina (219). In astrocytes, data on CICR are controversial; there is the one report that caffeine triggered a [Ca²⁺]_i increase in cultured embryonic cortical astrocytes (158). In contrast, several observations in cultured and freshly isolated astrocytes failed to detect an obvious caffeine-triggered [Ca²⁺]_i effect (60, 103). However, ryanodine and dantrolene, believed to be CICR antagonists, modulated [Ca²⁺]_i responses in astrocytes (60, 253). Finally, in Bergmann glial cells, studied in cerebellar slices, caffeine and ryanodine triggered a moderate [Ca²⁺]_i elevation and attenuated [Ca²⁺]_i transients evoked by kainate (S. Kirischuk and A. Verkhratsky, unpublished observations). In oligodendrocytes, caffeine and ryanodine did not affect [Ca²⁺]_i (238). In general (with several exceptions), glial [Ca²⁺]_i appears to be insensitive to caffeine, reflecting either an absence of ryanodine receptors in glial cells or the specific expression of "brain" ryanodine receptor isoform (RyR3), which is not modulated by caffeine (149, 395). This particular ryanodine receptor subtype could be activated by a newly discovered second messenger, cyclic ADP ribose (cADPR) (141); the possible effect of cADPR on glial [Ca²⁺]_i has not been investigated.

The amount of Ca²⁺ bound to internal stores may also regulate a distinct type of plasmalemmal Ca²⁺ permeability: it is widely recognized that the depletion of [Ca²⁺]_i pools activates a capacitative Ca²⁺ influx. This influx is associated with the activation of specific, store-operated plasmalemmal Ca²⁺ channels (75, 188). The existence of these channels in glial cells has not been clearly shown, although there are a number of suggestions that they might be important for Ca²⁺ homeostasis in gliomas (175, 362), astrocytes (418), and microglial cells (292, 293), although at least one group (347) reported that their attempts to find the capacitative Ca²⁺ entry in cultured astrocytes failed. In microglial cells, the long-lasting activation of capacitative Ca²⁺ entry after the maximal depletion of intracellular Ca²⁺ stores has been described recently (416). Once activated, the capacitative Ca²⁺ entry pathway in microglial cells remained operative for tens of minutes, creating a steady-state [Ca²⁺]_i elevation that dramatically outlasted the period of agonist action.

Mitochondria are another capacious Ca²⁺ storage site. However, their role in [Ca²⁺]_i homeostasis in glial cells is little understood. The dissipation of the mitochondrial electrochemical gradient by protonophores [carbonyl cyanide m-chlorophenylhydrazone (CCCP) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone] triggered Ca²⁺ release in oligodendrocytes (236, 238). However, CCCP treatment did not influence the kinetic parameters of the depolarization-triggered [Ca²⁺]_i transients (238). This suggests that mitochondrial Ca²⁺ accumulation does not play an important role in calcium signal termination under physiological conditions. Under pathological conditions, which may substantially disturb mitochondria, glial [Ca²⁺]_i homeostasis might be markedly affected. Alternatively, mitochondria could play an active role in Ca²⁺ signaling also under physiological conditions: in cultured oligodendrocytes, mitochondrial Ca²⁺ release/accumulation actively shaped intracellular Ca²⁺ waves and [Ca²⁺]_i oscillations originated from InsP₃-sensitive Ca²⁺ stores (388). In cultured astrocytes, histamine-evoked [Ca²⁺]_i oscillations were accompanied by oscillations in intramitochondrial free Ca²⁺ (209), also suggesting that mitochondrial Ca²⁺ store may play an active role in [Ca²⁺]_i homeostasis.

The intracellular distribution of active mitochondria is different in oligodendrocyte progenitors and mature oligodendrocytes. In the former, both rhodamine-123 staining and CCCP-induced Ca²⁺ release were confined to the tips of cellular processes, suggesting that active mitochondria were concentrated in these particular areas. Conversely, in mature oligodendrocytes, mitochondria were evenly distributed (236). Presumably, the preferential localization of active mitochondria in the processes of oligodendrocytic progenitors might be important to supply energy for protein synthesis during cellular growth; it could also be important for [Ca²⁺]_i handling in this subcellular compartment.

A low [Ca²⁺]_i and recovery from increases in [Ca²⁺]_i produced by receptors/channels activation is provided by plasmalemmal Ca²⁺ pumps (57) and an electrochemically driven Na⁺/Ca²⁺ exchanger (40). There is little information on the properties of glial Ca²⁺ pumps. However, it has been shown in oligodendrocytes that La³⁺-sensitive Ca²⁺-ATPases are primarily responsible for the restoration of [Ca²⁺]_i after a depolarization-triggered [Ca²⁺]_i increase (238). In contrast, substantially more information is available on the expression of a Na⁺/Ca²⁺ exchanger. Initial evidences concerning the existence of functional Na⁺/Ca²⁺ exchange in glial cells derived from radiotracer experiments demonstrating that transmembrane fluxes of ⁴⁵Ca²⁺ in glial cells are controlled by extracellular Na⁺ concentration ([Na⁺]_o) (254). In several astrocytic preparations, reduction of the Na⁺ gradient, by lowering [Na⁺]_o , increased [Ca²⁺]_i (96, 156, 158), reduced the Ca²⁺ efflux rate (178, 410), and affected the kinetics of the stimulus-evoked [Ca²⁺]_i (84, 203). Biochemical studies (both the presence of protein and specific mRNA) revealed the expression of a heart isoform of the Na⁺/Ca²⁺ exchanger in astroglial cells (156, 410). Astrocytic Na⁺/Ca²⁺ exchanger was inhibited by 30-min incubation with 0.1-1 mM ascorbic acid (409) and was stimulated by sodium nitroprusside and 8-bromoguanosine 3',5'-cyclic monophosphate (11, 411), suggesting that Na⁺-dependent Ca²⁺ transport in glial cells may be the target for nitric oxide. However, the relative importance of Na⁺/Ca²⁺ exchanger in regulation of [Ca²⁺]_i was questioned in several reports that showed only a minor effect of low [Na⁺]_o on [Ca²⁺]_i (103, 203). Recently, it was demonstrated (156) that a decrease in [Na⁺]_o , by itself, is not sufficient to increase Ca²⁺ influx via the Na⁺/Ca²⁺ exchanger in astrocytes. Nevertheless, under conditions of elevated intracellular Na⁺ concentration ([Na⁺]_i), in ouabain-treated cells, lowering [Na⁺]_o produced a dramatic increase in [Ca²⁺]_i . Interestingly, a ouabainlike compound has been proposed to act as a vertebrate adrenocortical hormone (163). Thus it is possible that the physiological effect of this compound might be to increase Ca²⁺ influx via an increase in [Na⁺]_i .

In Bergmann glial cells in situ, Na⁺/Ca²⁺ exchanger also seems to play a relatively minor role in regulating resting [Ca²⁺]_i . However, Ca²⁺ flux through the exchanger became significant under conditions of elevated [Na⁺]_i . Stimulation of Bergmann glial cells with kainate increased [Na⁺]_i to >30 mM, which turned the exchanger in the reverse mode, providing thus the additional pathway for Ca²⁺ influx. This Ca²⁺ influx significantly altered both the amplitude and kinetics of the kainate-triggered [Ca²⁺]_i signals (233).

An astrocytic Na⁺/Ca²⁺ exchanger may be an important means for glia to regulate the ionic content in the interstitium. Neurons, when being electrically excited, can decrease both [Ca²⁺]_o and [Na⁺]_o in the intercellular clefts (30). Under conditions of lowered [Na⁺]_o , the Na⁺/Ca²⁺ exchanger could reverse and supply the interstitium with Ca²⁺ by expelling it from adjacent astrocytes.

Finally, Na⁺/Ca²⁺ exchanger may be involved in mediating Ca²⁺ excitotoxicity under pathological conditions. In particular, reversal of Na⁺/Ca²⁺ exchanger was found to play an important role in the astrocytic injury due to Ca²⁺ reperfusion after periods of Ca²⁺ depletion (a phenomenon similar to the"Ca²⁺ paradox" well described in cardiac muscle). The reperfusion-induced Ca²⁺ excitotoxicity was significantly decreased in astrocytes in which expression of Na⁺/Ca²⁺ exchanger was inhibited by treatment with antisense oligodeoxynucleotides to the exchanger (282).

After entering the cytoplasm, Ca²⁺ binds to a number of proteins that trigger various intracellular signal transduction pathways (Fig. 2, see Ref. 147 for review). Probably the best known cytoplasmic Ca²⁺ sensor is calmodulin (CaM), which regulates the functional activity of at least three broad classes of enzymes, namely, CaM-dependent protein kinases, protein phosphatases, and adenylate cyclases. The latter either interact with cytoplasmic enzymes or transfer the signal further down to the nucleus, initiating other pathways responsible for gene expression. An alternative way to connect cytoplasmic Ca²⁺ signals and gene expression is associated with Ras proteins (small guanine nucleotide-binding proteins) which after being activated by Ca²⁺ trigger a cascade of phosphorylation events that lead to a modulation of gene expression (127). Finally, cytoplasmic Ca²⁺ signals may propagate to the nucleus, where they directly stimulate the synthesis of immediate early genes as well as structural genes. Unfortunately, little is known of the expression and role of these systems in glial cells; their characterization in glia is an important problem awaiting an experimental solution.

	IV. VOLTAGE-GATED CHANNELS AND DEPOLARIZATION-INDUCED CALCIUM SIGNALS

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Voltage-gated Ca²⁺ channels form an important pathway for Ca²⁺ entry in excitable cells; the latter have been found to express a variety of Ca²⁺ channels, differing in their voltage dependence, kinetics, and pharmacological properties (177, 190). Calcium channels are integral membrane proteins composed of five subunits, each playing a distinct role in channel function. The Ca²⁺ channel subunits are encoded by several gene families. The functional heterogeneity of Ca²⁺ channels arises mainly from differences in ₁-subunit proteins; at least six major subtypes of ₁-subunit have been cloned and characterized (177). On the basis of physiological properties and pharmacological profile, Ca²⁺ channels are classified as low-voltage-activated or T-type channels and several types of high-voltage-activated channels (code named as L, N, P, Q, and R types). Molecular classification based on the diversity of ₁-subunit distinguishes six types of Ca²⁺ channels (CaCh1-6). Glial cells, although being inexcitable from the classical point of view (they are not able to generate action potentials) are capable of expressing voltage-gated Ca²⁺ channels. These have been found in several populations of macroglial cells but so far not in microglia.

Several early attempts to identify Ca²⁺ currents (I_Ca) in cultured Schwann cells (see Ref. 392 for review) were unsuccessful. In 1991, Amedee et al. (5) discovered that Schwann cells are able to express Ca²⁺ channels only when cocultured with neurons. Later, it was shown that expression of Ca²⁺ channels in Schwann cells could be also induced by addition of a nonhydrolyzable analog of adenosine 3',5'-cyclic monophosphate (cAMP) to the culture media (28). Whole cell patch-clamp studies of Schwann cells in organotypic cultures of mouse dorsal root ganglia (DRG) revealed voltage-dependent Ca²⁺ currents. At 10 mM Ca²⁺ outside, the I_Ca had an activation threshold at 45 mV, current-voltage curve maximum at 10 mV, and was rapidly inactivated (complete decay of current took ~150-200 ms). The Ca²⁺ currents in cultured Schwann cells were insensitive to L-type Ca²⁺ channel modulators (nifedipine and BAY K 8644) but were blocked by 5 mM Co²⁺. In a minor cell subpopulation, a slowly decaying, nifedipine-sensitive current component was observed when using 89 mM Ba²⁺ as a charge carrier. The expression of voltage-gated Ca²⁺ channels should provide a means for generating [Ca²⁺]_i transients upon Schwann cell depolarization. However, a direct attempt to measure [Ca²⁺]_i elevation in Schwann cells in a similar DRG coculture (267) failed to detect any measurable [Ca²⁺]_i elevation in response to depolarization by 50 mM KCl.

Recently, voltage-gated Ca²⁺ channels were detected in perisynaptic Schwann cells at the frog neuromuscular junction (368). Calcium channel expression in these cells was visualized using either labeling with monoclonal antibodies against ₂/-subunit (monoclonal antibody 3007; Ref. 424) or fluorescent phenylalkylamine (242). Both markers clearly stained the Schwann cell membrane primarily on the processes close to transmitter release sites. The morphological observations were substantiated by confocal video imaging of [Ca²⁺]_i that demonstrated that perisynaptic Schwann cells respond to high-KCl depolarization with [Ca²⁺]_i transients sensitive to nimodipine (368). Thus it appears the perisynaptic peripheral glia express functional voltage-gated Ca²⁺ channels.

MacVicar (271) first demonstrated Ca²⁺ action potentials in cAMP-treated cultured cortical astrocytes when the K⁺ conductance was blocked and 10 mM Ba²⁺ was added. Subsequently, similar Ca²⁺ action potentials were recorded from Müller glial cells in freshly prepared retinal slices (311), and voltage-clamp experiments on enzymatically dissociated Müller cells revealed currents carried by Ca²⁺ (311).

There are essentially two techniques to detect the presence of voltage-gated Ca²⁺ channels, either by characterizing membrane currents using electrophysiological techniques or by recording [Ca²⁺]_i while activating Ca²⁺ channels with depolarization [commonly by elevating extracellular K⁺ concentration ([K⁺]_o)]. Calcium currents were characterized in detail in cultured cortical astrocytes (24, 71, 85, 273) and type 2 astrocytes from optic nerve (23, 25). A special treatment of cortical astrocytic cultures was necessary to record Ca²⁺ currents, namely, the addition to the culture medium of agents that increase intracellular cAMP. These include treatment with dibutyryl cAMP (71, 236, 273), forskolin, isoprotereneol, or certain types of sera (24, 156). Coculturing with neurons had the same effect (85). In untreated cortical astrocytes, Ca²⁺ currents were usually undetectable. In contrast, in both freshly isolated and cultured astrocytes from the optic nerve, Ca²⁺ currents could be recorded without such pretreatment (23, 25). These results suggest that certain intracellular metabolic processes (i.e., phosphorylation) are necessary to transfer Ca²⁺ channels between "silent" and functional pools and furthermore that neurons may regulate the availability of Ca²⁺ channels in certain types of astrocytes. This also indicates that astrocytes are heterogeneous with respect to Ca²⁺ channel expression.

The parameters of astrocytic Ca²⁺ currents and the types of Ca²⁺ channels expressed vary in cells of different origin. Using a double-microelectrode voltage clamp in cultured cortical astrocytes, MacVicar and Tse (273) recorded a Ca²⁺ current that closely resembled L-type currents described in neurons. This current inactivated slowly, had a typical voltage dependence (threshold at 20 mV and a maximum of the current-voltage curve at +10 mV while using 10 mM Ba²⁺ as a charge carrier), was completely blocked by 1 µM nifedipine, and was potentiated by -adrenergic agonists via increased intracellular cAMP. The amplitude of I_Ca in this preparation was quite substantial, reaching 4-6 nA at 5 mM extracellular Ba²⁺ and 10 nA at 10 mM extracellular Ba²⁺. In contrast, currents recorded from optic nerve astrocytes were much smaller, 200-400 pA, with 10 mM Ba²⁺ as a charge carrier (23). Furthermore, two components of I_Ca were recorded from optic nerve astrocytes (23, 25): inactivating (which was defined as a T-type I_Ca based on its kinetic and voltage dependence) and sustained, sharing properties of an L-type I_Ca (slow inactivation, sensitivity to low concentrations of Cd²⁺, and voltage dependence).

Similarly, small Ca²⁺ currents (<250 pA at 5 mM Ca²⁺ outside) were recorded recently from immature astrocytes in acutely prepared hippocampal slices (Fig. 3, Ref. 2). Hippocampal astrocytes in situ appear to express several types of Ca²⁺ channels as revealed by their sensitivity to various antagonists. The currents observed at voltages between 50 and 20 mV had parameters typical for T-type I_Ca (fast inactivation and sensitivity to amiloride). The I_Ca recorded at higher potentials were partially sensitive to nimodipine, verapamil, and -conotoxin, suggesting the coexpression of L- and N-type Ca²⁺ channels.

View larger version (20K): [in this window] [in a new window]   FIG. 3.   Ca2+ currents in glial cells. Left: low-voltage-activated (LVA) and high-voltage-activated (HVA) Ca2+ currents recorded from astrocytes in stratum radiatum of CA1 region of a hippocampal slice. Slices were prepared from 9- to 12-day-old mice. Ca2+ currents were recorded in Na+- and K+-free external solutions supplemented with 1 µM tetrodotoxin; intrapipette solution contained N-methyl-D-glucamine and tetraethylammonium as major cations. Left trace shows family of currents evoked by different depolarizing pulses after a conditioning hyperpolarization to 110 mV for 1.5 s. Current apparently represents superposition of both LVA and HVA Ca2+ currents. Middle trace shows HVA current in a pure form. To isolate HVA component, cells were held at 50 mV for 1.5 s before test depolarizations. Right trace shows LVA current obtained as a result of subtracting HVA component from total current. Corresponding current (I)-voltage (V) curves are shown at bottom. [From Akopian et al. (2). Reprinted by permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.] Right: 2 types of Ca2+ currents recorded from cultured oligodendrocyte. For Ca2+ current isolation, cells were perfused with Cs+, tetraethylammonium, and 4-aminopyridine solution while bathing in Na+-free media; 20 mM Ba2+ was used as a current carrier. Currents shown on left were evoked by test depolarizations to different voltages (indicated near traces) from holding potential of 75 mV. On right, I-V curves for total (LVA + HVA) Ca2+ current and net HVA current are presented. To separate HVA current, cells were held at a holding potential of 40 mV. [From Von Blankenfeld et al. (436) by permission of Oxford University Press.]

Finally, with the employment of whole cell and perforated patch-clamp recordings, L-type Ca²⁺ currents were recorded in cultured human Müller cells (357). These currents were inhibited by dihydropyridines, but insensitive to -conotoxins. The reverse transcription (RT)-polymerase chain reaction (PCR) examination of total RNA derived from cultured Müller cells revealed expression of mRNAs specific for _1D-, ₂-, and ₃-channel subunits, where ₂-subunit was represented by a splice variant distinct from skeletal muscle _2S- and brain _2B-isoforms.

An important question was to find out whether Ca²⁺ influx via voltage-gated channels alters [Ca²⁺]_i . The initial attempt to resolve this problem employed fura 2 and indo 1 [Ca²⁺]_i recordings from cultured embryonic (272) and neonatal (121) cortical astrocytes. In neonatal astrocytes, membrane depolarization with 25-100 mM KCl resulted in large increases in [Ca²⁺]_i (up to 1 µM) that were inhibited by nimodipine and D-600 (121). In embryonic astrocytes, maintained in confluent culture for 4-6 wk, application of 50 mM KCl generated [Ca²⁺]_i transients with an amplitude of 300-400 nM (272). The KCl-triggered [Ca²⁺]_i elevation was inhibited by nifedipine and significantly potentiated by BAY K 8644, suggesting the influx was mediated by L-type Ca²⁺ channels. In astrocytes kept in a culture for only 1-2 wk, 50 mM KCl did not raise (but rather lowered) [Ca²⁺]_i unless KCl was applied together with BAY K 8644. These results imply that astrocytes at early stages in culture have silent Ca²⁺ channels (272).

The variability of [Ca²⁺]_i channels in cultured cells raised questions as to the presence and function of voltage-gated Ca²⁺ entry in vivo. Freshly isolated mature hippocampal astrocytes (103) promptly respond to KCl application with [Ca²⁺]_i transients (400-800 nM in amplitude in response to 50 mM KCl). Potassium chloride-induced [Ca²⁺]_i transients were blocked by Co²⁺ and verapamil, but, in contrast to cultured astrocytes, they were resistant to dihydropyridines; depolarization-induced [Ca²⁺]_i transients in freshly isolated astrocytes were not affected by dibutyryl cAMP. The importance of voltage-gated Ca²⁺ channels for [Ca²⁺]_i regulation was further illustrated by recording [Ca²⁺]_i elevation evoked by depolarizing steps in voltage-clamped fura 2-loaded astrocytes from hippocampal slices (2). Thus the data available suggest that astrocytes in vivo express voltage-gated Ca²⁺ channels and that Ca²⁺ influx through these channels substantially affects [Ca²⁺]_i .

Oligodendrocytes are heterogeneous with respect to the expression of voltage-gated Ca²⁺ channels. In cultures from cortex, oligodendrocytes expressed both low-voltage (T type) and high-voltage (presumably L type) Ca²⁺ currents (431, 436). The amplitudes of Ca²⁺ currents in mature oligodendrocytes were up to about 200 pA with 20 mM Ca²⁺ as a charge carrier (Fig. 3). In contrast, voltage-clamp analysis of membrane currents in cultured oligodendrocytes isolated from rat optic nerve did not reveal Ca²⁺ currents (23). In situ recordings from oligodendrocytes in a white matter preparation also failed to detect voltage-gated Ca²⁺ currents (31). It could, however, not be excluded that such channels are present in membrane patches remote from the soma such as in the paranodal loops. It is unlikely that even large current injections into the soma will lead to significant membrane depolarization at such distant regions. In support of such an uneven distribution of voltage-gated channels is an observation by Waxman and colleagues (393), who found that voltage-gated Na⁺ channels in Schwann cells are concentrated in the membrane of the paranodal loops.

The expression pattern of Ca²⁺ channels undergoes considerable changes during development. Oligodendrocytic precursors from cortical cultures exhibited both T- and L-type Ca²⁺ currents. (431). Channel density was very low, and whole cell I_Ca were barely detectable (peak amplitudes <100 pA) even when Ba²⁺ was used to carry current. Cultured perinatal and adult oligodendrocyte progenitors from rat optic nerve (45) had only one component of Ca²⁺ current resembling the L type. In the cortical cultures, Ca²⁺ currents were substantially smaller in immature oligodendrocytes/late precursors and could not be detected in young oligodendrocytes. They were readily recorded from mature cells with complex morphology. Although it was not yet possible to detect Ca²⁺ channels in oligodendrocytes in situ, they were found in precursors from slices of mouse corpus callosum (31).

Despite the small amplitude of Ca²⁺ currents in oligodendrocytes, Ca²⁺ influx through voltage-gated channels was found to significantly increase [Ca²⁺]_i . Depolarization of cultured oligodendrocyte precursors and mature oligodendrocytes with KCl revealed substantial [Ca²⁺]_i increases that were sensitive to removal of [Ca²⁺]_o , inhibited by verapamil, and potentiated by BAY K 8644 (44, 45, 230, 238).

The depolarization-induced [Ca²⁺]_i transients in cultured oligodendrocytes were spatially heterogeneous, being in general more pronounced in oligodendrocytic processes (238). Furthermore, at the early developmental stages, T- and L-type Ca²⁺ channels were unevenly distributed over the cell membrane (Fig. 4). A moderate depolarization of the oligodendrocyte precursor by 20 mM K⁺ led to an increase of [Ca²⁺]_i in the processes only, whereas [Ca²⁺]_i levels in the soma remained unaffected. A further increase in [K⁺]_o resulted in a progressive fall in the amplitude of [Ca²⁺]_i elevations in processes, whereas in the soma, [Ca²⁺]_i transients became larger. Moreover, [Ca²⁺]_i signals in processes and in the soma of oligodendrocyte precursors can be dissected pharmacologically; Ni²⁺ (antagonist of low-voltage-activated Ca²⁺ channels) inhibited the depolarization-induced [Ca²⁺]_i transients only in the processes, whereas dihydropyridines preferentially affected somatic depolarization-triggered [Ca²⁺]_i responses (238).

View larger version (36K): [in this window] [in a new window]   FIG. 4.   Spatial heterogeneity of LVA and HVA Ca2+ channels in oligodenroglial precursors. A: differential effect of increasing extracellular K+ on intracellular Ca2+ concentration ([Ca2+]i) in an oligodenroglial precursor cell. Top: pseudocolor images that reflect [Ca2+]i distribution within cell in response to external application of solutions with increasing K+ concentrations. Bottom: changes in fluorescence ratio for fluo 3 (which is a function of [Ca2+]i) were simultaneously measured in soma and in processes. Stars correspond to images shown above. B: distinct pharmacological properties of [Ca2+]i transients in soma (a) and in processes (b) of an oligodendrocyte precursor. Application of 50 mM K+ triggered [Ca2+]i elevation in both regions. However, K+-induced [Ca2+]i elevation was blocked by 50 µM Ni2+ in processes but not in soma. Because Ni2+ preferentially blocks LVA Ca2+ channels, result indicates that these channels are present to a much greater extent in processes than in soma. [From Kirishuck et al. (238). Reprinted by permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.]

An uneven distribution of Ca²⁺ channels was also observed in mature oligodendrocytes; a depolarization-induced [Ca²⁺]_i increase was mainly confined to the processes, whereas [Ca²⁺]_i in the soma increased to a much smaller extent (238).

The opening of voltage-gated Ca²⁺ channels requires depolarization. This depolarization might normally result from local changes in K⁺ concentration that accompany neuronal activity; the [K⁺]_o can increase to 15 mM with intense neuronal firing (405). Such an increase in [K⁺]_o was found to trigger Ca²⁺ influx via voltage-gated channels in cultured oligodendrocytes (238). Much higher levels of depolarization can be achieved under pathological conditions (e.g., spreading depression), when interstitial K⁺ may rise up to 80 mM (313). Alternatively, glial cells can be depolarized by the opening of ligand-gated cationic channels (see sect. V).

	V. NEUROTRANSMITTER-INDUCED CALCIUM SIGNALING IN GLIAL CELLS

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One of the most surprising developments in glial research over the last 25 years has been the discovery that various glial cells express a heterogeneous pattern of functional receptors to a variety of chemicals previously known to affect neurons. These include not only the classical neurotransmitters but also neuromodulators and neurohormones. Table 2 summarizes many of the experimental results that demonstrated an effect of these substances to increase [Ca²⁺]_i . Their effect results from activation of various receptors linked via several pathways to [Ca²⁺]_i regulating molecular cascades.

Glutamate is the major excitatory neurotransmitter in the CNS of mammals, and its action is conducted via activation of highly diversified families of ionotropic and metabotropic receptors. The ionotropic glutamate receptors (GluRs) are ligand-gated cationic channels assembled from five subunits. There are three groups of ionotropic GluRs (according to their pharmacological properties), -amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), kainate, and N-methyl-D-aspartate (NMDA) (180). The recent advances in recombinant DNA techniques have given a precise characterization of the GluRs subunit structure and revealed the molecular determinants of GluRs permeability, gating mechanisms, and agonist specificity (180, 207). The GluR subunits A, B, C, and D (or 1-4) form AMPA-sensitive receptors, GluR5, -6, and -7, and subunits denoted as KA1 and KA2 assembled to form kainate-preferable GluRs. Finally, the NMDA-sensitive GluR subfamily is formed by NMDA R1 and NMDA R2A-D subunits (295). Various GluRs subunits can be differentially assembled forming homo- or heteromeric channels that bear different functional properties. The subunit structure of the GluRs determines their Ca²⁺ permeability, with a crucial role for the GluR B subunit; channels containing GluR B subunit are almost impermeable to Ca²⁺, and those lacking this subunit in the channel pentamer are highly Ca²⁺ permeable (52, 146).

Metabotropic glutamate receptors (mGluRs) also comprise a distinct gene family of at least eight members (mGluRs 1-8); the mGluRs belong to the so-called seven-membrane spanning domains receptors (307, 348). The mGluR1 and -5 are coupled (via G proteins) with PLC, being thus the activators of the InsP₃-mediated intracellular signaling pathway; other mGluRs are connected with adenylate cyclases.

View larger version (29K): [in this window] [in a new window]   FIG. 5.   Stimulation of ionotropic glutamate receptors (GluRs) in Bergmann glial cell in cerebellar slices triggers Ca2+ influx. A: pseudocolor image of a fura 2-loaded Bergmann glial cell (scheme of experiment is shown in inset on left) in control conditions and upon extracellular application of 100 µM kainate. Note preferential increase in [Ca2+]i in cell processes. B and C: [Ca2+]i and membrane current traces recorded from same cell. Both removal of extracellular [Ca2+]i (B) and blockade of ionotropic GluRs by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 µM) inhibited kainate-induced currents and [Ca2+]i elevation, suggesting a key role of Ca2+ influx. F340/380 , fluorescence ratio at 340/380 nm. (From T. Müller and H. Kettenmann, unpublished data.)

Adenosine 5'-triphosphate, adenosine, and related substances control a number of important physiological reactions and act as neurotransmitters in the peripheral nervous system and CNS (54, 102, 133). In recent years, clear evidence that ATP acts as an excitatory neurotransmitter in the CNS has been obtained (108, 109), and pharmacological and molecular characterization of ATP and adenosine receptors (named purinoreceptors) was achieved. Purinoreceptors are represented by a broad family of proteins classified into two major groups (88, 133): 1) adenosine receptors (or P₁ purinoreceptors codenamed also as A₁-A₄ receptors) coupled mainly with adenylate cyclases as well as with PLC (A₁ receptors) and 2) receptors for ATP and related nucleotides known as P₂ purinoreceptors. On the basis of pharmacological properties, the P₂ purinoreceptor family is subclassified into two groups: ionotropic receptors (P_2x and P_2z) and metabotropic receptors (P_2y , P_2u , P_2t and P_2d). Advances in molecular cloning extended this classification by showing that purinoreceptors are encoded by two distinct gene families (55). The family of P_2x receptors comprise several subtypes (labeled P_2x1-7) of ligand-gated ionic channels with a unique two transmembrane domain topology; the members of P_2x family differ in their ion selectivity and gating properties. Cloned P_2z receptors also belong to the P_2x gene family, and they are codenamed as P_2x7 subtype; P_2x7(z) receptors are large transmembrane pores that are activated in fact by a tetraanionic form of ATP (ATP⁴) and may pass molecules with a molecular mass up to 1 kDa. Cloned metabotropic receptors are classified as P_2y family, being represented by seven members (P_2y1-P_2y7). They all are similar to other G protein-linked metabotropic receptors by their seven-transmembrane domain structure and are often associated with PLC and hence InsP₃ turnover. The P_2y1 receptor pharmacologically matches P_2y subtype, P_2y2-P_2u , P_2y3 probably corresponds to P_2t receptor; P_2y4-7 are not yet assigned with known subtypes.

Purinoreceptors are unusually widely distributed among glial cells. A majority of glial cells studied (including peripheral glia, macro- and microglia from the CNS) appear to express various types of purinoreceptors. In many cases, activation of glial purinoreceptors leads to increases in [Ca²⁺]_i .

View larger version (25K): [in this window] [in a new window]   FIG. 6.   ATP-induced Ca2+ signaling in Bergmann glial cells results exclusively from inositol 1,4,5-trisphosphate (InsP3)-mediated Ca2+ release from intracellular Ca2+ pools A: ATP-induced [Ca2+]i transients were measured from "bulk-loaded" Bergmann glial cells (240) by incubating cerebellar slices in fura 2 acetoxymethyl ester (AM)-containing solutions. Addition of ATP triggered an increase in [Ca2+]i that persisted in Ca2+-free extracellular solution. B: in a similar experiment, incubation of slice in 500 nM thapsigargin completely and irreversibly blocked ATP-induced [Ca2+]i increase. C: addition of heparin via intracellular dialysis with a patch pipette inhibited [Ca2+]i increase induced by ATP. Control [Ca2+]i transient was recorded from fura 2-AM-loaded cells before starting intracellular dialysis. D: illustration of spatial distribution of [Ca2+]i at time of maximum ATP response. Note higher levels of [Ca2+]i in Bergmann glial cell processes as compared with cell body. [From Kirischuk et al. (235).]

Monoamines (epinephrine as an adrenal medullary hormone and norepinephrine as a neurotransmitter in both peripheral and CNS) exert their physiological action via a broad family of adrenoreceptors (AR). There are three basic subtypes of adrenoreceptors (₁-AR, ₂-AR, and -AR) that are coupled to different signal transduction systems and have distinct pharmacological profiles (56). The ₁-AR are coupled via G proteins with PLC and/or plasmalemmal Ca²⁺ channels and produce an increase in [Ca²⁺]_i in many tissues, whereas ₂-AR and -AR control the activity of adenylate cyclase. In glial cells, the expression of adrenoreceptors and monoamine-triggered Ca²⁺ signaling has been found exclusively in astrocytes.

Astrocytes express -AR (184, 276) and ₁-AR (256, 383) both in culture and in situ (see also Ref. 284 for review of AR in astrocytes). In cultured astrocytes, the expression of -AR was only found in type 1-like cells (51); ₁-AR are abundant in all types of astrocytes. Stimulation of ₁-AR with norepinephrine increases intracellular levels of InsP₃ (337, 339). Microfluorimetric [Ca²⁺]_i recording techniques revealed that norepinephrine and other monoamines increased cytoplasmic Ca²⁺ in cultured astrocytes (49, 193, 285, 317, 373). The pharmacological profile of the monoamine-induced [Ca²⁺]_i transients demonstrated a leading role for the ₁-AR receptor, although several reports point out that activation of ₂-AR can increase [Ca²⁺]_i (285, 315, 461), presumably involving PLC-driven production of InsP₃ (as revealed by the inhibition of ₂-AR-stimulated [Ca²⁺]_i elevation with the specific PLC blocker U-73122; Ref. 114). The density of ₂-AR linked to the generation of cytoplasmic Ca²⁺ signals was significantly upregulated by incubation of cortical astrocytic cultures with dibutyryl cAMP (114). The ₁-AR-mediated [Ca²⁺]_i transients are characterized by complex kinetics, often consisting of an initial peak followed by a long-lasting plateau or oscillations (e.g., Refs. 373, 450). The norepinephrine-induced [Ca²⁺]_i increase in cultured mouse astrocytes was substantially inhibited by chronic treatment (7-14 days) with 1 mM lithium (63). The latter is known to interfere with InsP₃ signaling pathway and has therapeutic potential as a mood-stabilizing drug. Thus it is not excluded that the psychological effects of lithium are due to modulating astrocytic rather than neuronal receptor.

Shao and McCarthy (379, 382) determined the relation between the amplitude of the [Ca²⁺]_i increase in cortical astrocytes and agonist concentration (379, 382). The threshold phenylephrine (PE) concentration was ~1 µM, but PE concentrations exceeding 5 µM triggered a maximal response. Moreover, using subsequent monitoring of ₁-AR-mediated [Ca²⁺]_i increase and the ₁-AR receptors density in the same cell, they (379) found that the amplitude of [Ca²⁺]_i response was independent of the density of ₁-AR (despite that the latter varied between 10 and 2,000 binding sites/1,000 µm² membrane area). They suggested that ₁-AR-mediated InsP₃ formation triggers all-or-nothing Ca²⁺ release from internal stores. The underlying mechanism is unclear but may involve facilitation of InsP₃-induced release by increased [Ca²⁺]_i . In contrast, experiments on cerebellar Bergmann glial cells (239) revealed a flatter dependence of [Ca²⁺]_i increase on agonist concentration, suggesting a gradual InsP₃-triggered Ca²⁺ release in these cells.

Recently, the expression of functional ₁-AR linked to [Ca²⁺]_i was found in situ in astrocytes from acutely prepared hippocampal stratum radiatum slices (104) and in Bergmann glial cells studied in isolated cerebellar slices (239). In both studies, norepinephrine- and epinephrine-evoked [Ca²⁺]_i responses exhibited ₁-AR pharmacology (the effects were mimicked by ₁-AR agonist PE, blocked by ₁-AR antagonist prazozin, and insensitive to ₂-AR-specific agents). The ₁-AR-mediated [Ca²⁺]_i responses were characterized by a long-lasting plateau phase that followed the initial [Ca²⁺]_i rise. Removal of [Ca²⁺]_o eliminated the plateau phase in Bergmann glia but not in hippocampal astrocytes.

-Aminobutyric acid (GABA) effects in the nervous system are mediated by three distinct subtypes of receptors: GABA_A and GABA_C are intrinsic ligand-gated Cl channels, whereas GABA_B receptors are coupled to their effectors (K⁺- or Ca²⁺-permeable plasmalemmal channels) via G proteins (206, 308, 386). In contrast to neurons, where GABA normally leads to a hyperpolarization, in macroglial cells GABA depolarizes the membrane (132, 221, 222, 435). The difference is not due to the receptor but rather due to the different intracellular Cl levels in the two cell types. Glial cells have a more positive Cl equilibrium potential (E_Cl) than neurons due to a higher intracellular Cl concentration. This is primarily due to the activity of two inwardly directed Cl transporters, a Na⁺-K⁺-2Cl cotransporter and a Cl/HCO₃ exchanger. The Cl reversal potential in glial cells is about 35 mV, and opening Cl channels led to a depolarizing Cl efflux (132, 435). The GABA-activated inward currents were recorded in cells from astrocyte and oligodendrocyte lineage employing both single cell (45, 131, 221) and brain slice models (32, 398).

In many cases, GABA-induced depolarization of glial cells exceeds the threshold for voltage-gated Ca²⁺ channels and thereby produces Ca²⁺ influx and measurable [Ca²⁺]_i transients. Such effects have been recorded from cultured (314) and freshly isolated (131) astrocytes as well as cultured cells of the oligodendrocyte lineage (230). Experiments with freshly isolated astrocytes and oligodendrocytes in culture demonstrated that GABA-induced [Ca²⁺]_i increases were exclusively due to Ca²⁺ entry via voltage-gated channels, since removal of [Ca²⁺]_o or Ca²⁺ channel blockers inhibited the [Ca²⁺]_i rise (131, 230). In experiments on cultured astrocytes, Nilson et al. (314) suggested that in addition to plasmalemmal Ca²⁺ entry, GABA triggered a release of Ca²⁺ from intracellular stores, suggesting a possible involvement of GABA_B receptors. Another piece of evidence suggesting the expression of GABA_B receptors coupled to Ca²⁺ entry pathway came from ⁴⁵Ca²⁺ flux measurements (3), although this observation has not been substantiated yet with physiological techniques.

Some glial cells have been reported to express glycine receptors that also increase Cl permeability (231). Activation of these receptors causes depolarization of oligodendrocytes and might also increase [Ca²⁺]_i , but this has not yet been shown.

Acetylcholine reacts with two distinct families of cholinoreceptors, nicotinic cholinoreceptors (NChRs) that contain an integral cationic channel and metabotropic muscarinic cholinoreceptors (MChRs) that are coupled to PLC and adenylate cyclase by means of G proteins (58, 122). The NChRs are assembled from a broad family of elementary subunits belonging to a distinct gene family; some NChR heteromeres have relatively high Ca²⁺ permeability (408, 433). There are five major subtypes of MChRs (M₁-M₅) that have been identified and cloned (41). They have the seven membrane spanning domain structure typical for metabotropic receptors. Type 1, 3, and 5 MChRs are coupled with PLC, thereby controlling InsP₃ turnover, and MChRs types 2 and 4 decrease adenylate cyclase activity.

In glial cells, NChRs have only been described in invertebrates. Stimulation of NChRs in leech neuropile glial cells causes depolarization (18, 376) and increased [Ca²⁺]_i due to both Ca²⁺ entry via NChRs and voltage-activated plasmalemmal channels (376). In insects, there is evidence of NChRs from immunochemical studies (255).

Muscarinic receptors are widely distributed in mammalian macroglial cells. Initially, MChRs were detected in both astrocytes (185, 366) and oligodendrocytes (366) by a variety of histochemical and immunocytochemical methods. Stimulation of MChRs leads to accumulation of InsP₃ in glial cell lines (250, 306) and macroglial cells (astrocytes, Refs. 302, 337, 339; oligodendrocytes, Refs. 78, 366). Thus InsP₃-mediated [Ca²⁺]_i increases were recorded from many types of glial cells treated with MChR agonists. This included astrocytoma cells (286, 320, 327), perisynaptic Schwann cells at the frog neuromuscular junction (200), primary cultured astrocytes (92, 382), and cultured oligodendrocytes (78, 213). In the latter, PCR was used to demonstrate the expression of both M₁ and M₂ MChRs. However, the carbachol-induced [Ca²⁺]_i increase was preferentially sensitive to the M₁ agonist pirenzipine, rather than the M₂ antagonist methoctramin. This suggests that InsP₃ formation and Ca²⁺ mobilization in oligodendrocytes involves M₁ muscarinic receptors (78). Interestingly, MChR-mediated [Ca²⁺]_i responses in cells of the oligodendrocyte lineage have two components, an initial rise associated with intracellular Ca²⁺ release and a long-lasting plateau resulting from Ca²⁺ entry (78) via an unknown mechanism. Both Ca²⁺ release and Ca²⁺ entry are important for stimulation of c-fos expression, which followed the activation of muscarinic receptors in oligodendrocyte precursors (79).

Pharmacological assays indicate that astrocytes also have both M₁ and M₂ receptors (8, 302) and that M₁ receptors are primarily responsible for InsP₃ production (8). In addition, in astrocytes from neonatal rat cerebral cortex, M₃ receptors coupled to phosphoinisitide hydrolysis were found (243). The expression of functional MChRs receptors varies considerably between astrocytes isolated from different brain regions; the carbachol-stimulated InsP₃ production mediated by M₁ receptors was much higher in astrocytes derived from mesencephalon than in cells from the medulla-pons and cerebral hemispheres, being another example of functional heterogeneity among astrocytes (8).

Histamine affects three types of receptors: H₁ (coupled with PLC), H₂ (positively connected with adenylate cyclase), and H₃ receptors that control histamine turnover and release (257). Histaminergic nerve terminals in the posterior hypothalamus release the transmitter nonsynaptically directly into the interstitial space (196). It is thus likely that adjacent glial cells equipped with the proper receptor are activated. Indeed, astrocytes as well as astrocytoma cell lines have both H₁ and H₂ receptors (187, 195, 196), and stimulation of H₁ receptors increased intracellular InsP₃ in these cells (10, 244, 305). Measurements of [Ca²⁺]_i revealed intracellular Ca²⁺ transients in both astrocytoma cell lines (286, 456) and primary cultured astrocytes (136, 193, 194, 285) mediated via H₁ histamine receptors. Experiments with cultured cortical astrocytes indicated that H₁ receptors and histamine-evoked [Ca²⁺]_i responses were mainly confined to type 2 astrocytes and a subpopulation of type 1 astrocytes (136, 244). The expression of H₁ receptors coupled to [Ca²⁺]_i was recently confirmed in experiments in situ in cerebellar Bergmann glial cells (239). In these cells, histamine induced [Ca²⁺]_i transients in a dose-dependent manner (threshold ~1 µM; and maximal response at ~100 µM). The histamine-induced [Ca²⁺]_i transients in Bergmann glial cells were sensitive to the H₁ specific antagonist chlorpheniramine but were not mimicked by H₂ and H₃ agonists (dimarit and -methylhistamine, respectively). Similarly, H₂ and H₃ antagonists (ranitidine and thioperamide) had no effect. Pretreatment of Bergmann glial cells with thapsigargin abolished the histamine-induced [Ca²⁺]_i response, suggesting that intracellular Ca²⁺ release was responsible for the H₁ receptor-sensitive [Ca²⁺]_i rise.

In cells of the oligodendrocyte lineage, histamine increased [Ca²⁺]_i in a subpopulation of cultured progenitors and mature oligodendrocytes (213).

Substance P belongs to a family of neuropeptides, the neurokinins, that affects neurons by stimulating neurokinin receptors widely distributed in the brain (275). These receptors are represented by three subtypes (NK₁-NK₃) identified by different molecular structures and a distinct pharmacological profile (89, 384, 458). Among them, NK₁ receptors preferably bind substance P. All three NK receptors are typical G protein-coupled metabotropic receptors with a seven transmembrane domain structure. Interaction of substance P with NK₁ receptors results in phosphoinositide hydrolysis (278, 279) in oligodendrocytes and type 2 astrocytes. Substance P and NK₁ agonists increase [Ca²⁺]_i by releasing Ca²⁺ from thapsigargin-sensitive stores in astrocytoma cell lines (43, 110, 353) in human glioma line (173), in primary cultured astrocytes (193, 278, 281), and in cultured cells of oligodendrocyte lineage (172, 213). In astroglia, substance P-dependent InsP₃ production and subsequent intracellular Ca²⁺ release was restricted to type 2 astrocytes (278). The NK₁-mediated [Ca²⁺]_i responses, found in experiments with both glial cells lines (43) and primary cultured oligodendrocytes (172), were relatively short and not affected by extracellular Ca²⁺ removal. This suggests that the Ca²⁺ is released from intracellular stores. Substance P-induced Ca²⁺ signals stimulate interleukin-1 production in cultured astrocytes (281). The activation of NK₁ receptors is accompanied by astrocyte depolarization (444) due to the closure of K⁺ channels (15). What is the mechanism of K⁺ channels inhibition upon substance P stimulation remains unclear; it could be mediated by an [Ca²⁺]_i increase. This depolarization, in turn, could activate additional Ca²⁺ influx through voltage-gated channels.

Two types of bradykinin (BK) receptors (B₁ and B₂) have been described (161). Both subtypes belong to the G protein-coupled metabotropic receptors linked to PLC and InsP₃ production. Bradykinin acts not only as an effective vasodilatator but also as a neurotransmitter. Bradykinin and its binding sites were found in the mammalian brain (135, 344), and various neurons express functional BK receptors. Initial evidence for the presence of BK receptors in glial cells came from biochemical experiments that identified BK binding sites on cultured astrocytes (68) and demonstrated stimulation of phopshoinisitide turnover in BK-treated cultured astrocytes (69, 278, 366) and oligodendrocytes (278, 400). In physiological experiments, using microfluorimetric [Ca²⁺]_i recordings, BK was found to increase [Ca²⁺]_i in cultured Schwann cells (330) and in cultured astrocytes (152, 399) and oligodendrocytes (213, 278). However, in freshly isolated Schwann cells, a BK-induced [Ca²⁺]_i rise was observed only in a minor cell population (267). The BK-triggered [Ca²⁺]_i elevation in cultured astroglia was mediated mainly by B₂ receptors as judged by its pharmacological profile, although a minor subpopulation of astrocytes may also contain B₁ receptors (152, 399). The relative importance of intracellular Ca²⁺ release/Ca²⁺ entry components in BK-induced [Ca²⁺]_i elevation remains unclear. In one series of experiments on cultured astrocytes, BK-triggered [Ca²⁺]_i transients consist of an initial Ca²⁺ release from ER stores followed by long-lasting Ca²⁺ entry component (152). Another study with the same type of cells found only the Ca²⁺ release component (399). Apart from an [Ca²⁺]_i rise, an inward current was observed in cultured astrocytes upon BK application (152). The BK-dependent membrane current was also sensitive to B₂ receptor antagonists. However, a clear disparity in the concentration dependence between changes in [Ca²⁺]_i and membrane conductance was found. The 50% effective concentration (EC₅₀) for [Ca²⁺]_i rise was ~10 nM BK, whereas EC₅₀ for BK-induced currents was ~1 µM. These results suggest that distinct concentration-dependent signaling pathways aimed at different targets are triggered by activation of B₂ receptors on cultured astrocytes. Bradykinin-induced Ca²⁺ signaling was observed in ~50-60% of cultured astrocytes and was a subject of developmental regulation in oligodendroglia, i.e., BK triggered [Ca²⁺]_i elevation in ~50% of mature oligodendrocytes compared with only 13% of oligodendrocyte precursors (213).

Endothelins, represented by three major isoforms encoded by distinct genes (ET-1, -2, -3; Ref. 197), were first found to be potent vasoactive compounds (455). Later, expression of both endothelin mRNAs and a peptide proper, as well as endothelin binding sites, were found to be widely distributed in the brain (148, 208), and physiological responses were detected in neurons challenged with endothelins (70, 299). Endothelin-induced cellular reactions are mediated via three major receptor subtypes (ETRs) bearing different sensitivity to endothelin isoforms. The most abundant form in the brain is the nonselective ET_BR (which is equally sensitive to all 3 endothelins). The ET_AR (preferably sensitive to ET-1 and ET-2, but not ET-3) is much less expressed. The ET_CRs have been described only in nonmammalian tissues (26, 372). All endothelin receptors have an overall topology similar to G protein-coupled seven transmembrane domain metabotropic receptors, and their activation involves various G proteins (either PTX sensitive or insensitive). The ETRs are significantly involved in regulation in [Ca²⁺]_i . They trigger the PLC-InsP₃-Ca²⁺ release transduction chain as well as stimulate transmembrane Ca²⁺ influx (372).

Initially, endothelin receptors were found with autoradiography in various subpopulations of brain astrocytes (186). Later, astrocytes were shown to express ET_A and ET_B receptors (111, 123, 162). Furthermore, in certain brain regions, expression of endothelin-binding sites was found to be glial specific. The endothelin binding in the cerebellum was quite similar in normal mice and in mutants lacking Purkinje neurons (pcd mutants), suggesting that glial cells carry the majority of ETRs (270). Stimulation with endothelin led to an increase of InsP₃ in astrocytes and astroglioma cells (70, 86, 263, 270, 459) and to increases in [Ca²⁺]_i in both glial cell lines (263, 456, 459) and cultured astrocytes (155, 183, 280, 396, 404); recently, ET-1-induced [Ca²⁺]_i transients were also detected in microglial cells (293). The glial [Ca²⁺]_i response to endothelins was found to be insensitive to PTX (183, 396). The endothelin-mediated Ca²⁺ mobilization in glioma cells was inhibited by opioid agonists presumably because they inhibit InsP₃ production (20). The expression of ETRs linked to [Ca²⁺]_i increases appears to be region specific, i.e., in cortical type 1 astrocytes, endothelin effects on [Ca²⁺]_i were mediated via ET_AR (183), whereas cerebellar astrocytes predominantly expressed ET_BR (396). The astrocytic expression of ETRs linked to [Ca²⁺]_i was recently demonstrated in experiments in situ on Bergmann glial cells, in which endothelin produced an elevation in [Ca²⁺]_i . The Ca²⁺ responses in Bergmann glial cells were mediated by ET_BR (418) as was revealed by singe-cell RT-PCR analysis.

The endothelin-induced [Ca²⁺]_i transients in both cultured and in situ astrocytes had heterogeneous kinetic properties varying from a simple peak response to a biphasic [Ca²⁺]_i rise, with a plateau lasting up to 20 min after agonist washout (155, 183). Sometimes endothelin triggered only a plateau without a defined peak (183). As in the case of other agonist-triggered [Ca²⁺]_i responses, the initial peak resulted from internal Ca²⁺ release, while the plateau phase was entirely dependent on extracellular Ca²⁺ (183, 263, 396, 404, 419). The nature of Ca²⁺ delivery pathway underlying the plateau phase of endothelin-induced responses remains controversial. Studying endothelin responses in cerebellar cultured glial cells, one study found it was completely (404) or partially (396) inhibited by nifedipine, suggesting an important role for voltage-gated Ca²⁺ channels, whereas in another study, nifedipine had only a marginal effect (183). The effects of ETR stimulation on Ca²⁺ channels might be mediated by diacylglycerol, since treating cultured astrocytes with a protein kinase inhibitor causes a nifedipine-sensitive plateau (183). Diacylglycerol might induce phosphorylation of Ca²⁺ channels and shift their threshold toward more negative membrane potentials. The participation of Ca²⁺ channels in the response may vary considerably from cell to cell. A second possible mechanism for producing a prominent plateau phase in endothelin-induced [Ca²⁺]_i transients might be either endothelin-activated plasmalemmal channels or capacitative Ca²⁺ entry. The capacitative Ca²⁺ entry pathway is likely to shape the plateau of endothelin-induced Ca²⁺ responses in Bergmann glial cells in situ (418). In the same cells, endothelin-triggered [Ca²⁺]_i transients exhibited a progressive rundown with repeated endothelin applications (418). This could be the result of the internalization of the ETRs (364).

Endothelin-induced [Ca²⁺]_i signaling might participate in the astroglial proliferative response. Increased levels of endothelins in brain tissue accompany various insults in the CNS (22, 153). This observation suggests the hypothesis that ETRs are involved in the transduction of a mitogenic signal which, at least partially, underlies the activation of astrocytes involved in brain pathology. Ischemic conditions are also accompanied by an increase in endothelin synthesis (22), and endothelin antagonists are neuroprotective during brain ischemia (125). It has been demonstrated that endothelin-induced [Ca²⁺]_i increases play a pivotal role in stimulation of DNA synthesis and proliferative response of cultured type 1 astrocytes (396, 404). In addition, endothelins were found to trigger immediate early genes expression; the level of Fos proteins increases significantly in endothelin-challenged glial cells from organotypically cultured cerebellar slices (403). This suggests that ET receptors can play a role in development or during pathological events when glial cells undergo phases of proliferation. In this context, the glial endothelin system has been implicated in a number of pathophysiological situations such as viral infections (269), Alzheimer's disease (460), and ischemia (454).

The data on the presence of neurotransmitter receptors presented above clearly demonstrate that glial cells can express functional receptors to almost all known neurotransmitters, neuromodulators, and neurohormones (Table 2). The important question is whether all these receptors are expressed in situ and which factors regulate their expression. Most of the experiments on glial receptors have been carried out in tissue-culture systems; our knowledge of the receptor pattern expressed by glial cells in vivo is far more limited. Glial cells in culture express a remarkable heterogeneity in their sensitivity to various neuroligands. Cultured astrocytes can respond at the same time to a number of ligands with an increase in [Ca²⁺]_i . The pattern of receptor expression can differ substantially between cells in the same culture (92, 285, 380, 381). Moreover, the same heterogeneity persists within clones of cultured astrocytes, suggesting that the receptor pattern is not inherited from the parent astrocyte (380). In a series of elegant experiments, Shao and McCarthy (380, 381) demonstrated that the [Ca²⁺]_i responsiveness to various neuroligands can differ between two sister cells immediately after astrocyte division. Thus astrocytes are not born with a fixed set of functional receptors but can vary the expression of a variety of neurotransmitter receptors depending on factors in their environment.

Similarly, neuroligand [Ca²⁺]_i responsiveness of cultured oligodendrocytes is heavily controlled by culture conditions. Even more interesting, the expression of neuroligand receptors linked to the Ca²⁺ signaling appeared to be controlled by oligodendrocyte-neuronal contacts (171). Preventing glial-neuronal contacts by transection of neurites in oligodenroglial-DRG cocultures significantly reduced the number of oligodendrocytes sensitive to a variety of neuroligands (ATP, carbachol, and histamine; responsiveness to BK was not affected; Ref. 171). Neuronal control of receptors expression in neighboring glia appeared to be sensitive to tetrodotoxin, suggesting the important role of neuronal activity.

An important question is whether this plasticity is present not only when astrocytes are in culture but also in vivo. Recently, this question has been investigated in studies of neurotransmitter-induced Ca²⁺ signaling in glial cells in situ, in brain slices. Glial cells (both oligodendrocytes and astrocytes) in corpus callosum slices demonstrated prominent developmental changes in their neurotransmitter receptor expression (33). In slices obtained from young (3-7 days old) mice, glial cells were rather promiscuous, responding with [Ca²⁺]_i increases to ATP, glutamate, histamine, GABA, norepinephrine, serotonin, angiotensin II, BK, and substance P. In contrast, in older (11-18 days old) animals, the expression of receptors was limited to glutamate, ATP, and norepinephrine. Another comprehensive study of Ca²⁺ signaling-linked neurotransmitter expression has been carried out in cerebellar Bergmann glial cells. These cells provide a unique model for studying glial cells in slices because their characteristic morphology makes identification easy. The study found that, contrary to cultured astrocytes, Bergmann glia always express a distinct set of receptors. These include ₁-adrenoreceptors, AMPA/kainate GluRs, mGluRs, H₁ histamine receptors, P_2y purinoreceptors, and ET_B endothelin receptors (232, 235, 239, 419, 429). Other substances known to induce Ca²⁺ signals in cultured macroglia were ineffective. Interestingly, the Purkinje cell layer, where Bergmann glial cells are located, receives afferents (323) using the following neurotransmitters: glutamate (parallel fibres), norepinephrine and ATP (terminals from locus ceruleus), and histamine (histaminergic terminals from tuberomammillary nucleus of posterior hypothalamus). Thus Bergmann glial cells express receptors that are appropriate to detect transmitters secreted in their vicinity. Furthermore, it appears that Ca²⁺ signaling-related receptors expressed by Bergmann glia almost exactly match receptors found on the closely apposed Purkinje neurons (Fig. 7; Refs. 234, 429). An exception was ET_BR. Although the mRNA for the latter is expressed in Purkinje neurons, as revealed by single-cell RT-PCR, the endothelins failed to trigger an [Ca²⁺]_i increase (418). Another example of similarity of glial and neuronal receptor expression came from the experiments on spinal cord slices. It appears that the spinal cord is the only region found so far where both oligodendrocytes and astrocytes express glycine receptors, which are known to be present on spinal cord neurons (231, 333). It seems fruitful to pursue comparable studies of the similarity of receptors on adjacent glial cells and neurons in other areas of the nervous system to test the hypothesis that the receptors expressed on glial cells enable the detection of transmitter substances released in the same anatomical region by neurons.

View larger version (45K): [in this window] [in a new window]   FIG. 7.   Bergmann glial cell and its neighbor Purkinje neuron bear a similar set of neurotransmitter receptors. See text for discussion and receptor nomenclature. NT, terminals from tuberomammillary nucleus of posterior hypothalamus which carry histamine innervation of cerebellar cortex; LC, terminals from locus ceruleus which utilize norepinephrine and ATP as neurotransmitters; CF and PF, climbing and parallel fibers, respectively, of major neurotransmitter glutamate; BA and ST, basket and stellate cells which deliver -aminobutyric acid (GABA) to Purkinje neuron layer. Glu, glutamate; AMPA, -amino-3-hydroxy-5-methylisoxazole-4-propionic acid; ET, endothelin; mGluR, metabotropic glutamate receptor; Hist, histamine; NA, norepinephrine. Other definitions are as in Fig. 2.

	VI. SPATIOTEMPORAL ORGANIZATION OF CALCIUM SIGNALS

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After stimulation, the temporal and spatial distribution of the increase in [Ca²⁺]_i in glial cells, especially astrocytes, is remarkably complex. At a given point, the time course of the increase may be a single spike, biphasic with an initial peak followed by a plateau, or oscillations (129, 429). Usually, simple monophasic increases follow activation of voltage-gated Ca²⁺ channels and/or ionotropic receptors (e.g., Refs. 103, 199), whereas stimulation of metabotropic receptors, releasing intracellular Ca²⁺, leads to complex biphasic/oscillatory [Ca²⁺]_i responses (61, 84, 225, 450). The initial phase of these complex [Ca²⁺]_i signals follows InsP₃-mediated Ca²⁺ release from intracellular stores. The plateau/oscillation phases requires extracellular Ca²⁺ (154, 155, 183, 203, 362, 419). The Ca²⁺ influx pathway activated with metabotropic receptor stimulation is unclear. By analogy with other tissues (35, 75), the most likely candidate is the store-operated Ca²⁺ channel, although the existence of this pathway in glial cells has not been demonstrated.

The appearance of biphasic/oscillatory [Ca²⁺]_i responses depends on agonist concentration; cultured astrocytes stimulated with low doses of metabotropic agonists responded with [Ca²⁺]_i spikes, whereas higher doses led to an additional plateau phase or [Ca²⁺]_i oscillated. Such a dependence was found for glutamate (1, 154), histamine (136), norepinephrine (373), and endothelin (155). For glutamate, the concentration dependence of [Ca²⁺]_i responses was even more complicated; at low concentrations, monophasic [Ca²⁺]_i transients were recorded, moderate concentration triggered [Ca²⁺]_i oscillations, and at high glutamate concentrations, the initial [Ca²⁺]_i peak was followed by a sustained [Ca²⁺]_i plateau (84).

View larger version (70K): [in this window] [in a new window]   FIG. 8.   Ca2+ wave propagation in glial networks. A: extracellular component in astrocytic Ca2+ wave propagation. Images show an electrically evoked astrocytic Ca2+ wave traversing a cell-free lane. a: Phase-contrast image of a bed of astrocytes in which a cell-free lane had been created by scratching with a micropipette 6 h earlier. Also in view is extracellular stimulating electrode 10 µm above astrocyte upper surface. Red square delineates field of fluorescent imaging of fluo 3 signals. b: Cellular outlines showing astrocytic bed, cell-free lane, and all of astrocytes participating in Ca2+ wave during observation time depicted on left. c: Pseudocolor images of change in fluorescent signal (F/Fo) in astrocytes at selected time points. Images were collected at 3, 16, 22, and 36 s after stimulation. Note that in third frame from left, Ca2+ wave jumps across cell-free lane and subsequently spreads to neighboring astrocytes (4th frame). Although magnitude of spread, amplitude of response, and number of participating cells can vary in each experiment, most preparations with lanes of <100 µm show responses similar to that shown. Bar = 50 µm. [From Hassinger et al. (167). Copyright 1997 National Academy of Sciences, USA.] B: propagation of Ca2+ waves in glial cells in acutely isolated rat retina. Spread of a Ca2+ wave initiated by a mechanical stimulus. Fluorescence image is shown in black and white. Superimposed yellow rings mark leading edge of Ca2+ wave (where change in fluorescence between successive panels exceeded a threshold value). Interval between panels = 0.93 s; scale bars in first frame = 50 µm. C: Ca2+ waves in retinal glia originate from intracellular stores. a: Ca2+ wave evoked by an electrical stimulus. b: Different region of retina shown in a, stimulated after 31 min in 0 mM Ca2+ and 0.5 mM EGTA. c: Ca2+ wave evoked by an electrical stimulus under control conditions. d: Different region of retina shown in c, stimulated 16 min after addition of 1.5 µM thapsigargin. Scale bar = 50 µm. [From Newman and Zahs (312). Copyright 1997 American Association for the Advancement of Science.]

In addition to a complex temporal pattern, glial Ca²⁺ signaling exhibits spatial heterogeneity. In many glial cell populations, [Ca²⁺]_i rises first in processes and then spreads toward the soma [e.g., in ATP or KCl-stimulated oligodendrocytes (237, 238), in histamine-stimulated astrocytes (194), or in ATP-challenged Bergmann glial cells (235); cf. Figs. 4-6]. Such a pattern is expected from considerations of surface-to-volume relations if the release or influx is constant for a given membrane area. However, subsequent analysis suggests the situation is more complex. The localization of Ca²⁺ release sites may reflect a higher concentration of Ca²⁺ signaling molecules in distal parts of glial cells in regions where they are in more intimate contact with neurons. Cultured astrocytes display more complex spatial [Ca²⁺]_i signals, developing long-lasting [Ca²⁺]_i waves (450, 451). These waves are associated with several intracellular loci, each characterized by its own oscillatory pattern with [Ca²⁺]_i waves propagating between them (450, 451). Several hypotheses have been proposed to explain [Ca²⁺]_i wave initiation and propagation. These include periodic fluctuations of cytoplasmic InsP₃ level, [Ca²⁺]_i-dependent regulation of InsP₃-gated Ca²⁺ release channel open probability, and an interplay between InsP₃-induced and Ca²⁺-induced Ca²⁺ release (see Refs. 6, 36, 259 for review).

In cultured astrocytes obtained from the visual cortex, the glutamate-evoked [Ca²⁺]_i oscillations demonstrated a long-lasting modulation in frequency. The second application of glutamate arriving 1-60 min after the first challenge always triggered [Ca²⁺]_i oscillation with higher frequency (332). These changes of frequency were exclusively confined to glutamate-induced Ca²⁺ signals; [Ca²⁺]_i oscillations triggered by other agonists did not show such long-term plasticity.

In cultured astrocytes, a stimulus-evoked intracellular Ca²⁺ wave can cross cell boundaries and travel within the astrocytic network. The seminal observation was made by Cornell-Bell et al. (83), who found that in the presence of glutamate, Ca²⁺ waves propagate through the astrocyte syncytium. The waves followed complex routes, without delays at cell borders, for hundreds of microns at a velocity of ~15-20 µm/s (83, 84, 428). The propagation of glutamate-triggered Ca²⁺ waves requires extracellular Ca²⁺. An alternative way to induce intercellular Ca²⁺ waves is with focal mechanical stimulation. These waves have different properties; they have a delay at cell borders and persist in Ca²⁺-free solutions (61). They are, however, dependent on Ca²⁺ release from the internal stores; the depletion of stores with thapsigargin blocks wave propagation (60). The mechanism of intercellular propagation also involves gap junctions; the waves are inhibited by octanol, halothane (115, 126), or by an endogenous derivative of arachidonic acid, anandamide (427). In the C6 glioma cell line, intercellular Ca²⁺ wave propagation was observed only in cells that were transfected with the gene for the gap junction protein connexin-43 (62). Thus a likely mechanism for Ca²⁺ wave propagation involves intercellular (via gap junctions) diffusion of InsP₃ , which generates Ca²⁺ release in one cell after another (126, 390); the degree of intercellular coupling would, presumably, determine the wave path. The important role of intracellular Ca²⁺ stores and InsP₃ production is substantiated by the finding that treatment of astrocytic cultures with thapsigargin or with PLC blocker U-73122 completely prevented the spread of intercellular Ca²⁺ waves (428). Interestingly, interglial communications via gap junctions may be regulated by various physiological stimuli, e.g., it increases upon glutamate and high K⁺ treatment (113, 150) or even after action potentials in adjacent axons (277). This susceptibility of interglial gap junctions to external regulation may influence interglial Ca²⁺ signaling.

Alternative mechanisms for Ca²⁺ wave propagation may involve an extracellular messenger released by stimulated cells. An extracellular pathway for Ca²⁺ wave propagation was observed by Hassinger et al. (167). To test the importance of the extracellular pathway, they created a cell-free lane in confluent astrocytic cultures by mechanically eliminating astrocytes with a glass pipette. As shown in Fig. 8A, electrically induced Ca²⁺ waves appeared to cross cell free lanes narrower than 120 µm. Moreover, the velocity of wave propagation through the cell-free lane did not differ significantly from the velocity of the wave spreading via the astrocytic network. The nature of the extracellular messenger was not clarified, although glutamate can be excluded; the wave spreading over cell-free regions was not modified by glutamate receptor antagonists. Whether the extracellular mechanism may work in concert with gap junction propagation or it may take the leading role in certain brain regions remains totally unclear.

The existence of interastrocytic [Ca²⁺]_i waves in brain tissue has only recently been demonstrated. A propagating wave was observed by Dani et al. (90) in organotypic hippocampal slice cultures. These waves were initiated by stimulation of mossy fibers, which are believed to utilize glutamate as a neurotransmitter. However, the intercellular Ca²⁺ waves observed in these experiments usually propagated for much shorter distances (2-3 astrocyte diameters) than observed in cultures. This might reflect a higher degree of coupling of cultured astrocytes than astrocytes in situ. More recently, propagating Ca²⁺ waves were recorded in glial cells from acutely isolated rat retina (312). These Ca²⁺ waves were initiated by either local mechanical or electrical stimulation or by focal application of ATP, carbachol, or phenylephrine (Fig. 8, B and C). Interestingly, focal applications of glutamate did not trigger Ca²⁺ waves in retinal glial networks, although incubation of the whole preparation in glutamate potentiated Ca²⁺ waves induced by other stimuli. The propagation velocity of Ca²⁺ wave in retinal preparation was ~25 µm/s for all types of stimulation. The retinal Ca²⁺ waves obviously originated from intracellular Ca²⁺ release, being preserved in Ca²⁺-free solution and blocked by thapsigargin as shown in Figure 8C.

	VII. GLIAL CALCIUM SIGNALING AND NEURON-GLIAL INTERACTIONS

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The coordination of neuronal and glial activity requires that appropriate signals pass from one cell type to the other. The study of these signals is a central theme in neuroglial research (82, 220, 389). These interactions might involve short-term functions such as ion regulation, release of substrates, or transmitter clearance or much longer term processes as might be involved in neuronal guidance during development, myelination, mitosis, and regeneration. The question of how changes in glial [Ca²⁺]_i serve to couple neuronal and glial functions is a major question raised by the work that has been reviewed and, unfortunately, largely unanswered. Most of the studies of [Ca²⁺]_i in glial cells have been carried out with astrocytes in tissue culture. These cells have membrane receptors for many neurotransmitters, and their response often includes changes in [Ca²⁺]_i . It is tempting to assume that when the transmitters are released from the neurons that they provoke responses not only in a postsynaptic cell but also in the closely apposed glial cells. Moreover, neurons release a variety of substances, e.g., amines, peptides, and hormones, which diffuse through the extracellular space bathing other neurons and glial cells. Among the questions that need to be addressed are the following: 1) Which receptors are expressed in glial cells in the intact nervous system? 2) Can a glial cell distinguish between stimulants that all increase [Ca²⁺]_i? 3) What is the functional response of the glial cell after it has undergone an increase in [Ca²⁺]_i? 4) What other changes within the glial cell result from neuronal activity in addition to an increase in [Ca²⁺]_i?

Because glial Ca²⁺ signaling appears to be a ubiquitous consequence of the activation of glial neurotransmitter receptor systems, it is of interest to explore whether neuronal activity leads to [Ca²⁺]_i fluctuations in glial cells. Indeed, experiments performed recently in both peripheral and central macroglial cells demonstrate that glia sense nervous activity, and the latter initiates Ca²⁺ signals in glial cells. The peripheral glia, the Schwann cells, generate [Ca²⁺]_i transients in response to electrical stimulation of peripheral nerves (200, 258, 363). In periaxonal Schwann cells, the Ca²⁺ signal probably results from depolarization occurring in response to increases in [K⁺]_o resulting from nerve activity; this depolarization, in turn, triggers Ca²⁺ entry via voltage-gated channels and subsequent activation of Ca²⁺-induced Ca²⁺ release from internal stores (258). In contrast, in perisynaptic Schwann cells, Ca²⁺ signals were mediated by either muscarinic or P₂ purinergic receptors (200, 363) activated by neurotransmitters released from the nerve ending.

Neuronal activity has been found to induce [Ca²⁺]_i elevation in glial cells from the leech nervous system (371). At least in part, these glial Ca²⁺ signals were mediated through activation of GluR. Similarly, in brain white matter, electrical stimulation of the optic nerve evoked oscillatory Ca²⁺ responses in periaxonal glial cells (65, 247); the frequency of [Ca²⁺]_i oscillations correlated with stimulation frequency. The possible mechanism underlying axon-glial communication in the optic nerve may involve glutamate release from the stimulated nerve due to reversal of Na⁺/glutamate transporter (65).

In astrocytes of the gray matter, neuronal-induced Ca²⁺ signaling has also been found in both neuronal-glial cocultures and in hippocampal slice preparations. In neuronal-glial cocultures, selective stimulation of neurons with NMDA triggered [Ca²⁺]_i spikes and [Ca²⁺]_i oscillations in the neighboring astrocytes (91). Using in situ preparation, Dani et al. (90) observed [Ca²⁺]_i transients evoked by mossy fiber stimulation in astrocytes in hippocampal cultured slices. Similarly, stimulation of Schaffer collaterals triggered [Ca²⁺]_i elevation in astrocytes located in CA₁ stratum radiatum region (351). In hippocampal and visual cortex slices, electrical stimulation of presynaptic afferents triggered [Ca²⁺]_i oscillations in astrocytes (331); moreover, the frequency of astrocytic [Ca²⁺]_i oscillations increased while increasing the intensity or frequency of afferent stimulation. In all cases, [Ca²⁺]_i transients in astrocytes were mediated by glutamate presumably released from neuronal terminals, since astrocytic Ca²⁺ responses were mimicked by (1S,3R)-ACPD (331) and were inhibited by either nonselective ionotropic GluR blocker kinurenic acid (90, 351) or by mGluR blocker -methyl-4-carboxyphenylglycine (351).

In cocultures, it has been found that not only may neurons initiate Ca²⁺ signals in glial cells, but also glial [Ca²⁺]_i waves may trigger Ca²⁺ signals in neurons. First, Nedergaard (310) found that [Ca²⁺]_i waves in astrocytes in rat forebrain cortical cultures triggered [Ca²⁺]_i spikes in neurons. This astrocyte-to-neuron Ca²⁺ signaling was sensitive to gap junction inhibitors, suggesting the direct spread of signal from astroglia to neuronal cells. As such, gap junctions have not been demonstrated in vivo; this result can be considered an artifact of the culture system. However, in neuronal-glial cocultures from visual cortex (329), hippocampus (166), and forebrain (59), glial [Ca²⁺]_i waves induced by mechanical (59, 166), electrical (166), or agonist (BK; Ref. 329) stimulation resulted in Ca²⁺ signals in neurons. In these experiments, glial-to-neuronal Ca²⁺ signaling was sensitive to ionotropic GluRs antagonists, suggesting a primary role of glutamate released from glial cells in this form of signaling.

Therefore, glial Ca²⁺ signaling is involved, at least in culture systems, in bidirectional neuronal-glial signaling. The questions of the physiological role of these signals, their precise cellular mechanism, and their occurrence in the intact nervous system remain unanswered and deserve further study.

	VIII. GLIAL CALCIUM AND BRAIN PATHOLOGY

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It is a well-established paradigm that cellular Ca²⁺ overload is a key trigger in causing cell death (66). Much is known about Ca²⁺ toxicity in neurons; however, the problem of [Ca²⁺]_i damage to glial cells is much less explored. Experiments on cultured cells demonstrated that Ca²⁺ overload of Schwann cells (291) and oligodendrocytes (29, 377) might severely damage or kill them. In neurons exposed to hypoxia or ischemia, the Ca²⁺ overload results mainly from overactivation of GluRs produced by excessive glutamate release (glutamate excitotoxicity). Astrocytes but not oligodendrocytes are generally believed to be more resistant to excitotoxic insults; astrocytes can tolerate a very high (up to 5 mM) glutamate concentrations, whereas oligodendroglia die when challenged with 200 µM glutamate (326). This result is probably because the most abundant astrocytic ionotropic GluRs are of AMPA type, characterized by rapid desensitization. Removal of AMPA receptor desensitization by cyclothiazide dramatically increased astrocytic vulnerability to glutamate (93). The high resistivity of astrocytes to glutamate coincides with their role as a major glutamate sink in the brain; it is well established that astrocytes are mainly responsible for glutamate removal from the extracellular space (212), being thus one of the important components of nerve tissue defense against glutamate excitotoxicity.

Injury or brain pathology leads to a complex reaction from the astrocytes and microglial cells that are an important part of the brain's defenses (145, 252). Astrocytes respond with a variety of biochemical, structural, and proliferative changes that transform them into so-called "active astrocytes." Microglia also undergo dramatic morphological and biochemical changes transforming them into phagocytotic macrophages. The initial signals producing these long-term changes are not well understood; there is some evidence that Ca²⁺ signaling is an important (or maybe even triggering) element in the glial response to these brain insults. Studies in both in vitro and in vivo experimental models used to study ischemic damage revealed that brief exposures of astrocytes to hypoxic/hypoglycemic conditions trigger a [Ca²⁺]_i increase as a result of both activation of voltage-gated Ca²⁺ channels and Ca²⁺ release from internal pools (105, 169). The ischemia-induced [Ca²⁺]_i increase was activated either by a propagating wave of raised [K⁺]_o , accompanying injury-induced spreading depression, or by a massive release of neurotransmitters. Interestingly, [Ca²⁺]_i was much more sensitive to ischemia in astrocytes in situ, in hippocampal slices, than in the same cells acutely isolated (105). This suggests that the ischemia-induced increase in [Ca²⁺]_i in astroglia results from factors released by the damaged tissue (e.g., excess of neurotransmitters or elevated K⁺ concentration). Intracellular Ca²⁺ increases in astrocytes induced by brain damage might change the functional state of the cell and lead to the release of growth factors or result in astrocytic volume changes.

Another potential pathological Ca²⁺ signal in glia is triggered by the human immunodeficiency virus-1 envelope protein gp120. It induces [Ca²⁺]_i increases in both cultured astrocytes and oligodendrocytes from cerebellum (76) and cortex (77), but not in neurons (72). These [Ca²⁺]_i responses varied between single spikes and [Ca²⁺]_i oscillations. An [Ca²⁺]_i elevation was also observed in cultured astrocytes attacked by a fragment (so-called PrP 106-126) of another pathogenic protein, infectious prion protein. This [Ca²⁺]_i increase was prevented by the dihydropyridine Ca²⁺ antagonist nicardipine or by Ca²⁺ removal from the incubation media, suggesting the leading role of Ca²⁺ influx through voltage-gated channels (130).

There have been only a few studies of changes in oligodendrocyte [Ca²⁺]_i after brain damage. A possible link between [Ca²⁺]_i in oligodendroglia and brain pathology was suggested by experiments demonstrating that the addition of complement triggered [Ca²⁺]_i oscillations in primary cultured oligodendrocytes (446). These oscillations were blocked by thapsigargin, indicating an important role of intracellular Ca²⁺ stores in this phenomenon.

Microglia detect and respond to brain pathology (246). Damage to the CNS triggers a complex cascade leading to a transformation of microglial cells from a resting form to an active form, termed a macrophage (145). The mechanisms of [Ca²⁺]_i homeostasis and the role of Ca²⁺ signaling in microglial function are not known in detail. Microglial cells do not respond to the "classical" neurotransmitters such as glutamate or GABA. They, however, are responsive to signaling molecules from the immune system such as cytokines or chemokines. For example, activation of complement receptors C5a or C3a triggers a Ca²⁺ increase in cultured microglial cells (292, 318). The microglial [Ca²⁺]_i transients were also observed in response to extracellular application of lipopolysaccaride, a potent activator of immune cells (16). The study of the Ca²⁺ signaling in microglial cells is at an early stage: the role of [Ca²⁺]_i in microglia activation remains obscure.

	IX. CALCIUM SIGNALS AND GLIAL FUNCTION

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Glial cells exhibit various mechanisms for controlling and varying [Ca²⁺]_i . As described above, modulation of a number of intracellular cascades and membrane pathways enables a variety of external stimuli to induce changes in [Ca²⁺]_i , the Ca²⁺ signals. Glial cells are able to sense and react to various neuroactive substances; their reactions almost always involve changes in [Ca²⁺]_i . What glial functions are modulated or initiated by such signals? A number of studies indicate that activation of glial Ca²⁺ cascades affects glial K⁺ channels (82, 359), thereby modulating ionic buffering properties of the glial syncytium. Furthermore, increases in [Ca²⁺]_i promote glycogen breakdown (343) and gene expression (264, 342) and trigger release of neuroactive substances (322, 330). Calcium influx into glial cell precursors triggers a phosphorylation of cAMP response element binding protein (341) that is known to be a key factor in Ca²⁺-dependent gene expression. Norepinephrine, which increases [Ca²⁺]_i , also regulates the uptake of glutamate and GABA into astrocytes (164). In addition, the propagation of Ca²⁺ waves through syncytial networks of neuroglia via gap junctions can serve to coordinate glial activity and to transfer information between neurons apposed to different parts of the network. This signal transfer can participate in the integrative functions of both glial and neuronal networks.

When a glial cell has multiple receptors that lead to changes in [Ca²⁺]_i , can it respond appropriately to each signal? There are experimental results to indicate that a single cell either in tissue culture (128) or in situ (235, 239, 419) responds to individual ligands with a cell-specific characteristic increase in [Ca²⁺]_i that has a different spatiotemporal pattern. Thus an individual cell may respond to ATP with a brief spike and to norepinephrine or endothelin with an oscillatory or sustained increase in [Ca²⁺]_i . The relative increase may vary in different cellular regions, e.g., processes vs. soma, depending on the stimulus and lead to quite different functional responses. Moreover, the exact source of the Ca²⁺, voltage-dependent channels versus release from the ER, may create large spatial Ca²⁺ gradients within the glial cell. Therefore, although the results require confirmation, it appears that Ca²⁺ signaling exhibits the flexibility to enable the cells to respond in an appropriate stimulus-specific manner. These considerations do not rule out the possibility that there are other important intracellular signaling substances in glial cells that have not been well characterized for lack of appropriate detectors. There can be no question but that the ready measurement of [Ca²⁺]_i with fluorescent probes has greatly accelerated the appreciation of the role of this ion in the signaling chain.

	X. CONCLUDING REMARKS: CALCIUM SIGNALS ARE A CONSEQUENCE OF GLIAL EXCITABILITY

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The classical view that glia are inexcitable refers to their inability to respond to electrical stimulation with a propagated electrical response, i.e., an action potential. However, as documented throughout this review, glial cells are not passive; they display diverse temporal and spatial increases in [Ca²⁺]_i in response to a variety of stimuli, e.g., chemical, electrical, or mechanical. These [Ca²⁺]_i increases may outlast the stimulus and exhibit agonist-specific spatiotemporal patterns, thus providing a possible means for information coding. The glial [Ca²⁺]_i signals are apparently also capable of propagating without decrement via gap junctions into neighboring cells and even to neurons.

The ability of glial cells to actively respond to external stimuli makes them excitable. To avoid confusing this excitability with electrical excitability, it is best to refer to glial cells as "internally calcium excitable." It may even indeed be appropriate to describe all electrically inexcitable cells that do respond to stimuli via second messengers as "internally second messenger excitable." As detailed above, there is quite a bit known about the physiology of glial Ca²⁺ signaling. A beginning has been made in the attempt to characterize in detail the molecular cascades involved in [Ca²⁺]_i homeostasis and shaping of [Ca²⁺]_i signals in glia. The questions that remain to be answered are as follows: How is Ca²⁺ signaling involved in the integrative function of glial cells? Is glial Ca²⁺ signaling involved in information processing in glial networks? Finally, how is glial Ca²⁺ signaling involved in brain function? These intriguing questions will be the subject of continued study in this area.

	ACKNOWLEDGEMENTS

  We are grateful to Professor R. C. Thomas and Dr. Frank Kirchhoff for helpful comments.

	FOOTNOTES

   A. Verkhratsky and H. Kettenmann's research is supported by grants from SonderForschungsBereich 1534. R. K. Orkand is supported by National Science Foundation (EPSCoR) and the National Institutes of Health (National Institute of Neurological Disorders and Stroke, Minority Institutional Research Development Program, and Fogarty Foundation).

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