Physiology of Astroglia

A. Proto-astrocytes in Caenorhabditis elegans

The nervous system of Caenorhabditis elegans is composed of 302 neurons, 50 supportive (glial) cells of ectodermal origin, and 6 supportive cells derived from mesoderm (1258, 1861, see also http://www.wormatlas.org/hermaphrodite/neuronalsupport/Neurosupportframeset.html). None of these glial cells expresses classical astroglial markers. The CNS of C. elegans is represented by the nerve ring located in the frontal part of the body; the nervous ring receives processes of sensory neurons that are located in the periphery. The central ring also contains cephalic and motor neurons, which send efferent signals through the ventral and dorsal nerve cords. Majority (46) of C. elegans glia are associated with the sensory system. These cells are subdivided into 26 socket cells and 20 sheath cells that (together with neuronal processes) compose the worm's sensory organs known as sensillas (1371). The remaining four glial cells known as cephalic sheath (CEPsh) cells are associated with the neural ring. These CEPsh cells are bipolar; the anterior processes enwrap cephalic neuronal dendrites and form the corresponding sensilla in the lips of the animal. Posterior processes of CEPsh cell have lamellar morphology; they ensheath the nerve ring and send processes to the neuropil, where they contact synapses (1258, 1683). Consequently, these CEPsh cells can be defined as proto-astrocytes (FIGURE 4). The C. elegans also contains six mesoderma-derived supportive cells (known as GLR cells), which are located around the nerve ring. These cells (rather uniquely) make gap junctions with neurons and muscle cells and possibly contribute to neuronal-muscular communications (1258). The function of proto-astrocytes in the round worms are yet to be fully characterized. Arguably they control ion homeostasis in perisynaptic regions and are involved in neuronal development and morphogenesis. Although artificial ablation of glial cells (by either exposure to laser beam, or by expressing the diphtheria toxin A gene under control of glia-specific promoter) renders a complex of morphological, developmental, sensory, and behavioral deficits, it is, nonetheless, compatible with survival of the worm (82). Incidentally, C. elegans glia display some intermediate neuronal/glial physiology; for example, they generate Ca²⁺ signals through activation of voltage-gated channels and do not have functional intracellular Ca²⁺ stores (1682).

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FIGURE 4.

Proto-astrocytes (cephalic sheath, or CEPsh glial cells) of roundworm C. elegans. A: a cartoon of an adult worm showing the four CEPsh glial cells (green) positioned in the anterior of the worm (inset). The CEPsh cell bodies with their velate processes are positioned around the central nerve ring (red), which they enwrap along with the proximal section of the ventral nerve cord. Additionally, each CEPsh glial cell possesses a long anterior process, projecting to the anterior sensory tip, which closely interacts with the dendritic extension of a nearby cephalic neuron (blue). Arrows indicate the dorsal (red arrow) and ventral (orange arrow) side of the worm. B: a confocal image showing green fluorescent protein expression driven by the hlh-17 promoter to visualize the four CEPsh glial cells (worm strain VPR839). The anterior (head) of a juvenile (larval stage 4) worm is shown; the worm is turned ~45 degrees from “upright” such that all four CEP sheath cells are visible. The sheath portion of the cells that form a tube around the dendritic endings of the CEP neurons are seen at the left of the image. The dorsal (red arrow) and ventral (orange arrow) CEPsh cell bodies are seen. The thin sheetlike extensions that surround and invade the nerve ring are seen in the rightmost part of the image. Scale bar, 20 µm. [From Stout et al. (1683).]

B. Homeostatic Glia in Annelida

The medicinal leech Hirudo medicinalis was employed for studying glial physiology in pioneering experiments of Stephen Kuffler and David Potter in the mid 1960s (920). The nervous system of the leech is composed of the anterior and posterior brains and the chain of 21 ganglia that lie in between. The anterior brain comprises six ganglia fused into two neuronal masses, while seven fused ganglia in the aft form the posterior brain. Somatic ganglia innervate corresponding segments of the leech body (342, 403). Every ganglion is composed of 400 neurons (with exception of the 5th and 6th ganglia innervating the reproductive system, which have ~700 neurons) and 10 glial cells. Each ganglion contains two connective glial cells, which ensheath axons, six packet cells covering neuronal cell bodies, and two giant glial cells (403). All three types of glia are connected through gap junctions formed by innexins [of which leech expresses 21 types (819), with the Hm-inx2 type seemingly being specific for glia (463)], creating a panglial syncytium (1005). The nervous system of the leech additionally contains some amount of microglial cells that proliferate in response to lesions (954). Both packet glia and giant glial cells perform homeostatic functions and hence resemble astrocytes. The packet glial cells contribute to the regulation of extracellular K⁺, especially at high extracellular K⁺ concentrations (1214, 1547). Somata of giant glial cells are 80−100 μm in diameter and are localized in the center of the ganglion; extensively branched processes of these cells extend (for 300−350 μm) through the entire neuropil and contact neuronal dendrites (1172). Processes of giant glial cells partition neuropil, thus segregating neuronal ensembles into functional domains; sometimes glial processes invaginate into neuronal somata creating a structure known as “trophospongium“ (717). Giant glial cells are characterized by large K⁺ permeability and relatively hyperpolarized resting potential (approimately −75 mV). They express multiple types of neurotransmitter receptors including ionotropic glutamate, acetylcholine, and serotonin receptors as well as metabotropic receptors to glutamate, serotonin, myomodulin, and possibly P_2Y-like purinoceptors and A₁-like adenosine receptors (403, 1166). Giant glial cells participate in homeostatic responses, such as regulation of pH involving several plasmalemmal Na⁺-HCO₃⁻ cotransporter, Na⁺-H⁺ and Cl⁻-HCO₃⁻ exchangers (399, 404, 405), as well as in regulation of neurotransmitters turnover through Na⁺-dependent glutamate and Na⁺-dependent choline transporters (407, 705, 1898). Giant glial cells respond to neuronal activity and to evoked behaviors by changes in membrane potential (400) as well as by generation of cytosolic Ca²⁺ signals that occur in both somata and processes, often in compartmentalised manner (1004). In contrast to mammalian glia, the main source of Ca²⁺ signal generation in leech glia is through opening of plasmalemmal Ca²⁺ channels. Termination of Ca²⁺ signal termination is mediated by plasmalemmal Ca²⁺ pump and Na/Ca²⁺ exchanger; the intracellular Ca²⁺ stores, although present, seem to play a minor role (408, 1004).

C. Astroglia-like Cells in Arthropods: The Case of Drosophila

The Arthropods (of which insects and crustaceans are by far the most numerous representatives) have well-defined CNS. The brain of Arthropods is divided into protocerebrum (mainly receiving visual information), deuterocerebrum (receiving sensory input), and tritocerebrum (which acts as an integrating center). The neuroglia is elaborated and highly diversified; neuroglial cells account for ~10% of the total number of cells (~90,000 cells) in Drosophila CNS. Neuroglia of Drosophila are classified (for details and further reading, see Refs. 27, 467, 528, 652, 1325) into the following classes: 1) wrapping glia of the peripheral nervous system; 2) surface glia, which makes the brain-hemolymph barrier. It is subdivided into perineural glia (relatively small cells lying on the ganglionic surface) and subperineural or basal glia (represented by large sheetlike cells connected with septate junction that forms the actual barrier). 3) Cortex glia contact neuronal cells somata in the CNS; each glial cell establishes contacts with many neurons. 4) Neuropil glia are located in the neuropil and cover axons and synapses. The neuropil glia are further subdivided into ensheathing or fibrous and astrocyte-like glia, which forms a perisynaptic glial cover. 5) Finally, tract glial cells cover axonal tracts connecting different neuropils (1751). Within these classes glial cells could be further subdivided into multiple subtypes with distinct morphology and function (282, 467, 1751); for example, the glia of the lamina (neuropil) of the optic lobe is classified into fenestrated glia, pseudocartridge glia, distal and proximal satellite glia, epithelial glia, and marginal glia. In the deep optic lobe glial cells are represented by giant optic chiasm glia, small outer optic chiasm glia, medulla satellite glia, and medulla neuropil glia; similarly, specific glial types were described in olfactory system and in antennal lobes. Recent cell mapping using glia-specific GAL4 drivers allowed for precise numerical calculation of various cell types in the Drosophila CNS. The perineural glia accounted for ~17%, subperineural for 2%, cortex glia for 20%, astrocyte-like glia for 34%, and ensheathing glia for 27% of total glial cells (903).

A similar degree of complexity is also found in other insects, for example, in housefly Musca domestica or in tobacco hornworm Manduca sexta in which some other types of glial cells, such as the “chandelier glia,” have been described (288, 1528). Drosophila glial cells that belong to classes ii to iv (i.e., surface glia, cortex glia, and neuropil glia) serve functions characteristic for astrocytes in mammalian systems; however, the degree of specialization seems greater with distinct cell types responsible for a distinct set of functions. The coverage of neuronal bodies is provided by highly ramified cortex glia that do not have much semblance to mammalian astrocytes [although they originate from bipolar cells somewhat similar to radial glia (462)], whereas the brain-hemolymph barrier is provided by similarly distinct surface glial cells. The barrier is sealed by septate junctions composed from several proteins (567, 1578) of which the most important are gliotactin, neuroglian, and α and β subunits of Na⁺-K⁺-ATPase (also known as Nervana 2). The glial barrier in the insects is functionally analogous to the endothelial blood-brain barrier in vertebrates. It selectively limits the exchange between hemolymph and the CNS, for example, protecting the brain from substantial fluctuations in K⁺ that occur after feeding (467). Glial cells in the insect CNS also plaster tracheoles, which deliver oxygen to the nerve tissue (1367). The neuropil glia similarly demonstrate specialization: the flat ensheathing glial cells line the borders of the neuropil, thus isolating it from the surrounding cortex. In addition, ensheathing glia delineate glomeruli in the antennae lobe, whereas the astroglia-like cells extend complex arborizations into neuropil and provide for synaptic coverage (441). The neuropil glia are connected through gap junctions formed by innexins 2 and 3 and optic ganglion reduced (ogre) protein (1662).

Glial cells in insects provide for a multitude of homeostatic functions, such as regulation of ionic balance in the CNS fluids and regulating clearance, recycling, and metabolism of neurotransmitters. These functions are fulfilled by a complement of pumps, transporters, and specific enzymes. In the retina, glial cells are the key element for recycling the principal neurotransmitter histamine. After being released by activated photoreceptors, histamine is (partially) accumulated by glial cells closely covering synaptic contacts (1099). In the glial cell histamine is converted into β-alanyl-histamine or carcinine by ebony (N-β-alanyl-biogenic amine synthetase)-catalyzed reaction (216), the ebony activity is further associated with black protein in a yet unknown way (1966). Subsequently, carcinin is transported back to photoreceptors through the specific solute carrier transporter carT from SLC22A family (1669). After entering photoreceptors, carcinin is hydrolyzed to histamine with catalytic help of the so-called Tan (acyltransferase) protein (1760), thus concluding the histamine-carcinine shuttle. Mutations in the components of this shuttle disrupt fly vision (311, 1966). The ebony amine synthetase expressed in Drosophila glia seemingly also controls circadian clock (1696). Genetic modifications of Drosophila glia that interferes with vesicle trafficking (by specific expression of temperature-sensitive dynamin) and ionic transport (by glia-specific expression of bacterial Na⁺ channel) affects circadian rhythmicity (1213).

The neuropil glia in the insects express excitatory amino acid transporters dEAAT1 and dEAAT2 (847, 1586) and glutamine synthetase (392) responsible for transport and recycling of glutamate. Glutamate transporters are preferentially localized to glial perisynaptic processes (1472), and their loss instigates neurotoxicity leading to neuropil degeneration and decreased lifespan (985). Glutamate homeostasis mediated by glial transporters contributes to insect behaviors, in particular to sexual behavior and courtship. This seems to be associated with glial-specific cystine-glutamate transporter, which regulates ambient glutamate concentration and hence the strength of glutamatergic transmission. Loss of function mutation of these transporters (known as genderblind, gb) results in homosexual courtship (610). Insect glial cells have been also found to take up γ-aminobutyric acid (GABA), thus regulating GABA levels in the neuropil (1259).

Glia in the insects are imperative for trophic and metabolic support of neurons and for neuroprotection; loss of glial cells by targeted ablation results in massive neuronal death (212). Similarly, neurodegeneration and neuronal loss occur in several mutants with malfunctional glia (such as drop dead, swiss cheese, and repo) (251, 905, 1900). In the retina of the honeybee, glial cells supply photoreceptors with alanine, which subsequently is converted to pyruvate for use in the Krebs cycle (1761). Finally, glial cells in insects contribute to CNS defense by mounting reactive gliosis (1037) and phagocytosis both in development and in the adulthood (441, 928).

D. Astrocytes in Chordata and Low Vertebrates

In the CNS of early Deuterostomia (which include Chordata, Hemichordata, and Echinodermata), the parenchymal glia common to invertebrates are substituted by radial glial cells. The radial glia, although being present at some developmental stages in the insects (see above) and being identified in some protostomes (in Annelida and Scalidophora; A. Reichenbach, personal communication), are mainly associated with vertebrates (1424), with their neuroepithelial, layered CNS contrasting the fused ganglia brain in the majority of invertebrates. In the Echinodermata (represented by sea urchin, star fishes, or sea cucumber), which are placed at the very base of Chordata, radial glial cells are the only glia in the CNS (the latter appears in the form of a circumoral nerve ring connected to radial nerve cords). These radial glial cells produce and secrete the Reissner's substance (1076, 1821), mainly comprising the glycoprotein known as SCO-spondin, which probably acts as cell adhesion modulator (580). The Reissner's substance is found in radial glia throughout Chordata from cephalochordates to Homo sapiens. Glia of Echinodermata have a characteristic radial morphology with elongated shape, long processes spanning the whole thickness of the neural parenchyma, perpendicular orientation to the surface of the neuroepithelium, and high level of expression of intermediate filaments in the cytoplasm (1075, 1076). Functional properties of these glial cells remain unknown.

Similarly, in the CNS of many early vertebrates, radial glia represent the main type of neuroglia, with almost complete absence of parenchymal glial cells. This in particular is characteristic for the brains with thin parenchyma. In elasmobranchii (chondrichthian fish, such as sharks and rays), the brains are subclassified according to their gross morphology. In type I, or “laminar” brain, neurons mainly concentrate in the periventricular zone; the brain walls are thin and ventricles are large. In the type II or “elaborate” brains, neurons migrate away from the periventricular zone and form nuclei; these brains have thicker walls and the ventricles are relatively small (62, 266). In the type I brains, radial glia dominate, whereas the elaborated brains of type II contain numerous well-developed astrocyte-like parenchymal glia (62). Emergence of parenchymal astrocytes in elaborate brains probably reflects an increased homeostatic challenge of the enlarged nervous tissue that cannot be met by radial glia. This constrains homeostatic capabilities of the radial glia and hence prompts an increase in numbers and complexity of parenchymal astrocytes (1452). Another possible factor could be associated with an increase in complexity of vascularization, which requires perivascular glial support that cannot be provided by radial glia (1879). In low vertebrates, parenchymal glia are the main component of the blood-brain barrier; in sharks for example, astrocytes completely enwrap vessels making them endocellular capillaries (254, 255). The radial glia seem to be the predominant glial cell type in all anamniotes (that is in fishes and amphibians). In zebra fish (the teleost), radial glial cells express GFAP and possess glutamine synthetase (indicating their possible role in glutamate homeostasis and metabolism) and aquaporin-4 (indicating their role in water homeostasis) (611, 1025). In contrast to elasmobranchii, the blood-brain barrier in teleosts is made by vascular endothelial cells, the arrangement common to most of the vertebrates (254). Similarly to higher vertebrates in the zebra fish and in amphibians, radial glia endfeet enwrap blood vessels (1025). Radial glial cells in zebra fish do not mount reactive response to brain injury; instead, they increase neurogenesis thus closing the lesions without scar formation (113).

The radial glia dominate the brains of reptiles (8, 814), although parenchymal cells bearing astrocytic morphology have been identified in the CNS of some snakes and lizards (1148, 1271). Parenchymal astroglia-like cells frequently appear in the brains of Cayman crocodiles where they intermingle with radial glia, the latter still being the predominant type (815). In adult birds where the astrocytes proper became a predominant cell type, the radial glia remains, although radial glial presence diminishes in postnatal development (30, 816). In mammals, the radial glia disappear almost completely with the exception of radial-like cells in retina (Müller glia), in cerebellum (Bergmann glia), in hypothalamus (tanycytes), and in subventricular zone.

B. Heterogeneity and Main Types

The class of astroglia embraces many cell subpopulations with radically distinct morphology and function; hence, the matter of astrocytic classification and definition according to structure and function has been always under debate (866). In this respect, we take a rather simplistic approach by recognizing all the cells of neuroepithelial origin that are responsible for regulation of any aspect of CNS homeostasis as astrocytes. According to this logic, the neural cells, the main function of which is myelination, belong to oligodendroglia (including mature oligodendrocytes and their precursors also known as NG2 glia), while microglia are clearly distinct in their myeloid origin.

3. Human astroglia

a) protoplasmic and fibrous astrocytes.

Human parenchymal astrocytes are much larger and far more complex than astroglial cells in lesser vertebrates (FIGURE 8). The average diameter of the domain belonging to a human protoplasmic astrocyte is ~2.5 times larger than the domain formed by a rat astrocyte (142 vs. 56 μm). The volume of the human protoplasmic astrocyte domain is ~16.5 times larger than that of the corresponding domain in a rat brain. Furthermore, human protoplasmic astrocytes have ~10 times more primary processes emanating from their somata, and correspondingly much more complex processes arborisation (1248). As a result, human protoplasmic astrocytes contact and integrate around 2 million of synapses residing in their territorial domains, whereas rodent astrocytes cover ~20,000–120,000 synaptic contacts (264, 1248). Likewise, the fibrous astrocytes, populating the white matter, are ~2.2 times larger in humans than in rodents.

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FIGURE 8.

Comparison of rodent and human protoplasmic astrocytes. A: typical mouse protoplasmic astrocyte. GFAP, white. Scale bar, 20 μm. B: typical human protoplasmic astrocyte in the same scale. Scale bar, 20 μm. C and D: human protoplasmic astrocytes are 2.55-fold larger and have 10-fold more main GFAP processes than mouse astrocytes (human, n = 50 cells from 7 patients; mouse, n = 65 cells from 6 mice; means ± SE; *P < 0.005, t-test). E: mouse protoplasmic astrocyte diolistically labeled with DiI (white) and sytox (blue) revealing the full structure of the astrocyte including its numerous fine processes. Scale bar, 20 μm. F: human astrocyte diolistically labeled demonstrates the highly complicated network of fine process that defines the human protoplasmic astrocyte. Scale bar, 20 μm. Inset: human protoplasmic astrocyte diolistically labeled as well as immunolabelled for GFAP (green) demonstrating colocalization. Scale bar, 20 μm. [From Oberheim et al. (1248).]

b) interlaminar astrocytes.

Interlaminar astrocytes [the name introduced by Jorge Colombo (350)] have been originally described by Carlo Martinotti, William Lloyd Andriezen (who named them “caudate neuroglia fibre cells”), and Gustav Retzius as cells with small cell bodies, residing in the upper cortical layer and sending long processes towards deeper layers (44, 1074, 1458). Many decades later it was found that these astroglial cells exist only in the brains of higher primates including old world monkeys, apes, and humans (347, 349, 350). Human interlaminar astrocytes (FIGURE 9) have a small spheroid cell body (~10 μm) localized in the supragranular layer (or layer I) of the cortex and several short and one or two very long (up to 1 mm) processes, which penetrate through the cortex, and end in layers II to IV (1248). The density of interlaminar astrocytes in human brain is quite high and is higher than in chimpanzee (1248). Processes of interlaminar astrocytes often demonstrate a spiriform or corkscrew pattern. The processes terminate in the neuropil and occasionally on the vasculature (1248); terminal portions of these processes end up with specific boutonlike structures also known as terminal masses or end bulbs, which usually contain mitochondria. The processes of interlaminar astrocytes run parallel to each other forming a “palisade.” The phylogenetic origin of interlaminar astrocytes remains unknown, although they were found in brown lemur, Eulemur fulvus (348), indicating that they could have appeared ~30 million years ago in prosimians. The interlaminar astrocytes appear postnatally and are thought to originate from some astroglial precursors and not directly from radial glia (349). The interlaminar astrocytes are specifically stained with CD44 antibodies; they have high immunoreactivity for GFAP and S100B and have low expression of glutamine synthetase and glutamate transporters (1655). Long processes of interlaminar astrocytes contact several blood vessels as well as the pia and traverse territorial domains of protoplasmic astrocytes. Electrophysiologically interlaminar astrocytes displayed passive currents similar to other types of astroglia (1655). The specific function of these cells remains unknown, although they might be instrumental in connecting distant cells and integrating cellular groups into wider structures.

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FIGURE 9.

Interlaminar and varicose projection astrocytes in human cortex. A: interlaminar astrocytes as seen by William Lloyd Andriezen (44). B: pial surface and layers 1–2 of human cortex. GFAP staining is in white; DAPI is in blue. Scale bar, 100 μm. Yellow line indicates border between layer 1 and 2. C: interlaminar astrocyte processes. Scale bar, 10 μm. [B and C from Oberheim et al. (1247), with permission of Springer.] D: varicose projection astrocytes reside in layers 5–6 and extend long processes characterized by evenly spaced varicosities. Inset: varicose projection astrocyte from chimpanzee cortex. GFAP, white; MAP2, red; DAPI, blue. Yellow arrowheads indicate varicose projections. Scale bar, 50 μm. E: diolistic labeling (white) of a varicose projection astrocyte whose long process terminates in the neuropil. Sytox, blue. Scale bar, 20 μm. F: high-power image of the yellow box in B, highlighting the varicosities seen along the processes. Scale bar, 10 μm. G–I: individual z-sections of the astrocyte in E, demonstrating long processes, straighter fine processes, and association with the vasculature. [D–I from Oberheim et al. (1248).]

c) polarized astrocytes.

These cells are similarly present only in the brains of primates. Their somata are positioned in the deep cortical layers very near to the white matter. These cells have one or two long (up to 1 mm in length) processes that penetrate into superficial cortical layers (1248).

d) varicose projection astrocytes.

The varicose projection astrocytes exist only in the brains of humans (FIGURE 9). These cells are characterized by several (up to 5) very long (up to 1 mm) unbranched processes that extend in all directions through the deep cortical layers. These processes are endowed with evenly spaced varicosities (1248). These cells are similar to atypical protoplasmic astrocytes, described in adult human brains. These cells acquire immunopositivity to CD44, while their numbers vary considerably between individual samples; they are never observed in neonatal brains (1655). Morphologically approximately one-third of these CD44-positive cells were similar to regular protoplasmic astrocytes; some (~10%) had decreased arborization, while ~60% had long and thin processes (1655). The atypical astrocytes phenotype was suggested to reflect individual age-dependent, possibly adaptive, changes.

e) human astrocytes and intelligence.

The possible contribution of astrocytes to the information processing remains a debatable question. It has been hypothesized that astrocytes represent the smallest computing unit of the brain, the microchip, for integration of synaptic input. This hypothesis is based on the observation that astrocytes in visual cortex generate Ca²⁺ signals to a shorter tuning range of light than neurons. This indicates that astrocytes are more selective than neurons to the visual input and that astrocytic Ca²⁺ spikes represent processed second level information (1581). Since the volume of human astrocytes is 15- to 20-fold larger than in the rodents, a single astrocytic domain covers many more synapses in the human brain. It is therefore tantalizing to ask whether integration of information by astrocytes constitutes an essential part of processing power and in part explains human intelligence. To start addressing this question, the humanized mice pups were developed by intra-brain implantation of human glial cell progenitors at postnatal day 1 (637). The human glial cells expanded, populated large parts of the chimerized mice brain, and replaced mouse glial progenitor cells (FIGURE 10). Further analysis demonstrated that the threshold for induction of long-term potentiation (LTP) in hippocampus was reduced, in a pathway partly driven by tumor necrosis factor (TNF)-α, but not by release of d-serine or adenosine. The humanized chimeric mice displayed enhanced memory and ability to learn in cognitive tests, including novel object recognition or auditory fear conditioning. Several parallel lines of work have similarly shown that human glial progenitors, independently of whether they are generated from embryonic tissue or from induced stem cells, exhibit a growth advantage and slowly replace the host murine glial progenitor cells and mature astrocytes (582, 1885).

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FIGURE 10.

Human astrocytes replace host glia in mice engrafted with human glial progenitors. A: schematic outlining the procedure for magnetic cell sort-based isolation (MACS) of human glial progenitors, tagging with EGFP and xenografting at P1. The chimeric mice brains were analyzed in 0.5- to 20-mo-old chimeric mice. B: representative dot map showing the distribution of human nuclear antigen (hNuclei)⁺ cells in 3 coronal sections from a 10-mo-old human chimeric mouse. C: the complex fine structure of human astrocytes in chimeric brain replicates the classical star-shaped appearance of human astrocytes labeled with hGFAP in situ. Most cells in the field are EGFP⁺/hNuclei⁺/hGFAP⁺ (hGFAP, red). D: at 5 mo, EGFP⁺ cells typically infiltrated corpus callosum and cortical layers V and VI. All EGFP⁺ cells labeled with an antibody directed against human nuclear antigen (hNuclei) and most of the human cells were also labeled with an antibody directed against human GFAP (hGFAP, red). E: at 11 mo, many areas of cortex were infiltrated by evenly distributed EGFP+ /hNuclei⁺ cells. F: the hippocampus was also populated with EGFP+/hNuclei⁺ cells in a 14-mo-old animal, with the highest density in the dentate. G: human EGFP⁺/hNuclei⁺/GFAP⁺ cells (green arrow) were significantly larger than host murine astrocytes (red arrow). The anti-GFAP antibody cross-reacted with both human and mouse GFAP (red). Inset shows same field in lower magnification. H: histogram compares the diameter of mouse cortical astrocytes to human cortical astrocytes in situ (freshly resected surgical samples) and xenografted human astrocytes in cortex of chimeric mouse brain. The maximal diameter of mouse and human astrocytes (in situ and in chimeric mice) was determined in sections stained with an anti-GFAP antibody that labels both human and mouse GFAP. In B–F, n = 50–65; **P < 0.01, Bonferroni t-test. EGFP, green; hNuclei, white and white arrow; DAPI, blue. Scale bars: 50 μm (C); 100 μm (D–F); 10 μm (G). Data graphed as means ± SE. [From Han et al. (637).]

C. Gender Differences

There are multiple indications for sexual dimorphism of astroglia (6). Gonadal hormones regulate the expression of GFAP, astroglial numbers, and morphology. Steroid deficiency causes astroglial atrophy in hippocampus, whereas treatment of organotypic cultures with estradiol and testosterone increases astrocyte complexity and extension of their processes (409). In young rats for example, the total number of GFAP-positive hippocampal astrocytes was higher in males, whereas feminization or masculinization of these animals reversed this difference (351). In adult rats, the GFAP immunoreactivity and astroglial numbers were highest in proestrus females compared with males or diestrus females; moreover, male astrocytes displayed more stellate, whereas female astrocytes had more reactive morphological appearance (63). In the somatosensory cortex, GFAP immunoreactivity and astroglial processes density are maximal in both proestrus and diestrus, while they decrease significantly during estrus (1685). In medial amygdala of adult rats, the number and complexity of astrocytes were higher in males, when compared with females or with testicular feminized mutant males lacking functional androgen receptors (798). Similarly, male astrocytes are more complex and have longer and more numerous processes in preoptic area (33), in nucleus arcuatus (1143), and in supraoptic nucleus (1692). In the cerebellum, Bergmann glial cells in males demonstrated more immunoreactivity for GFAP, whereas in females they showed more immunoreactivity for vimentin (1693). Sexual dimorphism was also extended to cellular functions; for example, female astrocytes in culture demonstrated more efficient glutamate uptake (1153).

D. Astroglial Networks or Syncytia

Macroglia (that is astrocytes and oligodendrocytes) represent the reticular portion of the brain being intracellularly connected into functional syncytia by gap junctions. The latter are ubiquitous structures responsible for intercellular integration in many tissues connecting, for example, epithelial cells of the gastrointestinal tract and kidney, providing metabolic coupling in the liver, electrical coupling in the heart, intercellular signaling in endocrine tissues, and defining cochlear physiology and hence hearing (217, 638, 1216, 1488). Gap junctions are specialized areas where two apposing membranes of adjacent cells come very close together so that the intercellular cleft is reduced to a width of ~2–3 nm (493). Within these areas, each gap junction is made of many hundreds of intercellular channels or connexons, which provide for intercellular transport of ions, second messengers, and other biologically active molecules with smaller than 1,000 Da (see sect. VIIH). In the grey matter pairs of astrocytes are connected with ~230 gap junctions on average, and injection of Lucifer yellow or biocytin into a single astrocyte results in staining of ~50–100 adjacent astroglial cells.

The earlier concept of panglial syncytium that connects all macroglia into single functional network, although being confirmed for invertebrates (1163), does not fully apply to mammalian CNS. First, the gap junctions between oligodendrocytes are rather restricted (1438). Second, in many brain regions anatomically segregated astroglial networks follow anatomical structures; for example, astroglial syncytia are confined to individual barrels of somatosensory cortex or to individual glomeruli in the olfactory bulb (571, 739, 1509). Coupling between adjacent astrocytes is also not ubiquitous; some (probably up to 15–20%) of the neighboring astrocytes are uncoupled as judged by either dye diffusion or by double patch-clamp recordings from the pairs of closely apposed cells (740, 1103). Thus astroglial coupling is defined not only by spatial proximity but also by some other factors, and astroglial networks may represent a wiring system parallel to that formed by neurons. The gap junctional connectivity may also represent a specialized “analog” intercellular signaling system (alternative to “binary” interneuronal communication), which by utilizing intercellular diffusion of multiple molecules, can provide a second level of information processing in the CNS.

Panglial syncytia, which connect astrocytes and oligodendrocytes, have been visualized in thalamus, neocortex, and hippocampus (604). Gap junction plaques between oligodendrocytes and astrocytes, localized on oligodendroglial somata, processes and outer (abaxonal) layer of the myelin sheath have been visualized with freeze-fracture electron microscopy (1079, 1437). Coupling between astrocytes and oligodendrocytes was also confirmed electrophysiologically (in pair recordings) and by the dye coupling (270, 856, 1337, 1477). The functional role of panglial connectivity remains largely unknown; an obvious suggestion links this to K⁺ homeostasis (1105). Mutations in oligodendroglial/astroglial connexins cause severe deficits in myelination in white matter (1281). Mutation in the gene GJB1, which encodes Cx32, underlies Charcot-Marie-Tooth disease associated with CNS white matter lesions (963, 1736), whereas mutations in GJC2 (encoding Cx47) are linked to leucoencephalopathies (253) and hypomyelinating leukodystrophies known as Pelizaeus-Merzbacher disease (1774). In mice, genetic deletion of either Cx32 or Cx47 does not result in an obvious phenotype; elimination of both genes however causes severe white matter demyelination (1104).

B. Membrane Potential

The most characteristic electrophysiological signature of mature astrocytes is their hyperpolarized resting potential that is set close to E_K (approximately −80 mV) and low input resistance (5−20 MΩ) indicative of high resting membrane permeability for K⁺ (379, 1125, 1126). This is also reflected by nearly linear current to voltage relationship, which is the most characteristic electrophysiological signature of astroglia (FIGURE 12; Refs. 11, 337, 436, 455, 772, 811, 1012, 1265). Incidentally neonatal astrocytes are even more hyperpolarized (V_m around −85 mV) (1951). Some variability of resting V_m (between −85 and −25 mV) reported for astrocytes in culture, in organotypic and acute slices, and in the optic nerve (208, 917, 1096), most likely reflect preparation artifact. These membrane properties are intrinsic for astrocytes as isolated cells are very similar to syncytially connected cells in the nervous tissue (454); astrocytes in the syncytia are isopotential (1027). Fluctuations of astroglial V_m generally reflect changes in extracellular K⁺ concentration (36, 379).

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FIGURE 12.

Passive membrane properties of astrocytes. A: voltage-clamp recordings from astrocytes freshly isolated from the cortex of transgenic mice expressing EGFP under control of the GFAP promoter. Astrocytes were identified by specific fluorescence; whole-cell currents were recorded in response to hyperpolarizing and depolarizing steps from −120 to +60 mV (step interval, 20 mV). To construct the current-voltage relationship, amplitudes of currents were normalized to the value measured at 0 mV; every point is mean ± SD for 20 cells. [From Lalo et al. (935).] B: voltage-clamp recordings from astrocytes in acute slices obtained from 3- and 20-mo-old GFAP-EGFP mice; astrocytes were identified by fluorescence. [From Lalo et al. (932).] C: voltage-clamp recordings from human astrocytes grafted into mouse brain. [From Han et al. (637).]

B. Sodium Channels

C. Calcium Channels

D. Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) Channels

The Na⁺/K⁺ HCN channels (HCN1−4, with Na⁺/K⁺ permeability ratio of 1: 4) have been detected in healthy astrocytes in situ; however, their expression increased significantly after ischemia (719, 1520). At transcript level, all four mRNA for HCN 1−4 were identified, whereas only HCN1−3 proteins have been detected in reactive post-stroke astrocytes; in these cells, the whole cell HNC-mediated currents have been characterized (719, 1520).

E. Acid-Sensitive Ion Channels, or ASICs

The acid-sensitive ion channels are rather ubiquitously expressed in the nervous system; ASIC1, ASIC2a, and ASIC3 were localized with immunocytochemistry in cultured rat cortical astrocytes. All three isoforms were predominantly localized in the nuclei and were not associated with membrane currents (746). In freshly isolated mouse hippocampal astrocytes, however, single-cell RT-PCR revealed transcripts neither for ASICs, nor for TRPV1 channels (1874). Expression of ASIC1a was reported to be increased in reactive astrocytes in the context of chronic epilepsy, and their activity may contribute to seizure generation (1911). To conclude, the functional importance of ASICs for astroglial physiology in vivo remains unknown.

F. Anion Channels

The whole cell outwardly rectifying voltage-dependent Cl⁻ currents were initially observed in cultured astrocytes depolarized to potentials more positive than −50 mV (166, 602). These findings, however, were not replicated in other studies, which demonstrated almost complete absence of anion conductance in astroglia at rest (868). Changes of astrocytic morphology (due to osmotic stress or treatment with cAMP, which induced cell stellation), however, activated the dormant Cl⁻ conductance (945). Apparently, Cl⁻ channels in astroglia are controlled by direct link to cytoskeletal actin (946). The actin connection may also explain earlier findings that single-channel Cl⁻ currents could be detected only in excised patches (106).

Several types of chloride (anion) channels [represented by the ClC channel family, by cystic fibrosis transmembrane conductance regulator (CFTR) channels, by voltage-dependent anion-selective channels (VDAC), by Ca²⁺-dependent Cl⁻ channels, and by volume-regulated anion channels (VRAC)] have been identified in astroglia (868). In cultured astrocytes, ClC-1, -2, and -3 channels were detected at transcript and protein levels (1326, 1944), while ClC-mediated currents were characterized in astrocytes in vitro as well as in hippocampal slices (1054, 1326). The immunoreactivity for ClC-2 was also found in Bergmann glia in cerebellum (192). Of note, ClC-2 channels were clustered in astroglial processes in the vicinity of GABAergic synapses, which prompted the hypothesis of astroglia-dependent regulation of intracleft Cl⁻ concentration and hence of GABAergic transmission (1618). The ClC channels are inwardly rectifying (i.e., promoting Cl⁻ efflux at potentials below E_Cl) and hence they mediate Cl⁻ secretion from the astrocyte. Furthermore, ClC-2 channel open probability is high at astroglial resting potential and is modulated by change in the cell volume. Thus these channels facilitate release of Cl⁻ in response to cell swelling (456, 1489).

The VRAC are fundamental for cell volume control and contribute to regulation of cell proliferation, cell motility, and apoptosis. Molecular identity of VRAC was discovered recently after identification of a plasma membrane spanning protein leucine-rich repeat containing 8A (LRRC8A) that was dubbed SWELL1 (1416, 1833). In astrocytes, VRAC are specifically important for regulatory volume decrease that invariably follows astroglial swelling due to osmotic disbalance (868). The VRAC channel currents induced by osmotic stress have been characterized in astrocytes in vitro (5, 138, 1326). The VRAC channels were also reported to form a conduit for excitatory amino acids; this was initially suggested by monitoring release of radioalbeled taurine, glutamate, and aspartate (867, 1333). Glutamate release, mediated by VRACs, has been subsequently observed in astroglial primary cultures (506, 991) and in astrocytes in situ (111, 1717). The activity of VRAC is claimed to be regulated by cytosolic Na⁺: increase in [Na⁺]_i suppressed VRAC activity (1122). The LRRC8A/SWELL1 are critical for swelling-activated release of glutamate and taurine from cultured rat astrocytes (757).

Astrocytes also express an anion channel of Bestrophin (Best) family. The Best gene, responsible for a dominantly inherited, juvenile-onset form of macular degeneration called Best vitelliform macular dystrophy, encodes a Ca²⁺-activated anion channel (655). In the brain, Best1 channels were reported to be largely expressed in astrocytes (1255, 1324), being concentrated in perisynaptic regions (1322, 1893). These Best1 channels were activated by relatively moderate increases in [Ca²⁺]_i at V_m −70 mV (i.e., close to astroglial resting potential), while Best1-mediated Cl⁻ currents could be blocked by broad-spectrum antagonists of anion channels such as niflumic acid, NPPB, and flufenamic acid (1324). Astroglial Best1 channels are suggested to form a conduit for secretion of glutamate and GABA (see sect. XIE).

G. Aquaporins

Astrocytes express three members of aquaporin family, the AQP1, AQP4, and AQP9 (84, 1546), although AQP4 has by far the largest presence (1184). The AQP4 is composed from eight intramembrane helical segments M1−M8 and two translation initiation sites designated as Met1 and Met23, which in turn define two AQP4 isoforms, M1 and M23 (1314). Hypothalamic tanycytes express solely AQP9 (474). The highest expression of AQP4 has been indentified in the cerebellum with lower AQP4 presence in the hippocampus, diencephalons, and cortex (750). Astroglial AQP4 channels are concentrated in the perivascular and subpial endfeet, where their density is at least 10 times higher than in other parts of the cell. In the endfoot membrane, the AQP4 channels are assembled into orthogonal arrays of particles (also known as square arrays) that are observed under electron microscopy of freeze-fracture replicas (411, 1439, 1889). High densities of AQP4 in the endfeet mediate direct interactions with pericytes (618), with endothelial cells and with extracellular matrix with a specific role for proteoglycan agrin (280).

Physiological roles of AQP4 are subject of intense investigations, as it seems to contribute to a wide variety of CNS functions. Rather surprisingly, at the time of generation, the AQP4 knockout mice did not demonstrate any obvious phenotype (1031). Subsequently, the gross alterations of nervous function have been revealed as AQP4^−/− mice displayed seriously impaired olfaction (1014), and even more seriously affected hearing: nearly all mice lacking AQP4 are deaf (1014, 1111). More detailed studies revealed a sevenfold decrease in astroglial water permeability (1641), deficient K⁺ buffering, compromised regulation of extracellular space (181), impaired hippocampal LTP/LTD without any changes in background synaptic transmission (1557, 1632), and altered spatial memory (1632, 1941). The AQP4 channels were found to be linked to astroglial Ca²⁺ signaling induced by osmotic stress (1749). Astroglial AQP4 channels also seem critically important for operation of the CNS glymphatic system (see sect. XIIH). Finally, genetic deletion of AQP4 significantly modifies various neuropathologies (1184).

H. Connexons

Connexons are fundamental for integrating astrocytes into interconnected syncytia. They form gap junctional channels spanning through the membranes of adjacent cells. Gap junctional channels are composed from two precisely aligned connexons from the two adjacent coupled cells (414, 1524). Assemblies of 100s of connexons into large cristalline-like structures make gap junction plaques between adjacent cells. When connexons fail to align with a congruent proteins from a neighboring cell, they may act as a stand-alone gated pores, known as hemichannels (490).

Connexons are assembled from six subunits, known as connexins (Cx), that represent a multigene family (with 21 members in human genome) which differ in molecular mass between 26 and 62 kDa; this underlies the nomenclature (e.g., Cx26 or Cx43). All connexins are similar in topology with four membrane-spanning domains. Functional connexons have a high conductance reflecting the pore with diameter between 6.5 and 15 Å; this pore is permeable not only for ions but also for hydrophilic molecules with molecular mass <1 kDa, including second messengers, nucleotides, glucose, various metabolites (397, 650, 1714), and even siRNA (1783). Of note, connexins have a high turnover rate with a half-life of several hours; degradation of connexins involves internalization into a specific double-membrane vacuoles known as annular junctions or connexosomes (888). Generally, connexons may assemble into homo- or heteromers, and gap junction channels can be made from identical connexons (making homotypic channel) or different (heterotypic channels). When the channel is composed from heteromeric connexons, it is classified as heteromeric. Finally, the gap junctions can connect the same type of cells (homocellular gap junction) or different type of cells (heterocellular gap junction). In astrocytes, connexons may also connect parts of the same cell through reflexive gap junctions (1890); these may create signaling and diffusional shortcuts in the highly arborized astroglia.

Astroglial expression of connexins is complex, and hence, astrocytes may contain multiple types of gap junctional channels. Astrocyte-astrocyte homocellular gap junctions are composed from Cx26, Cx30, and Cx43 (570, 923, 1187), of which Cx43 is the most abundant (1186). It seems that Cx30 and Cx43 may assemble as both homo- and heteromers, whereas Cx26 seems to make homomers only (28). Expression of Cx43 is ubiquitous, while the highest expression of Cx30 has been determined in thalamus and leptomeninges. Substantially lower expression of Cx30 is found in cortex and hippocampus, and no expression at all was detected in white matter (1186, 1640). Expression of Cx26 is restricted to astrocytes in subcortical areas, such as hypothalamus as well as reticular thalamic and the subtalamic nuclei (1188). Astrocyte-oligodendrocyte heterocellular gap junctions are composed from heterotypic channels which, in vitro, can be represented by four operational complexes: Cx47/Cx43, Cx47/Cx30, Cx32/Cx30, or Cx32/Cx26 (1049), although in situ the Cx32/Cx30, Cx47/Cx43 appear to dominate functional intercellular connectivity (28, 1282). Pairing Cx43 and Cx32 does not produce functional gap junctional channel (1878). There are some sporadic reports of astrocyte-neuronal heterocellular contacts (31, 1182, 1299), which are probably limited to developing brain and could not be considered as a significant pathway for glial-neuronal communication.

In cultured astrocytes obtained from Cx43 knockouts, expression of Cx26, Cx30, Cx40, Cx45, and Cx46 was detected (412, 1741), whereas Cx30 expression was almost doubled in Cx43-deficient animals (1742). These changes in expression explain why genetic deletion of Cx43 results only in partial (~50%) suppression of gap junctional astroglial connectivity (1742). Expression of connexins may be regulated by neuronal factors: coculturing neurons with astrocytes upregulates Cx43 and initiates Cx30 expression in the latter (899). Connexon-based gap junction channels are voltage dependent (being sensitive to trans-junctional potential, although some channels are also regulated by V_m); this voltage gating depends on the subunit composition (691). Conductance of Cx43-formed gap junctional channel in cultured astrocytes was around 50−60 pS (413, 570). Biophysical properties of connexons are regulated by multiple factors including pH, [Ca²⁺]_i, or phosphorylation state, which is controlled by protein kinases A, C, and G and by mitogen-activated protein kinases, etc. (473, 691). Connexons are also positively modulated by intracellular spermine and spermidine (134).

Unpaired connexons, the hemichannels, have been identified in astrocytes in vitro and in vivo; all three connexons expressed in astrocytes (Cx26, Cx30, and Cx43) can operate as hemichannels (572). As a rule, these hemichannels are closed in healthy resting cells, but can be activated by low external calcium concentration, by substantial depolarization, by some specific intracellular Ca²⁺ signals, or by exposure to proinflammatory agents (1273, 1274). The hemichannels are considered to provide conduits for astroglial secretion of various substances, including neurotransmitters and neuromodulators (see sect. XIC). Depolarization activates unpaired connexons, which generate voltage- and time-dependent transmembrane currents, biophysical parameters of which depend on channel subunit composition. In physiological conditions (i.e., at normal concentration of divalent cations), open probability of Cx channels is very low at resting membrane potential. Mouse Cx30 channels show prominent voltage dependence (1784). This is not the case for Cx43 (357), while human Cx26 hemichannels show bell-shaped voltage dependence (593). The unitary conductance for Cx26 hemichannels is ~320 pS (593), for Cx32 hemichannels ~90 pS (1535), and for Cx43 hemichannels ~220 pS (1457). Of note, the conductance of Cx43 gap junctional channel is about four times lower than that of unpaired one (413, 570), although theoretically it should be only half. This probably indicates some additional regulation of the channel in gap junctional configuration.

I. Pannexons

Pannexons have no homology with connexons and are classified as gap junction proteins because of some homology with innexons; pannexons never form intercellular channels and hence exist as stand-alone plasmalemmal gated pores (490).⁷ At the same time the overall topology (four transmembrane domains) and pharmacological properties of pannexins and connexins are rather similar, suggesting some common functional evolution and gating mechanisms (1002). Depending of the stimulation mode, pannexon opens as a large-conductance (~500 pS) pore or operates as ~50 pS anion channel (1032, 1856). Pannexon channels are assembled from six subunits, of which three subtypes pannexin1, -2, and -3 (or Panx1, -2,-3) are known. Transcripts for Panx1 were identified in astroglia in vitro and in situ (749, 1443), and Panx1 currents were electrophysiologically characterized in primary cortical astrocytes (758). Astroglial pannexons were activated by voltage and by stimulation of P2X₇ receptors; they were blocked by gap junctions blockers carbenoxolone (CBF) and mefloquine (MFQ) and were permeable to fluorescent tracer YoPro (758). The Panx1 channels in astrocytes could also be activated by an elevation of extracellular K⁺ concentration, which shifts activation potential of pannexons to more hyperpolarized values (1620). Astroglial pannexons were reported to be activated by fibroblast growth factor 1 (554). Pannexons, in high conductance state, can be permeable to molecules with molecular mass up to 1.5 kDa and hence may contribute to astroglial secretion (see sect. XIC). Panx1 in particular is considered as transmembrane conduit for ATP (375). The P2X₇-pannexon complex was also implicated in Ca²⁺-independent release of d-serine from cultured astrocytes (1306). The evidence for functional pannexons and their physiological role in astrocytes in vivo remain, at best, circumstantial.

A. Purinoceptors

Purinergic signaling system that utilizes purines and pyrimidines as extracellular signaling molecules is arguably the most ubiquitous being present in every organ and system without any obvious anatomical segregation. Correspondingly most of the living cells possess at least one (and often several) type(s) of purinoceptors (263). These latter are classified into adenine (P0) receptors, adenosine (A or P1) receptors, and nucleotide (ATP, ADP, UTP) receptors of the P2X (ionotropic) and P2Y (metabotropic) varieties (262, 1239, 1807). In the CNS, purines and pyrimidines are released from neurons (mainly from their terminals) and from neuroglia, with Ca²⁺-regulated exocytosis being probably the most widespread (and certainly the most studied) mechanism (2). After being released ATP is rapidly degraded by specific extracellular enzymes, known as ectonucleotidases (1968) to ADP, AMP, and adenosine that all act as purinergic agonists, which often can have opposite effects on target cells. The absolute majority of neuroglial cells studied so far express some purinoceptors (except P0 receptors, astroglial presence of which has not been studied yet). Neuroglial cells are also capable of releasing ATP and adenosine, which makes them an important source of physiologically released purines in the CNS and places purinergic transmission at the fore of gliotransmission (267, 1810). Purinergic signaling system also contributes to neuropathology; in particular, ATP released from damaged cells acts as a universal “danger” signal (often defined as damage-associated molecular pattern, DAMP) that controls glial defensive reactions such as reactive astrogliosis and activation of microglia (520).

2. Ionotropic P2X purinoceptors

Ionotropic P2X purinoceptors are archetypal ligand-gated cationic (Na⁺/K⁺/Ca²⁺) channels (FIGURE 13), assembled as homo- or heterotrimers from seven different subunits encoded by distinct genes that are classified P2X₁ to P2X₇ according to historical order of cloning (1239). Receptors formed through homo- or heteromeric assembly of P2X₁ to P2X₆ subunits (hitherto homomeric composition was shown for P2X_1–5 subunits, P2X₆ subunits apparently cannot olygomerize; heteromeric compositions are represented by P2X_1/2, P2X_1/4, P2X_1/5, P2X_2/3, P2X_2/6, and P2X_4/6 channels) are activated by low (submicromolar and micromolar) ATP concentrations, while homomeric P2X₇ receptors require millimolar ATP for activation (1240, 1705).

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FIGURE 13.

Main properties of ionotropic receptors functionally expressed in astroglia.

There is a substantial discrepancy between evidence for P2X subunits expression in astroglia and data on their functional manifestation. Primary cultured astrocytes contain transcripts for P2X_1–5 and P2X₇ receptors (435, 541). In tissue extracts from rat nucleus accumbens, RT-PCR revealed expression of all seven P2X mRNAs (516). Cortical astrocytes contained transcripts for P2X₁ and P2X₅ subunits (937). In acutely isolated rat retinal Müller cells, P2X₃, P2X₄, P2X₅ but not P2X₇ mRNAs were identified (776), whereas P2X₇ mRNA was detected in human Müller glia (1313). Immunocytochemistry also demonstrated expression of P2X subunits. In astrocytes from healthy nucleus accumbens, P2X_2,3,4 receptors proteins were detected (although all 7 subunits were present at mRNA level); mechanical lesion however triggered additional expression of P2X₁ and P2X₇ subunits (516). Immunoreactivity for P2X₁ and P2X₂ receptors was detected in astroglial cells in cerebellum (829, 1001), and P2X₂ receptors were found in spinal cord astrocytes (828), P2X₄ receptors were identified in astrocytes from the brain stem (69) and in Müller glia (708), whereas hippocampal astrocytes were immunoreactive for P2X_1–4, P2X₆, and P2X₇ receptors (922).

Electrophysiologically, P2X-mediated currents are much less characterized. In primary cultured astrocytes, ATP evoked inward currents and [Ca²⁺]_i rise, although subunit composition of the underlying receptors was not identified (1050, 1844). In hippocampal astrocytes, both acutely isolated or in slices, no P2X-mediated currents were found, contrasting RT-PCR and immunocytochemistry findings indicative of P2X subunits expression (778). No ATP-induced currents have been found in Bergmann glial cells in acute cerebellar slices (880). In astrocytes from acutely isolated optic nerves, ATP triggered large [Ca²⁺]_i transients sensitive to the P2X receptor antagonist NF023 (used at 100 μM concentration at which it blocks all P2X_1–4 receptors); the [Ca²⁺]_i response could be also mimicked by the broad P2X agonist α,β-methylene ATP (α,β-meATP) (631, 786). In cortical mouse astrocytes, expression of P2X_1/5 heteromeric receptor was reported (FIGURE 14) (937). These receptors are highly sensitive to ATP (EC₅₀ ~50 nM); they have specific biophysical properties represented by biphasic kinetics with distinct peak and steady-state components, activation of “rebound” tail current following the washout of the agonist, and a very little desensitization in response to the repetitive agonist applications (936, 937). The P2X_1/5 receptors contribute to “glial synaptic currents” triggered in astrocytes by stimulation of neuronal afferents in cortical slices (932, 936). The P2X_1/5 receptors also mediate spontaneous “miniature” postsynaptic currents in astrocytes in cortical slices (936). Astroglial P2X_1/5 have intermediate Ca²⁺ permeability (P_Ca/P_monovalent ~2.2), and their activation by endogenous agonists or by synaptically released ATP triggered transient cytoplasmic Ca²⁺ signals (1305). It is of course important to mention that most humans carry a single-nucleotide polymorphism at the 3′ splice site of exon 10 of the human P2X5 gene, which renders the P2X₅ subunit nonfunctional (898).

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FIGURE 14.

P2X_1/5 receptor-mediated currents in cortical astrocytes. A: the family of ATP currents evoked by repetitive applications of the agonist. The currents show no apparent desensitization. Current traces have a complex kinetics comprising the peak, the steady-state component, and the “rebound” inward current recorded upon ATP washout as indicated on the graph. B: concentration dependence of ATP-induced currents in cortical astrocytes. Membrane currents recorded from a single cell in response to different ATP concentrations are shown on the left. The right panel shows the concentration-response curves constructed from 9 similar experiments; current amplitudes were measured at the initial peak and at the end of the current, as indicated on the graph. C: inhibition of ATP-induced currents by PPADS. Currents recorded at various concentrations of PPADS are shown on the left, and the concentration dependence of inhibition for peak and steady-state components constructed for 7 individual experiments is presented on the right. The peak component of the response was more sensitive to PPADS. Application of PPADS started 2 min before application of ATP. All recordings were made at holding potential of −80 mV. [From Lalo et al. (937).]

Astroglial P2X₇ receptors have received much attention, mostly because of their pathological significance (i.e., initiation of apoptosis and cell lysis, regulation of astrogliosis or controlling processing and release of cytokines; see Refs. 520, 763 for relevant literature). The P2X₇ receptors are unique in their low ATP sensitivity (in all probability it is ATP^4- that acts as a true agonist), almost complete absence of desensitization, and ability to produce large transmembrane pores upon intense stimulation (1239). The very first in situ hybridization mapping (346) failed to detect P2X₇ receptors in the brain. Subsequent studies revealed P2X₇ transcripts and proteins in many areas including cortex, hippocampus, medulla oblongata, cerebellum, thalamus, and amygdala (1658). The specificity of antibodies used for labeling P2X₇ receptors is, however, far from ideal (1621), thus making many observations contentious.

In primary cultured astroglia, expression of P2X₇ receptors was frequently documented at both the transcriptional and protein levels (435, 457, 541, 752, 797, 1194, 1309, 1850). Freshly isolated astrocytes as well as astrocytes in slice preparations similarly demonstrated immunoreactivity for P2X₇ receptors (922, 1261, 1309). At the same time, in-depth analysis of the cellular distribution of P2X₇ mRNA in the rat brain using isotopic in situ hybridization detected these receptors in neurons, oligodendrocytes, and microglia but failed to identify P2X₇ receptors transcripts in astrocytes (1930). Astroglial expression of P2X₇ receptors, as a rule, increases after brain injury of various etiology (516, 517, 1194).

In cultured astrocytes, both P2X₇-mediated (as judged by pharmacology, activation by Bz-ATP, and inhibition by oxATP) [Ca²⁺]_i transients (93, 541, 1233) and membrane currents (457, 1238, 1515) were characterized. The P2X₇ currents were also detected in Müller cells freshly isolated from human retina (1313). In isolated optic nerve P2X₇ receptors mediated Ca²⁺ signaling, which was absent in P2X₇^−/− mice (631). The P2X₇ membrane currents were recorded from rat and mouse cortical astrocytes in acute slices. These currents demonstrated all characteristic properties of P2X₇ receptors; they were activated by millimolar ATP and 100 μM Bz-ATP; the agonist sensitivity was increased by removal of extracellular divalent cations; currents were blocked by Brilliant Blue G, as well as by a rather specific agonist A 438079, and they were absent in P2X₇ receptor knockout animals (1261).

Contribution of P2X₇ receptors to astroglial physiology is multifaceted, and seemingly, it may involve pathways and mechanisms not related to opening of the cationic channel. Indeed, activation of P2X₇ receptors in cultured astrocytes is linked to release of glutamate, GABA, ATP, and other purines by exocytosis, through P2X₇-associated transmembrane pore or through Cl⁻/HCO₃⁻-dependent mechanism of an unidentified nature (93, 457, 458, 1690). Sustained release of glutamate was also observed in astrocytes in situ in hippocampal slices following prolonged activation of P2X₇ receptors (503). Activation of P2X₇ receptors increased (by ~60 times) synthesis of endocannabinoid 2-arachidonoylglycerol in cultured astrocytes (1840), modulated release of TNF-α (915), stimulated nitric oxide (NO) production (1175, 1194), induced phosphorylation of AKT (783) and p38MAPK/ERK1/ERK2 (565), prompted transmembrane transport of NADH (1015), increased production of lipid mediators of inflammation cysteinyl leukotrienes (91), and regulated NF-κB signaling (797). Evidence also exists indicating P2X₇-mediated regulation of expression of P2Y₂ receptors (370) and aquaporin-4 (960) in cultured rat astrocytes.

All these functional outcomes of P2X₇ receptors activation are mediated through diverse intracellular signaling pathways. The P2X₇ receptor has a uniquely extended COOH terminus that interacts with several proteins and hence activates intracellular signaling pathways such as extracellular signal-regulated protein kinases (ERKs), serine-threonine kinase Akt (Akt), c-Jun NH₂-terminal kinases (JNKs), mitogen-activated protein kinase (MAPK), and p38 kinase (520). This P2X₇-dependent signaling may be activated by low physiological concentrations of ATP, reflecting non-channel mode of receptor function (426). There are also occasional data on heterogeneity of P2X₇ signaling. Stimulation of P2X₇ receptors led to pore formation in cortical but not in hippocampal astrocytes (173). This heterogeneity may reflect, for instance, expression of different splice variants of the receptor: the splice variant P2X₇A is linked to pore formation and cell death, whereas the P2X₇B variant does not form the pore and stimulates cell growth (12).

B. Glutamate Receptors

1. Ionotropic glutamate receptors in astroglia

a) ampa receptors.

Functional expression of ionotropic glutamate receptors of the AMPA type in astroglia has been well documented in vitro and in situ. In particular, these receptors were electrophysiologically characterized in astrocytes in hippocampal (777, 1589) and cortical slices (FIGURE 15; Refs. 932, 935), as well as in cerebellar Bergmann glia (1169, 1522). All four main subunits of AMPA receptors (GluA1–GluA4) have been identified in astrocytes, although combinations may vary in different brain regions. In hippocampus, for example, GluA2 and GluA4 subunits are predominantly expressed, which is reflected by linear I–V relation and low Ca²⁺ permeability (1589). In cortical astrocytes, GluA1 and GluA4 subunits are the most abundant (355). The AMPA receptor complex in Bergmann glia is devoid of GluA2 subunit which stipulates double-rectifying I–V relationship and some (P_Ca/P_monovalent ~1) Ca²⁺ permeability (563, 1169). In the spinal cord, astroglia immunoreactivity for GluA4 subunit was concentrated in the perivascular processes, whereas somata were stained with GluR2/3 antibodies (228). Conditional deletion of AMPA 1/4 receptors from Bergmann glia led to a retraction of glial perisynaptic processes and deficient fine motor coordination (1522).

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FIGURE 15.

AMPA and NMDA receptor-mediated currents in cortical astrocytes. A: NBQX inhibits the fast component of glutamate-induced current. Representative traces illustrate the current before, during, and after application of 30 μM NBQX (left panel) as well as the NBQX-sensitive current obtained by subtraction (right panel). The concentration dependence of the block of the fast component for four cells (IC₅₀ = 2.2 ± 0.4 μM, Hill coefficient = 1.9) is shown in the inset. B: d-AP5 inhibits the slow component of glutamate-induced current. Representative traces demonstrating the effect of 1 μM d-AP5 (left panel) and the d-AP5-sensitive component were obtained by subtraction (right panel). The concentration dependence of the block for five cells (IC₅₀ = 0.64 ± 0.1 μM, Hill coefficient = 1.6) is shown in the inset. C: NMDA-induced (2-s application) currents in a single astrocyte and concentration-response curve constructed from six such experiments (EC₅₀ 0.34 ± 0.06 μM, Hill coefficient = 1.5). D: glycine-dependent potentiation of astrocyte NMDA response. NMDA-induced currents in glycine-free normal extracellular solution are shown on the top; NMDA-induced currents in the presence of different glycine concentrations (30 nM and 1, 10, and 30 μM) are displayed on the bottom. The concentration-response curve (ΔI_norm represents the amplitudes of current increase normalized to the maximal increase at 30 μM glycine) constructed from seven experiments is shown on the right (EC₅₀ 1.1 ± 0.07 μM, Hill coefficient = 1.2). [From Lalo et al. (935).]

b) kainate receptors.

Functional expression of kainate receptors in astrocytes is yet to be identified, although the relevant subunits were detected at the mRNA and protein levels (228, 548). Expression of GluK1–5 was also found in reactive (but not in healthy) astrocytes in a model of temporal lobe epilepsy (1798).

c) nmda receptors.

Astroglial expression of the N-methyl-d-aspartate (NMDA) receptors was initially detected by EM immunocytochemistry of cortical preparations: the GluN1 and GluN2A/B labeling was found in peripheral astrocytic processes (52, 354). Transcripts for GluN1 and GluN2A/B subunits were identified in Bergmann glia (1023) and in cortical astroglia (1562). In human astrocytes, transcripts for all seven NMDA receptors subunits (i.e., GluN1, GluN2/A-D, and GluN3A,B) have been found (962). The GluN1 immunoreactivity was detected in adult rat astrocytes from the lateral and basal nuclei of the amygdala in the basolateral amygdala (497), in the bed nucleus of the stria terminalis (600), and in the locus coeruleus (1785). In humans, GluN1, GluN2A, and GluN2B proteins were detected in cortical astrocytes [where they predominantly concentrate in the processes (354)], in fetal astroglia (962), and in Müller glial cells (1414).

Exposure of slices to NMDA induced membrane currents and [Ca²⁺]_i transients in the cortical (935, 1261, 1562), in the spinal cord (1965), and in a subpopulation of hippocampal astrocytes (1400, 1593, 1666), whereas small NMDA-induced Na⁺ currents were observed in cerebellar Bergmann glial cells (1168). Evidence for functional NMDA receptors in hippocampal astrocytes remains controversial; several studies on slices or on acutely isolated cells failed to identify any NMDA receptor-mediated currents or [Ca²⁺]_i responses (276, 1589, 1600; see also Refs. 714, 1809). Cationic currents mediated by NMDA receptors were characterized in astrocytes acutely isolated (with nonenzymatic vibro-dissection procedure) from mice somatosensory cortex (FIGURE 15). These NMDA-induced currents were positively modulated by glycine and blocked by specific NMDA receptor antagonists MK-801 and d-2-amino-phosphonopentanoic acid (d-AP5) (935, 1809).

Astroglial NMDA receptors are assembled as heterotetramers from GluN1, GluN2 C or D, and GluN3 subunits. This composition defines their specific biophysical properties represented by weak Mg²⁺ block (which develops at about −120 mV) and relatively low Ca²⁺ permeability (P_Ca/P_monovalent ~3), as well as idiosyncratic pharmacology reflected by sensitivity to memantine and GluN2C/D subunit-selective antagonist UBP141 (464, 936, 1304, 1305). In cortical astrocytes NMDA receptors generate miniature spontaneous currents or they can be activated by neuronal synaptic inputs (932). Opening of astroglial NMDA receptors (in response to endogenous agonist application or synaptic stimulation) results in Ca²⁺ influx and in generation of Ca²⁺ signals (1305). In primary cultured adult rat astrocytes, NMDA receptors were found to be activated by mechanical stimulation delivered as fluid shear stress in the absence of agonists (1057). In cocultures of hippocampal astrocytes and neurons, application of NMDA-glycine mixture caused substantial (~30 mV) depolarization of astroglia; this depolarization was sensitive to MK 801 (972). In acute slices from astroglia-specific NR1 subunit knockout mice, application of NMDA with glycine caused much smaller astroglial depolarizations when compared with wild-type controls (972). Activation of astroglial NMDA receptors was also linked to antioxidant transcriptional activation nuclear factor-erythroid 2-related factor-2 (Nrf2), which stimulates release of glutathione precursors, thus boosting neuronal glutathione biosynthesis and providing for antioxidant protection (794).

C. GABA and Glycine Receptors

D. Acetylcholine Receptors

E. Receptors for Monoamines

1. Adrenergic receptors

The noradrenergic innervation of the brain is provided by numerous projections of neurons, the somata of which are localized in locus coeruleus. These projections do not form localized synaptic contacts; rather, norepinephrine is released from axonal varicosities into the neuropil and hence exerts neurohormonal action through volume transmission. Neurons from rostral and medium portions of locus coeruleus innervate the cerebral hemispheres and cerebellum; projections from the caudal part are sent to the spinal cord. Astrocytes throughout the brain abundantly express adrenergic receptors, which regulate metabolism, morphological plasticity, and physiological responses. The very first observation of catecholamine-mediated increase in cAMP in fetal rat cultured astrocytes was made in 1972 (577). The notion that astrocytes can be an important, if not the main, target of adrenergic innervation (FIGURE 16) appeared in late 1980s (1676) after discoveries that astroglial adrenoceptors control many aspects of cellular metabolism and trophic responses (for early studies, see Refs. 644, 689, 1530, 1757, 1786, 1787).

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FIGURE 16.

Direct stimulation of locus coeruleus elicits Ca²⁺ transients in cortical astrocytes. A: the scheme of experimental setup. B: average fluorescence of the image field in C over time showing Ca²⁺ response to locus coeruleus stimulation. C: fluo 4-labeled astrocytes taken at time points indicated by numbers in B. Insets are blown-up view of boxed area in pictures. Scale bar, 50 μm. D: images from coronal sections through somatosensory cortex illustrating the effect of N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) on locus coeruleus projection neurons as labeled by antibodies to TH (in red) and MAP-2 (in green) with DAPI labeling of nuclei for orientation. Scale bar, 100 μm (20 μm in inset). E: consistent with the significant reduction in locus coeruleus projection neurons and despite almost twice the stimulation intensity (216 ± 33 vs. 400 ± 39 μA; P = 0.004, t-test), there is a significant reduction in the cortical Ca²⁺ response to locus coeruleus stimulation in DSP-4-treated animals (P = 0.013, t-test). [From Bekar et al. (123).]

Both α- and β-adrenoceptors have been found in astroglia in vitro, in slices, and in vivo at mRNA, protein, and functional levels (687). Ultrastructural analysis revealed α- and β-adrenergic receptors in astrocytic processes (51, 53), which are often closely associated with varicosities and terminals of axons of noradrenergic neurons (53, 344, 949). Freshly dissociated and FACS sorted murine cortical astrocytes were found to express mRNA for α_1A-, α_2A-_, β₁-, and (albeit at much lower level) β₂-adrenoceptors (687).

The α₁-adrenoceptors are linked to PLC and InsP₃ cascades, and their activation elicits Ca²⁺ release from the endoplasmic reticulum, an effect well documented in astrocytes in culture (247, 1531, 1596), in freshly dissociated cells (1747), in slices (459, 882), and in vivo (FIGURE 17) (431). These receptors are activated by endogenous adrenergic agonists as well as by norepinephrine released from neuronal varicosities following stimulation of locus coeruleus (431). In neocortical slices from 8- to 12-wk-old mice, as well as in freshly isolated astrocytes from the same slices, norepinephrine caused robust [Ca²⁺]_i increase in all astrocytes; on the contrary, the effect on neurons was rather minor. This signaling was mediated by α₁ adrenoceptors (Ca²⁺ responses were mimicked by specific agonist A61603 and inhibited by specific blocker terazosin); the sensitivity of astroglial receptors was high, with K_D for norepinephrine of ~360 nM (1311).

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FIGURE 17.

Stimulation of α₁- and β-adrenoceptors triggers Ca²⁺ signaling in astrocytes in vivo. A: cortical astrocytes loaded with rhod 2-AM were imaged in layers I/II to detect changes in [Ca²⁺]_i. Agonists were injected using a microelectrode loaded with an artificail cerebrospinal fluid (aCSF) and the tracer Alexa 488. B: representative images of astrocytic [Ca²⁺]_i responses to the α₁-AR agonist methoxamine in awake Glt-1-EGFP transgenic mice. Astrocyte-specific loading of rhod 2-AM was confirmed by colocalization with Glt-1-EGFP. Scale bar = 100 μm. C: rhod 2-AM ΔF/F₀ traces from B, normalized to Glt-1-EGFP fluorescence. Representative aCSF-microinjection trace is shown at the bottom. D: astrocytic [Ca²⁺]_i responses to adrenergic and acetylcholinergic receptor agonists, measured by rhod 2-AM ΔF/F₀. Bar graph shows the percentage of astrocytes within the puff radius responding with a [Ca²⁺]_i increase. ***P < 0.001, one-way ANOVA, Bonferroni correction [n = 25 trials in 6 animals (methoxamine); 14 trials in 4 animals (isoproterenol); 24 trials in 5 animals (dexmedetomidine); 23 trials in 5 animals (carbachol)]. E and F: bar graphs showing average rhod 2-AM ΔF/F₀ and response duration from trials with responding cells. **P < 0.01; *P < 0.05, one-way ANOVA, Bonferroni correction [n = 25 trials (methoxamine), 13 trials (isoproterenol), 17 trials (dexmedetomidine), 20 trials (carbachol)]. Data are means ± SE. [From Ding et al. (431).]

The α₂-adrenoceptors in astrocytic processes have been identified immunochemically in the intact brain (53, 1118). Their functional expression has been corroborated in primary cultures of astrocytes by α₂-adrenoceptor binding (684), by mRNA and protein localization (1118, 1460), and by Ca²⁺ imaging (123, 1223, 1531).

All three types of β-adrenoceptors, i.e., β₁-, β₂-, and β₃-receptors have been identified in astrocytes in vitro and in vivo using radioligand binding, mRNA, and protein detection as well as immunocytochemistry (298, 997, 1060, 1596). The β₁-adrenoceptors are linked to regulation of glycogen synthesis and mediate cAMP-dependent inhibition of astrocytic K⁺ channels; β₂-adrenoceptors are mostly connected to adenylyl cyclase through G_s proteins and are implicated (together with β₃-adrenoceptors) in regulation of glucose uptake through modulation of GLUT1 glucose transporter and glucose metabolism through PKA signaling pathways (448, 684, 755, 1514). Individual astrocytes simultaneously express α- and β-adrenoceptors, and stimulation of both receptors triggers temporally distinct cellular events represented by Ca²⁺ oscillations developing within seconds and much slower tonic increase in cAMP, which saturates within 100s of seconds (723).

F. Bradykinin Receptors

Bradykinin receptors of the B₂ type (the G protein 7-transmembrane metabotropic receptors) have been identified in cultured astrocytes (330); activation of these receptors results in InsP₃ production and Ca²⁺ release from the endoplasmic reticulum (1328, 1672); the [Ca²⁺]_i increase was also concomitant with the activation of an inward current (578). This inward current likely reflects Cl⁻ efflux through Ca²⁺-activated anion channels, possibly of the VRAC variety (17, 990).

G. Cannabinoid Receptors

Cannabinoid receptors of CB₁ and CB₂ types are metabotropic receptors coupled to G_i/o proteins. The CB₁ receptors were identified in rat cultured astroglia (1667). They were also detected in brain tissue, in astrocytes, in the caudate putamen nucleus (1484), in the dorsal horn of the spinal cord (1529), and in olfactory and limbic structures where CB₁ immunoreactivity was concentrated in perivascular glia (1136).

Astroglial CB₁ receptors are linked to regulation of metabolism: their activation increases glucose oxidation and ketogenesis in vitro (196, 1534). In astroglial cultures stimulation of CB₁ receptors inhibits NO production (1137) and release of TNF-α and chemokines in LPS-activated astrocytes (1601). In astrocytes from mouse stratum radiatum pressure, application of 2-arachidonylglycerol (2AG), arachidonylethanolamide (AEA), (R)-(+)-methanandamide (MAEA), or (R)-(+)-WIN 55,212–2 (WIN) triggered [Ca²⁺]_i transients that were inhibited by 2 μM of the selective antagonist of CB₁ receptors AM251. The CB₁-mediated astroglial Ca²⁺ signaling was also reportedly triggered by depolarization-induced neuronal release of endocannabinoids (1197). Subsequently, it was shown that glutamate released from astrocytes acts on presynaptic mGluRs to increase neurotransmitter release in the CA1 and CA3 hippocampal regions (1198). A similar mechanism may also induce presynaptic LTP (592). At the same time, acute stimulation of the CB₁ receptors triggered LTD and impaired working memory in mice; these effects were eliminated in conditional astroglia-specific CB₁ knockout animals (634).

H. Neuropeptide Receptors

I. Receptors for Leptin and Insulin

Leptin is the “anorexic adipokine,” the cytokine produced by adipose cells and released into the circulation. Leptin acts on hypothalamic neural circuitry to curb food intake. The leptin receptors [LepR or Ob(ese)R; obese protein being another name for leptin] are membrane spanning receptors of class 1 cytokine receptors family linked to JAK/STAT, PI₃K-Akt, or ERK signaling cascades (1730). Probably the first observation of astrocyte-associated Lep-Rs was obtained by double anti-GFAP and anti-LepR immunostaining of the arcuate nucleus and median eminence (322). Subsequently, leptin receptors were immunolocalized in GFAP-positive astrocytes in the subcommissural organ of rabbits (377) and in GFAP-positive glia of the nucleus tractus solitarius (378). Strong astrocytic presence of leptin receptors (again revealed by specific immunoreactivity) was found in mice living on a high-fat diet (742) or in mutant mice predisposed to obesity (1307). The LepR mRNA was localized in astroglia in healthy rat hypothalamus with double-labeling fluorescent in situ hybridization; the LepR protein was colocalized with GFAP-positive astrocytic profiles (743). Incidentally, in adult-onset obesity hypothalamic astrocytes and not neurons show the most prominent increase in LepR expression (742, 1307), which possibly indicate a special role for astrocytes in regulation of food-related behaviors (see sect. XIIM). Leptin receptors contribute to regulation of astroglial glutamate transport and glucose uptake (539).

Insulin receptors (IRs) have been detected in rodent and human astrocytes in primary cultures (673, 1958). Astrocyte-specific (using GFAP and EAAT1 promoters) conditional deletion of astroglial IRs impaired brain glucose sensing, changed astroglial morphology, decreased astroglial coverage of hypothalamic neurons, and suppressed glucose transport into the brain (549).

J. Complement Receptors

Complement, which consists of ~20 plasma proteins and another ~10 membrane proteins, represents an important part of the humoral immune system. Complement activation produces C3 convertase, which in turn activates the third complement component (C3). Subsequent processes result in generation of small fragments with signaling properties, represented by C3a, C3b, and C5a molecules (1354). Receptors for C3a (557, 767, 1615) and C5a (555, 1196) have been detected in human fetal astrocytes and in rodent astroglia; the C5a-like receptor was also identified in rat astrocytes (559). The immunoreactivity for CR1 receptors, which regulate phagocytosis of various particles tagged with C3b, C4b, andC1q, has been found to preferentially colocalize with astroglia in the human brain (514, 556).

K. Platelet-Activating Factor Receptor

Platelet-activating factor (PAF) (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent biologically active molecule, discovered initially as an activator of platelets; it is operational in various tissues including CNS. Receptors for PAF were, for the first time, detected in cultured astrocytes using binding to specific ligand [³H]WEB 2086 (239). Subsequently, it was found that picomolar concentrations of PAF stimulate formation of InsP₃ in astroglia in vitro (1180, 1384) as well as promote concentration-dependent astroglial secretion of NGF (240) and release of prostaglandin E₂ from cortical cultured astroglia (1738).

L. Protease-Activated Receptors

Protease-activated receptors are G protein-coupled metabotropic receptors activated following cleavage of a part of extracellular domain by serine proteases; three members of this family (PAR-1, PAR-3, and PAR-4) are activated by thrombin, whereas PAR-2 is a trypsin receptor (1639). Existence of receptors for thrombin in astroglia was suggested following early observation of thrombin-induced stimulation of proliferation and morphological remodeling (599, 1374). Thrombin-activated PAR-1 and trypsin-activated PAR-2 receptors have been initially detected at mRNA level in primary cultured rat newborn astrocytes; activation of these receptors induced [Ca²⁺]_i transients reflecting endoplasmic reticulum Ca²⁺ release (1770, 1772). The astroglial PAR-1 receptor demonstrated a peculiar mode of desensitization, which involved cleavage of the NH₂ terminus (1771). Subsequent analysis revealed expression of transcripts and proteins for all four (PAR1−4) receptors in cultured astroglia, and all four receptors were linked to generation of Ca²⁺ signals (1855). Astroglial PAR receptors are linked to several intracellular signaling pathways, which include production of ROS and stabilization of hypoxia inducible factor-1α through ERK, JNK, and PI3K/Akt cascades (1962). Activation of PAR-1 receptors (for example, by selective peptide agonist TFLLR) is often used for selective stimulation of astroglia in situ (933, 1607), despite the fact that the very same TFLLR-sensitive receptors are known to trigger neuronal Ca²⁺ signaling (635).

M. Ephrin Receptors

Ephrins and their receptors (EphA and EphB) contribute to remodeling synaptic contacts (1174); ephrin receptors are tyrosine kinases activated by membrane-associated ephrin ligands. These Eph receptors (as well as ephrins) have been found in perisynaptic astrocyte processes (290, 510). Activation of EphA receptors induced formation of phylopodia and release of glutamate from astroglial cells in organotypic hippocampal slices (1207), while in cultured astrocytes activation of EphB3 and EphA4 receptors triggered secretion of d-serine (1963).

N. Succinate Receptors

The citric acid cycle intermediate succinic acid may rise quite high [from basal level of 5 μM up to 125 μM (709)] in tissues in physiology (for example during exercise) and in disease (for example in hypertension and metabolic diseases). Receptors for succinate known as SUCNR1 (GPCR91) are classical G protein-coupled metabotropic receptors, and they have been localized (at transcript level) in astrocytes (630). Stimulation of these receptors with succinate (with EC₅₀ ~50−60 μM) triggered [Ca²⁺]_i transients in ~15% of astrocytes from acute slices of nucleus accumbens (1138).

O. Toll-like Receptors

Toll-like receptors (TLRs), deeply involved in various defensive mechanisms associated with neuroglia, are type 1 transmembrane glycoproteins containing a leucine-rich repeat (LRR) domain and a Toll/IL-1 receptor (TIR) domain. These receptors are linked to multiple intracellular signaling systems through five intracellular “adaptor” signal transmitting proteins, which, for example, include myeloid differentiation factor 88 (MyD88) that links TLRs to NF-κB, TNF-α, or interleukin cascades (391, 1759). Although expression of TLRs 1−9 was found in cultured mouse astrocytes, the TLR3 (at least in physiological conditions) seems to be the most abundant (292, 1097). Astroglial expression of TLR3 was detected in vitro, in situ, and in vivo (498). In human astrocytes expression of a TLR1−5 and TLR9 has been reported (250, 779); of these TLR2−4 were identified also in vivo, especially in pathological conditions (250, 779, 1695). In general, expression of TLRs remarkably increases in diseased CNS and in reactive astroglia (1759). In the latter, TLR3 mediate production and secretion of numerous neuroprotective factors such as ciliary neurotrophic factor, neurotrophin-4, and vascular endothelial growth factor, consequently, activation of TLR-3 boosts neuronal survival in organotypic brain cultures (250).

P. Steroid Receptors

Classical nuclear estrogen receptors of α- and β-types (ERα or ERβ) have been found in astrocytes in primary and explant cultures from several brain regions (303, 730, 1545); ERβ receptors were localized by immunostaining in the processes and perikarya of astrocytes of hippocampal tissue (78). Estradiol triggered Ca²⁺ signaling in cultured astroglia (303) through transactivation of mGluR1 and subsequent InsP₃-induced Ca²⁺ release from the endoplasmic reticulum (925). The ERα and ERβ may localize not only intracellularly, but also at the plasma membrane; in addition specific membrane ERs, classified as G protein-coupled estrogen receptor (GPER or GPR30), the Gq-membrane ER (the Gq-mER) and the ER-X have been characterized (6). The GPER and Gq-mER have been detected in astrocytes (26, 924), where they were linked to generation of cytoplasmic Ca²⁺ signals (924) or modulation of expression of EAAT2 glutamate transporters (836). In cultured astrocytes activation of membrane α-ER receptors by estradiol and related compounds was shown to either inhibit glutamate uptake (1545) or stimulate it by increasing EAAT1/2 expression (836). Astrocytes also express nuclear progesterone receptors (PR), membrane progesterone receptors of the PR_A type, and progesterone receptor membrane component-1 (Pgrmc1) (930, 1866) as well as androgen receptors, which were found in primary cultured astroglia (806) and in minor populations of astrocytes in situ (446).

B. Secondary Transporters of Solute Carrier Family

1. Glutamate transporters

a) Na⁺-dependent glutamate transporters.

Astroglial glutamate transporters (known as excitatory amino acid transporters or EAATs) are members of SLC1 family of high-affinity glutamate and neutral amino acid transporters (see TABLE 4). Astrocytes specifically express EAAT1/SLC1A6 and EAAT2/SLC1A2, rodent versions of which (respectively) are often referred to as GLAST1 (glutamate-aspartate transporter 1; Ref. 1678) and GLT-1 (glutamate transporter 1; Ref. 1392). The EAAT2 transporter was the first to be identified in the rat brain (381); shortly after the EAAT1 was discovered (1727), and characterization of human variants followed (68). Both EAAT1 and EAAT2 are assembled as homotrimers, and several functional splice variants have been described (for a comprehensive account of the molecular structure and structure-function relations of glutamate transporters, see Ref. 1792). Astroglial glutamate transporters show differential expression in various brain regions: the EAAT1 is predominant in cerebellum (966), in the retina (1442), and in circumventricular organs (148); whereas EAAT2 prevails in all other parts of the brain. Expression of both transporters is rather high: the EAAT1 and EAAT2 account for 0.3 and 1.3% of total tissue protein in hippocampus and 1.8 and 0.3% for all tissue protein in cerebellum. The average density of EAAT1 is ~4,700/μm² in Bergmann glia and ~2,300/μm² in the CA1 region, whereas the density of EAAT2 is ~8,500/μm² in hippocampus and ~740/μm² in the cerebellum (966). The actual densities of both transporters are certainly much higher in perisynaptic membranes. The EM immunogold experiments clearly demonstrated that EAAT1 as well as EAAT2 are preferentially concentrated in astroglial membranes facing neuropil and their density is lower in the membrane portions facing neuronal somata or other astrocytes (313). Of note, ~10% of total EAAT2 is expressed in neurons (1956).

Table 4.

Astroglial SLC transporters

Transporter Type	SLC Classification	Stoichiometry	Properties and Function	Reference Nos.
Excitatory amino-acid transporters 1 and 2: EAAT1, EAAT2; in rodents known as GLAST and GLT-1	SLC1A3, SLC1A2	3Na+: 1H+: 1Glu (In): 1K+ (Out). Electrogenic	EAAT1/2 provide for ~80% of glutamate uptake in the CNS. Erev >10 mV; do not reverse in physiological conditions. Operation of EAATs results in substantial increase of [Na+]i.	380, 966, 1290, 1494, 1792, 1939
Sxc− cystine/glutamate antiporter	SCL7A11	1 Glu (Out): 1 Cys (In). Electroneutral	Exchanges glutamate for cystine; the latter is a main precursor for glutathione. Regulates extracellular glutamate in extrasynaptic regions. Electroneutral	23, 326, 844, 1162
Na+-coupled neutral amino acid transporters: SNAT 3, SNAT 5	SLC38A3, SLC38A5	1Na+ (In): 1Gln: 1 H+ (Out). Electroneutral	Exports glutamine from astrocytes.	680, 1552
GABA transporter: GAT-1/GAT-3	SLC6A12	2Na+:1Cl−: 1GABA (In), (?) 3Na+:1Cl−: 1GABA (In). Electrogenic	Normally removes extracellular GABA. Erev is close to resting Vm, can reverse in physiological conditions.	121, 356, 1044, 1121
Glycine transporter: GlyT1	SLC6A9	2Na+: 1Cl−: 1Gly (In). Electrogenic	Removes extracellular glycine. Erev is close to resting Vm, can reverse in physiological conditions.	491, 1932
Cationic amino acid transporter: Acs-1	SLC7A10	Electroneutral	Na+ -independent, high-affinity transport of small neutral d- and l-amino acids. In physiological conditions may transport glycine, l- and d-serine and l-cysteine.	470
Sodium-calcium exchanger: NCX1, NCX2, NCX3	SLC8A1, SLC8A2, SLC8A3	3Na+ (In): 1Ca2+ (Out). Electrogenic	Electrogenic exchanger; Erev close to Vm, operates in both forward and reverse modes.	195, 588, 878, 1303, 1461, 1725
Sodium-proton exchanger: NHE1	SLC9A1	1Na+ (In): 1H+ (Out). Electroneutral	Removes H+ from astrocytes; Erev >60 mV, hence cannot reverse in physiological conditions.	321, 402
Sodium-bicarbonate transporter: NBC	SLC4A4	1Na+: 2/3 HCO3− (In). Electrogenic	Erev close to Vm, operates in both forward and reverse modes.	244, 321, 402, 603, 1743
Sodium-potassium-chloride co-transporter: NKCC1	SLC12A2	1Na+: 1K+: 2Cl− (In). Electroneutral	Erev is determined by [Cl−]i, in physiological context; transport is always inward. May participate in clearing large K+ loads.	789, 818, 942, 1039
Mitochondrial sodium-lithium-calcium exchanger: NCLX	SLC8B1	3Na+ (In): 1Ca2+ (Out)	Main pathway for Ca2+ extrusion from mitochondria. In cultured astrocytes interacts with store-operated Ca2+ entry.	1327
Na+-dependent ascorbic acid transporter: SVCT2	SLC23A2	2Na+: 1Asc− (In). Electrogenic	Accumulates ascorbic acid into astrocytes; part of antioxidant defense.	149, 553, 896
Concentrative nucleoside transporter: CNT2, CNT3	SLC28A2, SLC28/A3	1 Na+: 1 nucleoside (In). Electrogenic	Accumulates adenosine into astrocytes. Part of adenosine homeostatic cycle.	975, 1363
Equilibrative nucleoside transporters: ENT-1, ENT-2, ENT-3, ENT-4	SLC29A1, SLC29A2, SLC29A3, SLC29A4	Electroneutral	Transports adenosine across plasmalemma according to its concentration gradient.	205
Na+-dependent dopamine transporter: DAT1	SLC6A3	1/2 Na+:1 DA: 1Cl− (In)	Uptake of dopamine; data on astroglial functional expression are controversial.	834, 886, 1722
Neutral amino acid transporter subtype ASCT2	SLC1A5	1Na+: 1d-Ser (In)	Transmembrane transport of d-serine.	1070
Norepinephrine transporter: NET/SLC6A2	SLC6A2	1NA: 2Na+ and 1 Cl−(In). Electrogenic	Uptake of norepinephrine and dopamine; has higher activity for dopamine.	765, 1577, 1722
Vesicular glutamate transporters: VGLUT1, VGLUT2, VGLUT3	SLC17A6, SLC17A7, SLC17A8	Unknown; is H+ and Cl− dependent	Glutamate accumulation in secretory vesicles.	153, 169, 368, 529, 902, 1144, 1279
Vesicular nucleotide transporter: VNUT	SLC17A9	Unknown	ATP accumulation in secretory vesicles.	1550
Vesicular polyamine transporter: VPAT	SLC18B1	Unknown	Spermine and spermidine accumulation in secretory vesicles.	693
Glucose transporters: GLUT1, GLUT2	SLC2A1, SLC2A2	Electroneutral	GLUT1 is the main astroglial glucose transporter.	22, 967, 1151
Sodium-dependent glucose transporter		1Glucose: 2 Na+ (In)	Detected only in astrocytes in vitro.	1799
Monocarboxylate transporters: MCT1, MCT4	SLC16A1, SLC16A3	Electroneutral	Lactate-exporting transporters.	151, 1422
Monocarboxylate transporter: MCT2	SLC16A7		Lactate-importing transporter. Was identified in astroglial endfeet in the brain stem and in the corpus callosum.	1356, 1422
Zink transporter: ZnT	SLC30		Main Zn2+ transporter.	1590

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Astroglial glutamate transporters utilize the transmembrane Na⁺ gradient as the driving force. Translocation of a single glutamate molecule is coupled with cotransport of 3 Na⁺ and 1 H⁺ and with countertransport of 1 K⁺ (FIGURE 18; Refs. 1290, 1939). With the use of this stoichiometry, both the equilibrium potential for the transporter and glutamate concentrating capacity could be calculated using modified Nernst equation:

EEAAT=RT/ F[3(Na)+1(H)−1(K)−1(Glu)]×ln{3ln([Na+]o / [Na+]i)+ln([H+]o / [H+]i)+ln([Glu−]o / [Glu−]i)−ln([K+]o / [K+]i)}

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FIGURE 18.

Stoichiometry of astroglial solute carrier (SLC) neurotransmitter transporters. EAAT1,2 , excitatory amino acid transporters 1 and 2; Sxc, cystine-glutamate exchanger; GAT1,3, GABA transporters 1 and 3; GlyT1, glycine transporter; ENT, equilibrative nucleotide transporters; CNT, concentrative nucleotide transporters; NET, norepinephrine transporter.

The E_EAAT is defined by transmembrane ion gradients and by cytosolic and extracellular glutamate concentrations (877, 1939). The extracellular concentration of glutamate in resting conditions is around 25 nM (677), the cytosolic concentration of glutamate in neurons is usually assumed to be in a range of 1−10 mM. In astrocytes, however, glutamine synthetase catalyzed conversion of glutamate into glutamine ascertains much lower glutamate levels determined at around 0.3 mM (227). Hence, at the resting state, the E_EAAT lies at a positive membrane potential around ~9 mV, and reversal of the transporter is possible only in conditions of substantial increase in extracellular K⁺ and intracellular Na⁺. Such a reversal can be achieved in experimental conditions (1713), which however reflect a severe pathology associated with a loss of cellular ion homeostasis. In physiological conditions, the stoichiometry of the transporter is capable of supporting ~10⁶ transmembrane glutamate gradient and can operate as an uptake system even when transmembrane ion gradients are seriously perturbed. Transmembrane Na⁺ gradient contributes the most to setting E_EAAT: 10-fold changes in [Na⁺]_o shift the equilibrium potential by 100 mV, whereas 10-fold changes in [Glu⁻]_o, [H⁺]_o, and [K⁺]_o shift E_EAAT by 31, 25, and −31 mV, respectively (1939). Nonetheless, in the physiological context, the major role belongs to glutamate, the concentration of which in the synaptic cleft may increase by 5−6 orders in magnitude during synaptic activity. Increase in extracellular glutamate shifts E_EAAT toward positive values; at 1 mM of glutamate in the cleft, the transporter reverses at 145 mV. This is a strong safety factor that ensures sustained glutamate uptake regardless of ionic perturbations. Transporter stoichiometry also confers electrogeneity to the EAATs, which generate transmembrane current mainly carried by Na⁺ ions (877, 1792). This Na⁺ influx may elevate [Na⁺]_i by 10−20 mM (1777), thus contributing to cytoplasmic Na⁺ signals (see sect. XB).

Operation of astroglial glutamate transporters is also associated with thermodynamically uncoupled Cl⁻ flux through an aqueous pore (similar to a classical ion channel) that is an intrinsic part of the transporter molecule (1541, 1791). The Cl⁻ current reverses at E_Cl, and Cl⁻ flux is relatively minor for astroglial transporters, in contrast to EAAT4/5 which have a substantial Cl⁻ permeability. Nonetheless, activation of glutamate uptake was described as the major pathway for Cl⁻ efflux in cerebellar Bergmann glia (1778). The Cl⁻ flux pathway also allows movement of water that is accompanying operation of the transporter to compensate for redistribution of osmolytes (1790). It has been estimated that water flux through EAATs attains ~10% of that through aquaporins (1034, 1036).

Kinetics of the glutamate translocation by EAATs is relatively slow; both EAAT1 and EAAT2 transport ~30 molecules of glutamate per second (1286, 1956). The glutamate binding to the transporters (K_m ~20 μM) is however much faster, and hence glutamate transporters concentrated at the perisynaptic processes act as almost instant buffers for glutamate. The higher is the density of transporters, the higher is their buffering capacity (1792). The EAAT2 in cultured hippocampal astrocytes has a remarkable lateral mobility regulated by glutamate, possibly allowing a continuous exchange of glutamate-bound and unbound transporters to maintain high buffer capacity of perisynaptic zones (1177).

b) sxc⁻ cystine/glutamate antiporter.

The cystine/glutamate antiporter, in contrast to Na⁺-dependent glutamate transporters, exports glutamate from the cell in exchange for cystine (FIGURE 18). This exchanger, known as Sxc⁻, is a heteromeric amino acid transporter composed of xCT/SCL7A11 and 4F2hc/SLC3A2 proteins (243). In the CNS, Sxc⁻ is mainly expressed in astroglia (23, 326, 844), although it was also detected in some immature neurons, in microglia, in endothelial cells, in ependymal cells, in the choroid plexus, and in the leptomeninges (232). Expression of Sxc⁻ in cultured astrocytes is substantially (up to 7 times) upregulated by dibutyryl-cAMP (581, 1587) and by depletion of intracellular glutathione (1587). The Sxc⁻ antiporter provides cystine required for production of glutathione and contributes to regulation of extrasynaptic level of glutamate (see sect. XIID).

9. Sodium-proton exchanger, NHE

The Na⁺-H⁺ exchanger expressed in astrocytes is represented by NHE1/SLC9A1 transporter; this is the main system expelling H⁺ from astroglia (321, 402). The electroneutral stoichiometry [1Na⁺ in exchange for 1H⁺ (1278)] is reflected by a positive reversal potential, which ascertains that NHE1 operates as H⁺ extruder across the whole range of physiological membrane potentials. An important task for NHE1 (FIGURE 19) is to export protons generated by metabolism and acquired by astrocytes in the process of glutamate uptake (each glutamate brings a single H⁺ ion) and Ca²⁺ extrusion (PMCA exchanges 1 or 2 H⁺ for each Ca²⁺ ion expelled). Activity of astroglial NHE1 is positively modulated by phosphorylation mediated by several protein kinases associated with metabotropic signaling cascades (1454). Acidification of the cytosol by metabolic inhibition triggers substantial influx of Na⁺, which supports extrusion of protons; this in turn triggers Na⁺ elevation, Ca²⁺ entry through the reversed Na⁺/Ca²⁺ exchanger, aberrant signaling, abnormal release of glutamate, and cell death (209, 301, 872).

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FIGURE 19.

Astrocytes and pH homeostasis. Astrocytes regulate extracellular pH by supplying H⁺ through operation of NHE, EAAT1/2, MCT, and possibly V-H⁺ pump, by removing H⁺ by PMCA and by supplying extracellular space with HCO₃⁻ through NBC. EAAT1,2, excitatory amino acid transporters 1 and 2; NHE, Na⁺-H⁺ exchanger, PMCA, plasmalemmal Ca²⁺-ATPase; VH⁺ pump, vacuolar H⁺ pump localized in plasmalemma; MCT, monocarboxylate transporter; NBC, Na⁺-bicarbonate exchanger.

A. Calcium Signaling in Astroglia

The concept of glial Ca²⁺ excitability was instigated by a series of observations on primary cultures which demonstrated that chemical or mechanical stimulation of astrocytes almost invariably triggers changes in [Ca²⁺]_i either in the form of [Ca²⁺]_i transients or [Ca²⁺]_i oscillations (385, 479, 842, 864, 1093). Discovery of [Ca²⁺]_i waves traveling over astroglial syncytium (306, 360, 361, 382) following focal stimuli bestowed long-range communication capabilities on Ca²⁺ signals. Studying astroglial [Ca²⁺]_i become popular among gliobiologists with a host of papers published; for comprehensive list of references, we direct the reader to relevant reviews covering 25 years of glial calcium signaling (15, 115, 408, 512, 1518, 1610, 1808, 1814, 1817, 1831).

Calcium ions are arguably the most widespread and ubiquitous signaling molecules operating in all types of living cells (160, 284, 666, 1378, 1394). Calcium homeostasis and calcium signaling in astroglia (simialrly to other eukariotic cells) are maintained by coordinated activity of Ca²⁺ fluxes created by plasmalemmal and organellar channels, pumps, and transporters and Ca²⁺ binding to intracellular proteins which serve as Ca²⁺ buffers and Ca²⁺ sensors (FIGURE 20; for extended account on Ca²⁺ signaling, see Refs. 161, 260, 285, 929, 1377, 1379, 1661).

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FIGURE 20.

Molecular pathways of astroglial Ca²⁺ signaling. Cytoplasmic calcium signaling is driven by the concentration gradients for Ca²⁺ ions between extracellular space and the cytosol and between the lumen of intracellular organelles (mainly endoplasmic reticulum) and the cytosol. Physiological stimulation opens plasmalemmal or intracellular Ca²⁺ channels, thus creating Ca²⁺ fluxes which translate into [Ca²⁺]_i dynamical changes in intracellular compartments. In astroglia, Ca²⁺ signals originate from both InsP₃-induced Ca²⁺ release from the ER calcium store (which can be also amplified by Ca²⁺ release through ryanodine receptors) and Ca²⁺ entry via plasmalemmal Ca²⁺ channels (see sect. VIIC for detailed description), ionotropic receptors (see sect. VIII), or reversed Na⁺-Ca²⁺ exchanger. In the cytosol, Ca²⁺ interacts with Ca²⁺-bindng proteins, which are represneted by Ca²⁺ buffers and Ca²⁺ sensors. The latter are Ca²⁺-regulated enzymes, which initiate various cellular reactions. Cytosolic Ca²⁺ signals are terminated by Ca²⁺ extrusion into extracellular space by Ca²⁺ pumps and Na⁺-Ca²⁺ exchanger and Ca²⁺ uptake into the ER (by SERCA pumps) and mitochondria (by mitochondrial uniporter). Mitochondrial Ca²⁺ entry acts as the main link between cell activity and energy production by regulating mitochondrial electron transport and ATP synthesis. CBP, Ca²⁺ binding proteins; ER, endoplasmic reticulum; GPCR, G protein-coupled metabotropic receptors; InsP₃R2, InsP₃ receptor type 2; NCX, Na⁺-Ca²⁺ exchanger; ORAI, Ca²⁺ release-activated Ca²⁺ channels; PMCA, plasmalemmal Ca²⁺-ATPase; RyR, ryanodine receptors; SERCA, sarco(endo)plasmic reticulum Ca²⁺-ATPase; SOCE, store-operated Ca²⁺ entry; TRP, transient receptor potential channels; VGCCs, voltage-gated calcium channels.

Recent advances in Ca²⁺ imaging revealed a highly variable spatiotemporal organization of Ca²⁺ signals across complex morphological landscape of astroglial cell. Introduction of genetically encoded Ca²⁺ indicators and new microscopic techniques demonstrated that astroglial compartments have specific mechanisms controlling [Ca²⁺]_i and hence are capable of generating distinct Ca²⁺ signals. In physiological (in situ or in vivo) settings, Ca²⁺ signals in astroglial processes often develop independently from somatic Ca²⁺ signals and demonstrate singular kinetic profiles and underlying mechanisms. In the distal processes, Ca²⁺ signals occur as localized microdomains either spontaneously without obvious link to neuronal activity (821, 1208, 1609) or in response to synaptic stimulation (423, 609, 821, 1225, 1660). Local Ca²⁺ signals in astroglial processes may be linked to specific morphological structures such as “appendages” in Bergmann glial cells (FIGURE 21; Ref. 609) or perisynaptic leaflets (1610). As a rule, local Ca²⁺ signals in astroglial processes developed independently from the somatic [Ca²⁺]_i transients and had distinct kinetics. Usually [Ca²⁺]_i transients were larger and longer in the soma; in the processes both frequent and relatively fast [Ca²⁺]_i fluctuations (423, 1308, 1831) as well as very slow (~40 s) events, defined as “Ca²⁺ twinkles” (821) were reported.

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FIGURE 21.

Ca²⁺ microdomains in Bergmann glia. Stimulation of parallel fibers triggers local calcium signals in Bergmann glial cells. A: experimental protocol. Parallel fibers (PF) were stimulated via a pipette connected to a stimulator (STIM) while calcium-dependent fluorescence responses were recorded in a Bergmann glial cell (BG). PCL, Purkinje cell layer. B: confocal fluorescence intensity image of a patch-clamped Bergmann glial cell dialyzed with the calcium indicator Oregon green 488 BAPTA-1 (right). Two processes were distinguished (indicated as 1, 2). C: calcium signals in response to PF stimulation were measured independently for each process (left). Time of PF stimulation is marked by arrows. D: the responding process (1) was further subdivided into regions of interest, in which calcium signals were measured separately (right). Time of PF stimulation is marked by an arrow and a dotted line. (From Matyiash, Grosche, Reichenbach, Verkhratsky, and Kettenmann, unpublished data.)

Spatial heterogeneity of astroglial [Ca²⁺]_i dynamics reflects different molecular mechanisms of Ca²⁺ signal generation. Genetic deletion of InsP₃R type 2 (InsP₃R2^−/−) led to an almost complete disappearance of spontaneous and evoked somatic [Ca²⁺]_i responses, (14, 820, 1381, 1382), indicative of the predominant role for metabotropic receptors/PLC/InsP₃ signaling cascade. In hippocampal astroglial processes, the fastest (half time of recovery ~700 ms) and most localized (microdomain size ~4 μm) [Ca²⁺]_i transients coexisted with longer (recovery half time >3 s) and larger (~12 μm in size) [Ca²⁺]_i microdomains. Both types of localized Ca²⁺ dynamics in astroglial processes were associated with InsP₃R-mediated Ca²⁺ release; they were inhibited by intracellular injection of heparin and were almost completely eliminated in InsP₃R2^−/− mice (423). This observation was not universally confirmed, and in many experiments local evoked and spontaneous Ca²⁺ signals in distal astroglial processes were only partially reduced in the type 2 InsP₃R^−/− mice (663, 821, 1660). In hippocampal astrocytes studied in situ, spontaneous Ca²⁺ tranients confined to fine processes persisted in InsP₃R2^−/− KO mice and were entirely dependent on plasmalemmal Ca²⁺ influx (1516). Moreover, in type 2 InsP₃R^−/− animals, astroglial [Ca²⁺]_i transients evoked by metabotropic agonist endothelin were almost completely inhibited in the soma, while they were left unchanged in the processes (1660). Anatomical segregation of Ca²⁺ signaling suggests alternative routes for Ca²⁺ mobilization in different parts of the astrocyte. These alternative molecular cascades may involve Ca²⁺ entry through ionotropic receptors and plasmalemmal channels, or Ca²⁺ influx mediated by reverse mode of NCX (115, 1610, 1817, 1831). Reversal of NCX may readily occur in response to rise in [Na⁺]_i accompanying operation of major neurotransmitter transporters; such NCX-mediated [Ca²⁺]_i events have been, for example, observed in astrocytes in slices of the olfactory bulb (439). Heterogeneity of Ca²⁺ mobilization may also arise from alternative intracellular signaling associated, for example, with cAMP (663).

In depth analysis of resting [Ca²⁺]_i in hippocampal astrocytes in situ and cortical astrocytes in vivo with fluorescence-lifetime imaging microscopy revealed a [Ca²⁺]_i gradient between peripheral processes and soma (1950). Basal [Ca²⁺]_i levels in the distal processes of 21-day-old mice were ~125 nM, whereas [Ca²⁺]_i in the soma was ~100 nM. These gradients were even higher in cells from younger animals ([Ca²⁺]_i in processes and soma were ~200 and 130 nM, respectively; Ref. 1950). Incidentally, these experiments also pointed out that astroglial resting [Ca²⁺]_i was ~1.5−2 times higher compared with their neighboring neurons (1950). The gradient of basal [Ca²⁺]_i aimed from the distal processes to the soma highlights regional differences in [Ca²⁺]_i regulation and possibly further corroborates the importance of transmembrane Ca²⁺ entry in peripheral structures of astrocytes. Compartmentalization of Ca²⁺ signaling in fine astroglial processes may involve specific positioning of mitochondria which tend to immobilize themselves in sites of focal [Ca²⁺]_i spikes (423). Similarly, functional compartmentalization can occur around plasmalemma-endoplasmic reticulum junctions characterized in astrocytes in vitro; it appeared that key plasmalemmal (NCX1, NKA) and endoplasmic reticulum (SERCA2 and InsP₃Rs) molecules coimmunoprecipitated, indicating the link between plasmalemma and endomembrane (968). Finally, localized [Ca²⁺]_i dynamics may reflect specific distribution of various types of receptors or channels at different cellular locations (1831). The physiological relevance for heterogeneous basal [Ca²⁺]_i in astroglia remains to be explored; local differences of [Ca²⁺]_i homeostasis may reflect functional specialization of subcellular compartments.

Further probing of local Ca²⁺ signaling in astrocytes using membrane-tethered Ca²⁺ indicator revealed even smaller submembrane [Ca²⁺]_i transients (“spotty Ca²⁺ signals”) associated with Ca²⁺ influx through TRPA1 channels (1608). Importantly, Ca²⁺ mobilizing mechanisms differ between astrocytes from different brain regions: Ca²⁺ microdomains in Bergmann glia and in the main processes of hippocampal astrocytes were mediated solely by InsP₃ receptors (423, 879). The near-membrane [Ca²⁺]_i transients were mediated by TRPA1 in astrocytes in CA1 hippocampal area, whereas TRPA1 contribution was less prominent in stratum radiatum and absent in CA3 region (1608). In neocortical astrocytes, [Ca²⁺]_i transients may be amplified by RyR-mediated [Ca²⁺]_i-induced Ca²⁺ release (1311), which however seems to be absent in hippocampal astroglia (118). Sporadic evidence even suggests the role for voltage-dependent Ca²⁺ channels in generating astroglial [Ca²⁺]_i responses (972, 1332). In the in vivo settings, sensory stimulation triggered global [Ca²⁺]_i transients in astrocytes in somatosensory cortex. These Ca²⁺ signals were initiated in the processes and then spread towards the soma with a speed of 15 μm/s; genetic deletion of type 2 InsP₃R eliminated this type of signaling (821). In the olfactory bulb, however, physiological stimulation of the olfactory sensory neuron terminals triggered [Ca²⁺]_i transients only in the astroglial processes, and left the soma idle (1288). In cortical astrocytes from adult (7−20 wk) mice examined in culture, in slices, and in vivo, spontaneous Ca²⁺ microdomains in fine processes were linked to Ca²⁺ release from mitochondria mediated by brief openings of mitochondrial permeability transition pore (13).

Further adding to the complexity, astroglial [Ca²⁺]_i dynamics and Ca²⁺ signaling mechanism undergo developmental regulation (1702, 1950). In the brain of adult nonanesthetized animals, astrocytes in the CNS are under control of almost ubiquitous noradrenergic innervation; sensory stimulation, arousal, and locomotion are associated with release of norepinephrine from locus coeruleus projections which trigger large global [Ca²⁺]_i signals in astrocytes in multiple brain regions (FIGURE 22, Refs. 431, 1341). These global Ca²⁺ signals were mediated by α₁-adrenoreceptors, and inhibition of α₁-adrenoceptrs almost completely blocked all spontaneous Ca²⁺ activity in awake behaving mice (431). A similar role for monoaminergic input has been recently described in Drosophila glia, where Ca²⁺ signals in vivo were driven by norepinephrine analogs tyramine or octopamine (1033). Similar global astroglial signaling is observed in attention and vigilance state, when widespread astrocytic responses are evoked by acetylcholine release from projection of the nucleus basalis of Meynert and are mediated through mAChRs-InsP₃ signaling cascade (316, 1720). Finally, global astroglial [Ca²⁺]_i signaling that occurs throughout the entire cortex was observed in response to transcranial direct current stimulation; this type of Ca²⁺ signaling was again mediated solely through α₁-adrenoceptors (1141). How all these globalized Ca²⁺ signals interact with Ca²⁺ microdomains remains hitherto unknown.

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FIGURE 22.

Startle stimulation triggers widespread astroglial Ca²⁺ signaling. A: astrocytic Ca²⁺ transients measured by rhod 2-AM were detected in response to 30 s of 3-Hz whisker stimulation. Representative local field potential LFP and electromyogram (EMG) recordings are shown. B: representative images of rhod 2-AM fluorescence increases during whisker stimulation in a Glt-1-EGFP animal. Scale bar = 100 μM. C: selected cells from B. Rhod 2 ΔF/F₀ was normalized to Glt-1-EGFP fluorescence. Electrocorticogram (ECoG) traces corresponding to rhod 2 ΔF/F₀ are shown below. D: air pulses were directed at the face or tail of the animal to elicit a startle response. Representative ECoG and EMG traces are shown with no apparent evoked ECoG response and strong EMG activity, indicative of a startle response. E and F: representative images and corresponding rhod 2 ΔF/F₀ traces show stable and repeatable astrocytic [Ca²⁺]_i transients after startle stimulations. Bottom: representative ECoG traces from startle stimulation are shown. G: bar graph showing the response rate of cortical astrocytes to whisker and startle stimulation, averaged across animals. *** P < 0.001, one-way ANOVA, Bonferroni correction [n = 11 animals (parietal cortex startle and whisker stimulation), 4 animals (frontal cortex startle)]. H: scatter diagram with superimposed mean and SE for the delay from the onset of whisker/startle stimulation to the beginning of astrocytic rhod 2 ΔF/F₀ responses. Average delay from each successful trial is shown. *** P < 0.001, one-way ANOVA, Bonferroni correction [n = 28 trials in 14 animals (whisker stimulation), 38 trials in 13 animals (parietal cortex startle), and 9 trials in 4 animals (frontal cortex startle)]. Data are means ± SE. [From Ding et al. (431).]

Another fundamental aspect of astroglial [Ca²⁺]_i signaling is linked to the propagating intercellular Ca²⁺ waves that have been widely considered as the substrate for long-range information transfer in astrocytic syncytia (388, 1553, 1741). Propagating Ca²⁺ waves have been characterized in detail in astrocytes in vitro (360, 361, 619, 1635, 1935) and in situ (649, 1560, 1622, 1873, 1883), and the mechanisms of propagation have been discerned. The spread of Ca²⁺ wave was found to be mediated either by intercellular diffusion of InsP₃ (or possibly also cyclic ADP-ribose, cADPR) through gap junctions (20, 712, 974) or through regenerative paracrine ATP-mediated signaling (40, 61, 366, 659) or through the combination of both (1553). Operation of these mechanisms seems to be anatomically segregated: for example, ATP-mediated propagation dominates in corpus callosum, whereas in the neocortex Ca²⁺ waves depend entirely on gap junctions (620).

Appearance, physiological role, and mechanisms of propagating astroglial Ca²⁺ waves in vivo are yet to be investigated and apprehended. Propagating Ca²⁺ waves were detected in cerebellum in syncytia of velate astrocytes; they were sensitive to purinoceptor antagonists and spread for ~50 μm at a speed of 4−11 μm/s (722). Spontaneous Ca²⁺ waves were also observed in Bergmann glia (1081). In contrast, Ca²⁺ waves have not been detected in the in vivo cortex in both awake and anesthetized animals. In cortical astrocytes Ca²⁺ signals seem to be restricted to individual cells or to a limited number of adjacent cells, without much spreading to neighboring regions. For example, stimulation of sensory inputs by mechanical displacement of whiskers in mice induced [Ca²⁺]_i transients in astrocytes in the barrel cortex; these Ca²⁺ signals were confined to individual cells and did not produce propagating Ca²⁺ waves (1858). Sensory stimulation of astrocytes in the visual cortex of ferrets evoked [Ca²⁺]_i transients in single astrocytes but did not trigger propagating Ca²⁺ waves (1581). Synchronized somatic [Ca²⁺]_i transients were similarly observed in cortical astrocytes imaged in vivo in response to focal application of α/β-adrenoceptors agonists (123). All in all, the extent of propagation and the velocity of astroglial Ca²⁺ waves seem to be substantially limited in vivo compared with experiments in the dish.

Propagating fast (at 60 μm/s) Ca²⁺ waves were found to spread through thousands of astrocytes in mouse hippocampus; these waves were called somewhat romantically “glissandi” from a musical term denoting a rapid series of ascending or descending notes on the musical scale (921). These Ca²⁺ waves were sensitive to inhibitors of gap junctions and purinoceptors, and somewhat unexpectedly, they were blocked by 2 μM TTX (921). These observations, however, were performed on mice heavily anaesthetised with urethane, while cortices were removed by aspiration to gain the access to hippocampi; consequently these unusually fast Ca²⁺ waves might reflect the traumatic or toxic injury of the brain tissue.

To conclude, hierarchy of astroglial Ca²⁺ signals ranges from local microdomains to propagating Ca²⁺ waves and synchronized global Ca²⁺ signals (FIGURE 23). Characterization of physiological targets and coding principles of astroglial Ca²⁺ signaling remain the ultimate and yet unanswered question. Fluctuations in [Ca²⁺]_i obviously impact on gene expression and on astroglial energy metabolism (through accumulation in mitochondria); these however are generic targets of Ca²⁺ signals in all eukaryotic life forms. Which astroglia-specific processes are governed by [Ca²⁺]_i? How do intracellular proteins decipher incoming Ca²⁺ signals? The Ca²⁺-regulated secretion is the most popular target, yet [Ca²⁺]_i may regulate many other astroglial mechanisms from expression of membrane transporters to regulation of K⁺ uptake (1853). The remarkable heterogeneity in parameters and mechanisms of astroglial Ca²⁺ signaling most likely reflects an extensive adaptive potential of astrocytes, which depending on the context may employ distinct Ca²⁺ signaling toolkits or rapidly remodel the existing ones to meet the homeostatic requirements of their immediate environment.

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FIGURE 23.

Hierarchy of astroglial Ca²⁺ signaling.

B. Sodium Signaling in Astrocytes

Stimulation of astrocytes, either mechanical or chemical or synaptic, triggers [Na⁺]_i transients with distinct spatiotemporal parameters; these transients are the substrate of the cytosolic Na⁺ signaling (881, 1499). The [Na⁺]_i transients in response to physiological stimulations have been observed in astrocytes both in vitro (851, 1496, 1498) and in situ (877, 878, 940, 1495). Regulation of [Na⁺]_i has its own idiosyncrasies: 1) it relies solely on plasmalemmal Na⁺ transport because of absence of intracellular storage compartments and 2) cells do not have (or we are not yet aware) specific Na⁺ buffers, which may spatially organize cytosolic Na⁺ dynamics. The signaling function of Na⁺ is mediated through the transmembrane concentration gradients which drive multiple plasmalemmal transporters and through “Na⁺ sensors” represented by Na⁺-sensitive enzymes. These are by far less characterized compared with Ca²⁺ sensors, although some of them have been identified. Several metabotropic receptors including class A G protein-coupled receptors (to which adenosine or dopamine receptors belong) have a conserved binding site for Na⁺, which negatively modulates their affinity to agonists (845). Changes in Na⁺ promote the dissociation of trimeric G proteins, thus affecting the activation or inhibition of Gβγ-gated ion channels (202, 1471). The Na⁺-dependent K⁺ channels, which mediate slow afterhyperpolarization, operate in neurons (170), whereas in astrocytes increases in [Na⁺]_i facilitate spermine-induced inhibition of inward rectifier K_ir4.1 channels (916).

Astroglial Na⁺ homeostasis and Na⁺ signals are governed solely by transmembrane Na⁺ fluxes (FIGURE 24); Na⁺ ions enter the cytosol through plasmalemmal channels and Na⁺-coupled SLC transporters and are extruded mainly by NKA and possibly by NCX operating in the reverse mode (881, 1499). Resting [Na⁺]_i in astrocytes is relatively high (15−20 mM, see sect. VIA); it defines driving force and sets the equilibrium for many Na⁺-dependent transporters critical for homeostatic activity of astroglia. Inhibition of NKA with ouabain or by decreasing extracellular K⁺ results in rapid increase in [Na⁺]_i (586, 762, 869, 1496, 1645, 1846), which indicates significant constitutive Na⁺ influx at rest. This influx may reflect background channel activity or Na⁺ entry mediated by plasmalemmal transporters. The NKCC1 seems to contribute to setting resting [Na⁺]_i in astrocytes in vitro, as its inhibition by the diuretic bumetanide decreases resting [Na⁺]_i by several millimolar (851, 1689). The role of NKCC1 in regulation of astroglial ionic fluxes in vivo remains, however, questionable (942). Resting Na⁺ influx may be also mediated by Na⁺/bicarbonate (NBC) transporter (851).

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FIGURE 24.

Na⁺ entry pathways. Astroglial Na⁺ signaling is generated by Na⁺ entry through plasmalemma; the Na⁺ influx occurs through Na⁺ channels, ionotropic receptors, and TRP channels. Na⁺ entry also accompanies operation of plasmalemmal SLC transporters and Na⁺-Ca²⁺ exchanger. Termination of Na⁺ signals is mainly mediated by Na⁺ extrusion via Na⁺-K⁺ pump. The TRPC channels are the molecular substrate of astroglial store-operated Ca²⁺ entry; as these channels pass both Na⁺ and Ca²⁺, they establish the link between metabotropic activation of Ca²⁺ release and Na⁺ signaling. AMPAR, AMPA receptors; EAAT1,2, excitatory amino acid transporters 1 and 2; ER, endoplasmic reticulum; GAT1,3, GABA transporters 1 and 3; GlyT1, glycine transporter; GPCR, G protein-coupled metabotropic receptors; NCX, Na⁺-Ca²⁺ exchanger; Na_v, voltage-gated Na⁺ channels; Na_x, Na channels gated by extracellular concentration of Na⁺; NKA, Na⁺-K⁺-ATPase; NMDAR, NMDA receptors; P2XR, P2X puriunoceptors; SOCE, store-operated Ca²⁺ entry; TRP, transient receptor potential channels.

Operation of glutamate transporters (which cotransport 3 Na⁺ with 1 glutamate) creates substantial Na⁺ entry, which may elevate [Na⁺] by 10−20 mM, both in response to endogenous glutamate or to synaptic stimulation (FIGURE 25). Glutamate transporter-associated [Na⁺]_i transients have been observed in cultured astrocytes (310), in situ in Bergmann glia (140, 877), in astrocytes from neocortex (938, 1776), and from calyx of Held (in these [Na⁺]_i increased by ~3.5 mM; Ref. 1780). Uptake of GABA produces smaller Na⁺ entry (reflecting the 2Na⁺: 1GABA stoichiometry of GAT) and may increase [Na⁺]_i by 4−6 mM (310, 1777). Sodium entry can also be mediated by ionotropic receptors (878, 940), by TRP channels (1461), or by NCX operating in the forward mode (878). Relative contribution of Na⁺ entry pathways differs between different astrocytes: in hippocampal astrocytes Na⁺ influx occurs solely through glutamate transporter, whereas in Bergmann glia the contributions of transporter and AMPA glutamate receptors are almost equal (940). In response to synaptic stimulation, [Na⁺]_i transients are directly proportional to the stimulation strength. At low stimulation intensity, [Na⁺]_i increased only in astroglial processes; increase in the intensity of stimulation resulted in larger [Na⁺]_i transients occupying the whole cell from processes to soma (940).

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FIGURE 25.

Astroglial Na⁺ signaling associated with glutamate uptake in cerebellar Bergmann glial cells. A: voltage dependence of glutamate-transporter current as compared with kainate-induced (AMPA-mediated) currents. The current traces are shown on the top (transporter current on the left and receptor-mediated current on the right), while current-voltage curves for glutamate- and kainite-induced currents are displayed at the bottom. For each cell membrane, currents at different voltages were normalized to the amplitude of current recorded at the holding potential of −70 mV. Note that glutamate-induced responses do not reverse at +20 mV, whereas kainate-induced currents show a reversal potential close to 0 mV. *P < 0.05 and **P < 0.01, significant difference between glutamate- and kainite-induced currents. B: simultaneous recordings of glutamate-induced inward current and [Na⁺]_i elevation in normal conditions in Na⁺-free (Na⁺ substituted by NMDG⁺) solution. C: stimulation of parallel fibers activates membrane currents and transient [Na⁺]_i elevation in Bergmann glial cell. The [Na⁺]_i transient is not affected by inhibitors of ionotropic glutamate receptors. [From Kirischuk et al. (877).]

Astroglial [Na⁺] transients evoked by either chemical or synaptic stimulation are quite long-lasting with recovery times in a range of tens of seconds (310, 878, 940, 1780), which argues against free Na⁺ diffusion in the cytosol. Mechanisms shaping kinetics of [Na⁺]_i transients are enigmatic; possibly long-lasting [Na⁺]_i responses may reflect prolonged periods of Na⁺ entry or Na⁺ binding/unbinding to plasmalemmal transporters (1494). Similarly unknown is the possibility for generation of [Na⁺]_i microdomains, especially in small subcellular compartments; these diffusion-limiting regions may be associated with perisynaptic processes or with astroglial endfeet (1243). Astrocytic Na⁺ signals can propagate through individual cells and through astroglial syncytia in the form of spreading [Na⁺]_i waves with a velocity ~60 μm/s; in hippocampal slices, these Na⁺ waves expand through all neighboring astrocytes (941). Astroglial Na⁺ waves propagate mainly through Cx30 or Cx43 gap junctions (941).

The existence of spatially restricted localized [Na⁺]_i microdomains is indirectly supported by specific colocalization of Na⁺-dependent transporters, pumps, and channels in astroglial processes. Electron microscopy of cortical and hippocampal astrocytes revealed concentration of NCX1–3 in distal processes enwrapping excitatory synapses (1120), which coincided with the localization of NMDA receptors (354). In cultured astrocytes, NCX was colocalized with NKA at specific domains of plasma membrane directly opposing outposts of the endoplasmic reticulum (194); local Na⁺ signaling may occur in these compartments (589). The NKA are also spatially linked to glutamate transporters as indicated by colocalization and coimmunoprecipitation of α2 NKA subunit and EAAT1/2 in cortical astrocytes (329, 1475, 1501). Local Na⁺ signals associated with glutamate transport in astroglial perivascular endfeet in hippocampal slices increase ATP consumption possibly reflecting local homeostatic adaptation (939).

Astroglial Na⁺ executes the signaling function through several mechanisms (FIGURE 26). Changes in transmembrane Na⁺ gradient control multiple transporters responsible for neurotransmitter homeostasis, and relatively small increases in [Na⁺]_i can reverse for instance GABA and glycine transport. Cytosolic Na⁺ also regulates glutamine-glutamate(GABA) shuttle through direct action on glutamine synthetase (139) and regulation of glutamine transporters (1752). These regulatory mechanisms are of great potential importance in perisynaptic processes, where local [Na⁺]_i transients may control local homeostatic responses. Fluctuations of [Na⁺]_i contribute to regulation of K⁺ buffering through affecting NKA transport; similarly, [Na⁺]_i is fundamentally important for pH homeostasis by regulating NBC and NHE. By controlling reversal potential of NCX, astroglial [Na⁺]_i transients may contribute to Ca²⁺ signaling, for example, by initiating local Ca²⁺ influx in distal processes. Changes in [Na⁺]_i are directly linked to astroglial metabolism, through controlling glycolysis and lactate production and possibly regulating ATP synthesis (309). Cytosolic Na⁺ signals can invade mitochondria (156); moreover, astroglial mitochondria can generate spontaneous Na⁺ spikes (77). This mitochondrial connection may further implicate Na⁺ into regulation of astroglial energy metabolism, which however remains hypothetical (1494).

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FIGURE 26.

Na⁺ signals regulate multiple molecules and multiple homeostatic functions (see text for further details). ASCT2, alanine-serine-cysteine transporter 2; ASIC, acid sensing ion channels; CNT2, concentrative nucleoside transporters; EAAT, excitatory amino acid transporters; ENaC, epithelial sodium channels; GAT, GABA transporters; GS, glutamine synthetase; GlyT1, glycine transporter; iGluRs, ionotropic glutamate receptors; Na_x, Na⁺ channels activated by extracellular Na⁺; NAAT, Na⁺-dependent ascorbic acid transporter; NBC, Na⁺/HCO₃⁻ (sodium-bicarbonate) cotransporter; NCX, Na⁺/Ca²⁺ exchanger; NCLX, mitochondrial Na⁺/Ca²⁺ exchanger; NHE, Na⁺/H⁺ exchanger; NKCC1, Na⁺/K⁺/Cl⁻ cotransporter; NET, norepinephrine transporter; MCT1, monocarboxylase transporter 1; P2XRs, ionotropic purinoceptors; SN1,2, sodium-coupled neutral amino acid transporters which underlie exit of glutamine; TRP, transient receptor potential channels; ROS, reactive oxygen species; VRAC, volume-regulated anion channels.

In summary, the sodium signaling system may be considered as the pathway for fast coordination of neuronal activity with “homeostatic” response of astroglia mediated through Na⁺-dependent transporters, concentrated in perisynaptic processes. Influx of Na⁺ into these perisynaptic processes, characterized by exceedingly high surface-to-volume ratio, may also trigger local depolarization, which will further regulate plasmalemmal transporters.

B. Exocytosis

Exocytosis utilizes specific membranous organelles carrying heterogeneous cargo and capable of fusing with plasmalemma, hence providing the release of their luminal content. Exocytosis is an evolutionary conserved (appearing probably in Archea) and ubiquitous property of most eukaryotic cells (1657). Exocytotic vesicular release (that underlies both constitutive and regulated secretion) is regulated by an extended family of SNARE (soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptor) proteins. Of these, R(arginine)-SNAREs (also known as vesicle-associated membrane proteins or VAMPs) are associated with the membranes of secretory vesicles, while Q(glutamine)-SNAREs are associated with plasmalemma (784, 839). In the process of excitation-secretion coupling, high [Ca²⁺]_i instigates formation of a ternary SNARE complex from R/Q-SNAREs, which mediates fusion of vesicles with plasma membrane (1694).

1. Astroglial secretory organelles

Astrocytes possess intracellular (synaptic-like microvesicles, dense-core vesicles, and lysosomes) and extracellular (ecto- and endosomes) secretory organelles (FIGURE 28). Intracellular vesicles are cellular organelles that may completely fuse with cellular membranes, whereas extracellular vesicles are membranous compartments released into the surrounding environment.

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FIGURE 28.

Astroglial secretory organelles.

a) synaptic-like microvesicles, or SLMVs.

Astroglial SLMVs have a diameter of 30−100 nm; they usually appear in pairs or in groups (up to 15) of vesicles, thus being much less numerous than neuronal synaptic vesicles, 100−1,000 of which are clustered in presynaptic terminals (153, 169, 804). The SLMVs were identified in processes and somata of astrocytes from various brain regions (including hippocampus, cortex, and cerebellum) both in culture and in situ (153, 169, 1145, 1280). The role for large (1−3 μm) SLMVs identified in astroglial processes in hippocampal slices (825) remains unclear. Astroglial SLMVs contain vesicular neurotransmitter transporters VGLUT1–3 and VNUT (see sect. IXB12). In astrocytes in culture, SLMVs were found to store glutamate or costore glutamate and d-serine (1071, 1160). In contrast, analysis of astrocytes in situ revealed distinct populations of glutamate and d-serine-containing vesicles (153). Also, intracellular mobility of astroglial vesicles has its peculiarities: for example, glutamate-containing vesicle movement accelerates with an increase in [Ca²⁺]_i (1671), whereas peptidergic vesicles and endolysosomes slow down (1402, 1404); such differences have never been seen in neurons.

b) dense-core vesicles, or DCVs.

Astroglial DCVs usually have a diameter of 100−600 nm, and their population is substantially smaller than SLMVs (279, 368, 753, 1407), even smaller DCVs (~50 nm) were also reported (1404). In astrocytes in culture, DCVs were found to carry secretory proteins secretogranin II (279, 1295, 1407) and secretogranin III (1296), chromogranins (753), ANP (902, 1295), neuropeptide Y (1407, 1425), and ATP (341, 1310). The chromogranin and secretogranin II-containing DCVs were also found in human astrocytes from tissues obtained during surgery (753).

c) secretory lysosomes.

Lysosomes are the essential intracellular organelle of eukaryotes generally responsible for degradation and recycling of macromolecules and autophagy (909), although lysosomes may also participate in secretion. There are several reports indicating the role of lysosomes in astroglial secretion, in particular in Ca²⁺-dependent release of ATP in astrocytes in culture (785, 979, 1947). Astrocytic secretory lysosomes have diameters of 300−500 nm; they can be tagged with various fluorescent conjugated dextrans (1793), with FM-dyes or with [2'/3′-O- (N'-methylanthraniloyl)adenosine-5′-O-triphosphate], or MANT-ATP (1947); they coexist (and can be coreleased) with other types of secretory vesicles in the same cell (993). Astroglial secretory lysosomes are in possession of VNUT, which mediate accumulation of ATP (1292), and they express lysosomal specific markers such as cathepsin D, lysosomal-associated membrane protein 1 (LAMP1), and ras-related protein Rab7 (1947). Lysosomal exocytosis is mediated by tetanus toxin-insensitive vesicle-associated membrane protein VAMP7 (304), and downregulation of VAMP7 suppresses ATP release (1804). Lysosomal fusion was also reported to be triggered by distinct slow and localized Ca²⁺ signals (1804). Similarly to other cells, secretory lysosomes in astrocytes are likely to play a role in membrane repair (43).

d) extracellular vesicles.

The extracellular vesicles represented by exosomes and ectosomes carry a wide spectrum of bioactive substances including cytokines, signaling proteins, mRNA, and microRNA. Exosomes are formed intracellularly from multivesicular bodies (1083), whereas ectosomes are produced through direct budding of the plasma membrane (1744). Astroglial exosomal secretion has been observed in response to oxidative and heat stress (1735) or in pathology, for example, in astrocytes surrounding amyloid plaques in a mouse model of familial Alzheimer's disease (1854). Some astroglia-released exosomes were also containing mitochondrial DNA (613). Despite high expectations of exosome-mediated intercellular signaling in the CNS (537), the astroglial component of it remains largely uncharacterized.

Shedding of ectosomes from astroglial membrane is triggered following activation of P2X₇ purinoceptors. It occurs because of rapid activation of acid sphingomyelinase that moves to the plasma membrane outer leaflet and modifies membrane structure/fluidity leading to vesicle blebbing and shedding (174, 175). Astroglial ectosomes are heterogeneous with diameters between 100 and 1,000 nm (174, 1411). These ectosomes were reported to carry fibroblast growth factor-2 and vascular endothelial growth factor (1411), IL-1β (174), nucleoside triphosphate diphosphohydrolases (302), matrix metalloproteinases (1551), and acid sphingomyelinase and high levels of phosphatidylserine on their membrane outer leaflet (174).

C. Diffusion Through Membrane Channels

Secretion by diffusion through plasmalemmal pores is driven by transmembrane concentration gradient, which for many biologically active molecules could be rather high. For example, cytosolic ATP concentration (~5 mM) exceeds ambient extracellular concentration (low nM) by ~10⁷ times, whereas glutamate ([Glu]_i ~0.3 mM, [Glu]_o ~25 nM) exceeds by 10⁴ times. Any conductive pore large enough to accommodate the secreting molecule can therefore act as a conduit, and nonvesicular release of various signaling molecules have been identified in different tissues [for instance in taste buds (871) or erythrocytes (1415)]. Connexin hemichannels (see sect. VIIH) were probably the first (711) and are certainly the most studied (572) diffusion secretion pathway in astrocytes. Activation of connexin hemichannels occurred in response to lowering extracellular Ca²⁺ (711) or metabolic inhibition (358), or treatment of cell cultures with proinflammatory cytokines (1457). Opening of these hemichannels was associated with the release of glutamate or ATP (143, 1681, 1919), and hence, the role of unpaired connexons in release of astroglial signaling molecules was considered (61, 365, 1553). In particular, connexon-mediated release of ATP has been recognized as a paracrine signal critical for the generation of intercellular Ca²⁺ waves in cultured astroglial cells (365, 1681). Pannexons also have been implicated in glutamate and ATP release from astroglia (572) and were shown to mediate release of d-serine (1306).

The repertoire of hemichannel-mediated secretion could be much wider. There are indications that numerous signaling molecules such as NAD⁺, adenosine, InsP₃, cyclic ADP-ribose, nicotinic acid adenine dinucleotide phosphate, lactate, and NO (249, 509, 596, 833, 1147, 1643) all can pass through Cx hemichannels in either direction (i.e., serving for secretion or uptake depending on concentration gradients). In addition, hemichannels could support uptake or release of several metabolites such as glucose, ascorbate, or glutathione (572, 1431). Both connexons and pannexons are also permeable to several synthetic molecules including Lucifer yellow, ethidium, calcein, propidium, 5 (6)-carboxyfluorescein and 2-(4-amidinophenyl)-1H-indole-6-carboxamide, and DAPI, which are often used for monitoring the activity of these channels (1525). Connexons are densely expressed at perivascular astroglial endfeet and may therefore contribute to astroglia-vasculature signaling. Release of ATP (with subsequent degradation to adenosine) from glia limitans endfeet through Cx43 hemichannels was claimed to induce dilatation of pial arterioles (1904). Release of ATP from astrocytes through Cx43 gap junctions was also shown to affect synaptic activity in hippocampal slices; Cx43 were activated by local drop in [Ca²⁺] following photoactivation of exogenous extracellular Ca²⁺ buffer (1754). The Cx43 hemichannels seem to be active in resting conditions when they provide constitutive ATP secretion to regulate basal excitatory transmission (324). Astroglial connexons (and possible pannexons) have been also implicated in release of lactate in acute slices from the brain stem, hippocampus, hypothalamus, and cortex (833). Inhibition of astroglial Cx43 channels in vivo by injection of selective inhibitor TAT-L2 peptide into basolateral amygdala impaired memory consolidation and resulted in amnesia for auditory fear conditioning. This can be rescued by coinjection of a somewhat eclectic mixture of neuroactive molecules (glutamate, glutamine, lactate, d-serine, glycine, and ATP) (1663). Opening of astroglial hemichannels is caused and regulated by many factors, which include membrane depolarization, mechanical stimulation, decrease in extracellular or increase in cytosolic [Ca²⁺], fluctuations in pH, reactive oxygen species, or intracellular phosphorylation of connexins (572). Activity of connexons is also regulated by growth factors, proinflammatory cytokines, or CB₁ cannabinoid receptors (535, 1275), whereas in tanycytes Cx43 channels are activated by extracellular glucose (1273).

A second route for secretion by diffusion is associated with plasmalemmal ion channels. The most characterized of these channels are P2X₇ purinoceptors operating in dilated (pore-forming) mode (see sect. VIIIA). The P2X₇ receptors have been implicated in the release of ATP and excitatory amino acids (457, 503, 1690). There is also evidence for glutamate release through TREK-1 channels following activation of metabotropic receptors. Apparently, G protein βγ-subunits directly activate TREK-1 channels by interaction with the NH₂ terminus of the latter (1893). Glutamate and GABA may also diffuse through anion channels, including volume-regulated anion channels and Best-1 chloride channels (1893). Glutamate released from astrocytes through these channels was claimed to activate synaptic NMDA receptors in CA1 pyramidal neurons (636), thus modulating synaptic plasticity (1323). Evidence about Best-1-mediated secretory pathway, however inspiring, is yet in need of independent confirmation.

D. Transporter-Mediated Release

Plasmalemmal transporters widely contribute to astroglial secretion; they are involved in release of many of the neuroactive molecules originating from astroglia. Specific roles of plasmalemmal transporters are summarized in TABLE 5 and discussed below.

E. Signaling Molecules Secreted by Astrocytes