Morphology and Anatomic Distribution

of Astrocytes The interstitial tissue between neurons and blood vessels which contain stellate and spindle-shaped cells was named as "neuroglia" or "nerve glue" by Virchow in 1860 (2). About 100 years ago, use of the gold sublimate staining method showed well-developed processes to emerge from many sides of astrocytes giving them their stellate shape and allowing the distinction of astrocytes from other glia (3). Cajal also noted that the tips of astrocytic processes having bulbous dilatations or end-feet terminated on vessel walls and that astrocytes could form a physical bridge between neurons and vessels. The Weigert technique (4) demonstrated the cytoplasmic fibrils in astrocytes and allowed the distinction between fibrillary astrocytes in the white matter, which have abundant fibrils, and protoplasmic astrocytes in the cerebral cortex which have fewer fibrils. Astrocytic processes also combine at the surface of the brain to form the glia limitans (Fig. 1a). The Bergmann astrocytes of the cerebellum have processes predominantly oriented in one direction and extending all the way from their cell bodies in the Purkinje layer to the surface of the molecular layer, where their end-feet form the glia limitans.

Ultrastructural studies show that astrocytes have relatively "clear" cytoplasm containing small, highly electron-dense granules that are glycogen, and all the usual organelles and lipid droplets. Microtubules are rarely seen in mature astrocytes. Their nuclei are oval and contain evenly distributed moderately abundant DNA components. A feature of the cytoplasm is the presence of 9-nm intermediate filaments which may also occur in parallel bundles in their processes (Figs. 2 and 3). Thin short extensions appear to interconnect some of these filaments.

Adjacent astrocytes are separated by a 15-20 nm extracellular space along which two types of junctions are present. The first type of junction is the puncta adhaerentia where adjacent astro-cyte membranes are parallel being separated by a wider space of 25-30 nm (5). Slightly increased electron density of the gap and adjacent cytoplasm is also present. The second type of junction is the gap junction where adjacent membranes are separated by a 2-to 3-nm wide gap (Fig. 3). Both junctions allow the penetration of tracers such as horseradish peroxidase and lanthanum (6).

Astrocyte Morphology

Z0-1/AQP4

Fig. 1. Merged confocal images from rat brain dual-labeled for GFAP (red) and Claudin-5 (green) (a, c, e, and g), for caveolin-3 and GFAP (d), and zonula occludens-1 (ZO-1) and aquaporin4 (AQP4) (f ) are shown. (a) The glia limitans formed by astrocytic processes is present at the brain surface and astrocytic end-feet surround the entire circumference of two intracerebral arterioles. (b) One-month-old postnatal rats showing two protoplasmic astrocytes, one filled with Lucifer Yellow (green) and the other with Alexa 568. Astrocytic domains are well established and the fine spongiform processes of adjacent astrocytes intermingle in an area only a couple of microns wide. (c) Fibrillary astrocytes of the white matter have few processes and the long axes of these cells are parallel to the white matter axons. (d) Normal rat brain shows colocal-ization (yellow) of caveolin-3 and GFAP in the white matter and in hippocampal astrocytes. (e) A large cortical vessel shows the termination of end-feet in the form of looped processes (arrowhead). (f ) Vessel segment shows endothelial ZO-lfibrils (green) surrounded by astrocytic foot processes which are immunoreactive for AQP4 (red, arrowhead). (g) Reactive astro-cytosis adjacent to a cortical cold lesion is shown. The density and size of astrocytes are increased. Note also that their processes terminate into multiple mini processes which form a network (arrowheads). (f) Scale bar=50 mm (a, c-e, g); 10 mm (b) and 20 mm (f ). (b) Reproduced with permission (13); (f ) reproduced with permission (64).

Z0-1/AQP4

Fig. 1. Merged confocal images from rat brain dual-labeled for GFAP (red) and Claudin-5 (green) (a, c, e, and g), for caveolin-3 and GFAP (d), and zonula occludens-1 (ZO-1) and aquaporin4 (AQP4) (f ) are shown. (a) The glia limitans formed by astrocytic processes is present at the brain surface and astrocytic end-feet surround the entire circumference of two intracerebral arterioles. (b) One-month-old postnatal rats showing two protoplasmic astrocytes, one filled with Lucifer Yellow (green) and the other with Alexa 568. Astrocytic domains are well established and the fine spongiform processes of adjacent astrocytes intermingle in an area only a couple of microns wide. (c) Fibrillary astrocytes of the white matter have few processes and the long axes of these cells are parallel to the white matter axons. (d) Normal rat brain shows colocal-ization (yellow) of caveolin-3 and GFAP in the white matter and in hippocampal astrocytes. (e) A large cortical vessel shows the termination of end-feet in the form of looped processes (arrowhead). (f ) Vessel segment shows endothelial ZO-lfibrils (green) surrounded by astrocytic foot processes which are immunoreactive for AQP4 (red, arrowhead). (g) Reactive astro-cytosis adjacent to a cortical cold lesion is shown. The density and size of astrocytes are increased. Note also that their processes terminate into multiple mini processes which form a network (arrowheads). (f) Scale bar=50 mm (a, c-e, g); 10 mm (b) and 20 mm (f ). (b) Reproduced with permission (13); (f ) reproduced with permission (64).

Fig. 2. An electron micrograph of an astrocyte in layer 3 of the cerebral cortex shows relatively electron-lucent cytoplasm in which the normal complement of organelles is present. Groups of 9-nm intermediate filaments are present in the cytoplasm (arrowheads ). x18,500.

Fig. 3. An electron micrograph taken at the level of layer 3 of the cerebral cortex shows an arteriolar segment which consists of endothelial, smooth muscle, and attenuated leptomeningeal cell layers. The latter is separated from two astrocytic end-feet (A ) by a basement membrane. Note that in well-fixed vessels, a perivascular space is not present. The end-feet show mitochondria and the cross-sections of intermediate filaments (arrowheads) in the cytoplasm and are separated by a gap junction (*). Note the proximity of the end-feet to pre- and postsynaptic terminals which together form a tripartite synapse. x50,000.

Fig. 3. An electron micrograph taken at the level of layer 3 of the cerebral cortex shows an arteriolar segment which consists of endothelial, smooth muscle, and attenuated leptomeningeal cell layers. The latter is separated from two astrocytic end-feet (A ) by a basement membrane. Note that in well-fixed vessels, a perivascular space is not present. The end-feet show mitochondria and the cross-sections of intermediate filaments (arrowheads) in the cytoplasm and are separated by a gap junction (*). Note the proximity of the end-feet to pre- and postsynaptic terminals which together form a tripartite synapse. x50,000.

Gap junctions are evenly distributed along the astrocytic processes, often interconnecting adjacent astrocytic processes derived from the same cell referred to as autocellular junctions (7, 8). Only in the narrow interface of adjacent cells do gap junctions couple processes from different cells (9). At the gap junction, each of the joined cells contributes a hemi-channel or connexon to each cell-cell channel. Each hemi-channel comprises a hexamer of connexins arranged around a central pore, and the cell-cell channels are gated by several stimuli, including transjunc-tional voltage, low pH, and various pharmacological agents. Connexin30 and connexin43 colocalize at gap junctions (10). Zonula occludens-1 (ZO-1) has also been localized at astrocytic gap junctions where it is found to colocalize with connexin30 and 43 and ZONAB (ZO-1-associated nucleic acid-binding protein) (11). Connexins are known to have adhesive properties and the autocellular junctions may stabilize the complex network of astro-cytic processes and may also facilitate intracellular diffusion of energy metabolites and possible signaling molecules, such as Ca2+ and inositol (1,4,5)-triphosphate (IP3), between fine astrocytic processes (8).

2.1. Astrocyte Astrocytes typically extend between five to eight major processes,

Domains each of which is highly ramified into innumerable delicate leaflet like processes, which are insinuated between and around the various components of the nervous tissue (12). Microinjection of single hippocampal astrocytes with fluorescent dyes demonstrates that each astrocyte occupies a discrete area that is free of processes from any adjacent astrocytes thus defining its own anatomical domain (Fig. 1b). Only the most peripheral processes interdigi-tate with one another in a narrow interface within which <5% of the volumes of adjacent astrocytes overlap (13, 14). Glial fibrillary acidic protein (GFAP) labels only the major processes of astrocytes, many of the smaller processes being nonreactive with GFAP. These smaller processes fill a volume that is best defined as a polyhedron (12-14). These fine processes have a significantly higher density of mitochondria as compared with the surrounding neuronal processes, synapses, other glial processes, and endothelial cells, supporting the concept that oxidative metabolism is a major part of the energy metabolism in protoplasmic astrocytes (15).

Within a single astrocyte domain, 300-600 neuronal dendrites (16) and 105 synapses are present in the rodent cortex and hippocampus. In contrast, in the human cortex, a single astrocyte might sense the activity and regulate the function of more than one million synapses within its domain (17). The distribution of astrocytes throughout the brain and spinal cord is highly organized being evenly distributed, such that their cell bodies and larger processes are not in contact with each other (18).

The functional significance of these nonoverlapping astrocytic domains is unknown, although all synapses lying within a given volumetrically defined compartment may be under the sole influence of a single astrocyte (19).

Neurons are dispersed among the astrocytic domains, with the innumerable fine neurites penetrating each astrocytic domain and being surrounded by its processes. The ratio of glia to neurons is higher in humans than most other species (19-21).

2.2. Types of Astrocytes in the Human Cerebral Cortex

Based on GFAP immunostaining, human cortical astrocytes are reported to have four distinct morphologies being named protoplasmic, interlaminar, polarized, and fibrous or fibrillary astrocytes (17).

2.2.1. Protoplasmic Astrocytes

Protoplasmic astrocytes are the most abundant type in human cortex, being present in cortical layers 2-6. Human protoplasmic astrocytes are larger and more elaborate than their rodent counterparts (22). Although the cell body of human astrocytes is only ~10 mm in diameter, their processes span 100-200 mm, giving them a 27-fold greater volume than their rodent counterparts (17). The synaptic density in the rat cortex has been estimated to be 1,397 million synapses/mm3, while that of human cortex is ~1,100 million synapses/mm3 (23). This suggests that synaptic density alone does not account for the increased capacity of human brain. The majority of the GFAP-positive processes of protoplasmic astrocytes do not overlap indicating a domain organization.

2.2.2. interlaminar Astrocytes

Interlaminar astrocytes were first described in cortical layer 1 of primate cortex (24, 25) where they extend striking long, frequently unbranched, processes extending through the cortical layers, terminating in layers 3 or 4 (26). The cell bodies of these astrocytes are ~10 mm in diameter and extend two types of processes: three to six fibers that contribute to the astrocytic network near the pial surface, and another one or two that penetrate deeper layers of the cortex. The latter have a constant diameter and can extend up to 1 mm in length (26). These processes are tortuous and, although largely unbranched, occasionally send collaterals to the vasculature (26). The endings of these interlaminar fibers deep in the cortex might be in the form of a "terminal mass" or end bulb containing a multilaminar structure and mitochondria (27). The function of these interlaminar astrocytes is unknown. Their interlaminar fibers clearly violate the domain organization and might serve as a nonsynaptic pathway for long-distance signaling and integration of activity within cortical columns (17).

2.2.3. Polarized Astrocytes These unipolar cells are relatively uncommon and are present in layers 5 and 6 of the cortex, near the white matter, and extend one or two long GFAP-positive processes, which are up to 1 mm in length, away from the white matter (17, 22). These long processes are straight, frequently unbranched or branch once, have a constant diameter of ~2-3 mm, and have numerous "beads" or varicosities. Occasionally, polarized astrocytes extend processes to the vasculature, but most terminate in the neuropil (22). Polarized astrocytes do not respect the domain boundaries of their neighbors, because the long processes from these cells travel directly through other protoplasmic astrocytic domains. The function of these cells has not been investigated, but these cells might serve as an alternative pathway for long-distance communication across cortical layers, perhaps forming links between functionally related domains in different laminae, or between gray and white matter (17).

2.2.4. Fibrous Astrocytes These white matter astrocytes have fewer primary GFAP-positive processes and their fibers are straighter and less branched than those of other glia (22, 28) (Fig. 1c). These cells are roughly equidistant from one another. In contrast to protoplasmic astrocytes, the processes of adjacent fibrous astrocytes intermingle and overlap (22, 28). The simple morphology of these cells and their relative uniformity suggest that their function might be limited to metabolic support and not extend to information processing and modulation of neural activity.

Transcriptome databases are available for genes expressed in acutely isolated mouse astrocytes from postnatal days 1-30 (29) and genes expressed in protoplasmic astrocytes in adult mouse cortex (15). Postnatal mouse astrocytes have high expression of many transcription factors, signaling transmembrane receptors, and secreted proteins (see supplemental material in www.jneurosci.org). Astrocytes were found to be enriched in specific metabolic and lipid synthetic pathways, including draper/Megf10 and Mertk/ integrin avb5 phagocytic pathways, suggesting that astrocytes are professional phagocytes. Since the gene profiles of astrocytes and oligodendrocytes were found to be very dissimilar, it was suggested that they should no longer be classed together as "glia."

Genomic expression profiling was used to identify metabolic pathways in protoplasmic astrocytes and neurons from adult mouse cortex (15). The analysis showed that both astrocytes and neurons express transcripts for oxidative metabolism of glucose; however, the expression of the majority of enzymes in the tricarboxylic acid cycle was higher in astrocytes than neurons. These findings support the presence of robust oxidative metabolism in astrocytes.

3. Properties of Astrocytes

3.1. Molecular Properties of Astrocytes

3.1.1. Genes Expressed by Astrocytes

3.1.2. Molecules In GFAP and S100P are well recognized and widely used specific

Astrocyte Cell Bodies markers of astrocytes (30). Dye injection in adult rodents shows that GFAP expression occupies only 15% of the total volume of the cell (12, 14). Other markers of astrocytes are the gap junction proteins connexin30 (10) and connexin43 (31). Immunohistochemistry shows diffuse connexin43 localization in brain, while localization of connexin30 is more heterogeneous being present in gap junctions of gray, but not white, matter astrocytes (10). Caveolin-3 is expressed in both cortical and white matter astrocytes (Fig. 1d). Basic fibroblast growth factor (FGF)-2 (32) is present in adult astrocytes and expression of FGF receptor -2 and 3 is reported in astrocytes and oligoden-drocytes (33).

Aldehyde dehydrogenase 1 family member L1 (Aldh1L1) has been identified as a new astrocyte-specific marker (29) in mouse brain. The mRNA signal for this gene is present throughout the CNS in a pattern consistent with pan-astrocyte expression. Immunohistochemistry shows Aldh1L1 localization in both the gray and white matter astrocytes with labeling of the cell body and its extensive processes, while GFAP only labels the thick main processes of some astrocytes (29, 34). Dual labeling demonstrates that Aldh1L1 does not label other cell types in the brain making it a useful astrocyte-specific marker (29).

High-affinity astrocyte-specific glutamate transporters, excitatory amino acid transporter (EAAT)1 (GLAST) and EAAT2 (GLT1), and subtypes are enriched in astrocytic processes and play a major role in glutamate clearance in the adult CNS (35, 36). Localization of P-gp is described in Subheading 3.1.3. Astrocytes also express urea transporter 3 (37), nucleoside transporters (38), and ryanodine receptors (39). Other efflux transport proteins including Multidrug Resistance Proteins 1-6 and the Breast Cancer Resistance Protein (ABCG2) have been reported in astrocytes, although expression varies depending on the species examined and the age of the rat, whether embryonic or adult (40).

3.1.3. Molecules in Cerebral capillaries are typically positioned along the interfaces

Astrocytic End-Feet between adjacent astrocytic domains and the end-feet of adjacent astrocytes provide a contiguous but nonoverlapping sheath around the capillaries bordering its domain (41). The end-feet of astrocytes cover >99% of the vascular surface facing endothelial cells or pericytes, but are not always GFAP positive, giving the false impression that astrocytic coverage of the vasculature is incomplete (41, 42). Penetrating arterioles are normally surrounded by GFAP-positive end-feet (Fig. 1a), whereas end-feet around capillaries are GFAP negative. The significance of GFAP expression in end-feet is not clear, but GFAP expression is upregulated by mechanical stress and may be induced by arterial pulsation (43). Confocal microscopy shows that astrocyte end-feet terminate into multiple looped processes lying on the vessel wall forming a rosette-like structure (42) (Fig. 1e). This morphology greatly increases the surface area of the end-feet in contact with the blood vessel. Each astrocyte has at least one process with end-feet surrounding a blood vessel (41), although individual astrocytes can contact several endothelial cells via multiple end-feet, even those lying some distance away. Thus, vessels can be covered with many end-feet derived from distinct astrocytes (Fig. 1, Chapter 1).

Astrocyte polarity refers to the molecular and structural heterogeneity of specific membrane domains on the astroglial surface. Vascular end-feet are highly polarized expressing several specialized proteins at their luminal surface, including glucose transporter-1 (45-kDa form) which facilitates rapid transfer of glucose to metabolically demanding dendrites (42), P-glycoprotein (44) which is involved in the removal of lipo-philic molecules and BBB differentiation, the purinergic receptors P2Y(2) and P2Y(4) which are mediators of astrocytic Ca2+ signaling and colocalize with GFAP around larger vessels in the cortex (41), and functional a1 and b adrenergic receptors which, when activated, cause prominent elevations in end-foot Ca2+ (45). The gap junction protein connexin43 is also highly expressed in astrocytic end-feet (41).

AQP4, the principal AQP in mammalian brain, is expressed in astrocytes at the borders between major water compartments and the brain parenchyma (46, 47). Expression of AQP4 in astrocytic foot processes brings it in close proximity to intracerebral vessels and thus the blood-brain interface (Fig. 1f). Water molecules moving from the blood pass through the luminal and abluminal endothelial membranes by diffusion and across the astrocytic foot processes through the AQP4 channels. AQP4 is also expressed in the basolateral membrane of the ependymal cells lining the cerebral ventricles, in subependymal astrocytes which are located at the ventricular cerebrospinal fluid (CSF)-brain interface, and in the dense astrocytic processes that form the glia limitans at the subarachnoid-CSF fluid interface.

Freeze-fracture studies have shown orthogonal arrays of intramembranous particles (OAPs) in astrocytic end-feet (48-52) (Fig. 4). A positive relationship between the OAP-based polarity and an intact BBB has been postulated (50, 53). When the BBB becomes leaky as occurs in brain tumors, the OAP-related polarity of astrocytes decreases (49).

The Kir 4.1 K+ channel and AQP4 are located in the OAPs anchored to a-syntrophin, an adaptor protein associated with the dystrophin-dystroglycan complex (DDC) (54, 55). Restriction of AQP4 immunoreactivity to the astrocytic end-feet membrane is dependent on the extracellular heparin sulfate proteoglycan, agrin, which is deposited by both astrocytes and endothelial cells

Fig. 4. Freeze-fracture images from mouse brain showing astrocytic end-feet at the glia limitans in low (a) and high magnification (b). (a) The borders between astrocytic end-feet are marked by arrows. (b) High magnification of (a) shows orthogonal arrays of intramembranous particles, some of which are circled. Scale bar=(a) 1 mm, (b) 100 nm. Contributed by Dr. H.Wolburg, University of Tuebingen, Germany.

Fig. 4. Freeze-fracture images from mouse brain showing astrocytic end-feet at the glia limitans in low (a) and high magnification (b). (a) The borders between astrocytic end-feet are marked by arrows. (b) High magnification of (a) shows orthogonal arrays of intramembranous particles, some of which are circled. Scale bar=(a) 1 mm, (b) 100 nm. Contributed by Dr. H.Wolburg, University of Tuebingen, Germany.

(56). Agrin binding to a-dystroglycan couples to AQP4 through a-syntrophin. If agrin is absent from the basal lamina, AQP4 immunoreactivity becomes diffuse, being present across the entire cell surface (57).

Altered expression of AQP4 in brain injury has been reported in experimental autoimmune encephalomyelitis (EAE) (58). Areas showing perivascular accumulation of inflammatory cells show loss of the polarized localization of AQP4 to end-feet and localization becomes diffuse over the entire astrocytic cell surface associated with loss of OAPs as observed in freeze-fracture replicas (58). Loss of ß-dystroglycan immunoreactivity is also present in these areas suggesting that loss of ß-dystroglycan-mediated astrocyte foot process anchoring to the basement membrane leads to loss of polarized AQP4 localization in astrocytic end-feet, and thus to edema formation in EAE.

3.1.4. K+ Channels At least four K+ channels have been identified in cortical astro cytes, namely Kir 2.1, 2.2, 2.3, and 4.1 (59). Based on studies in transgenic mice, it appears that one member in particular, Kir 4.1, is the predominant K+ channel in mature astrocytes and almost solely responsible for establishing the astrocyte negative resting membrane potential (60, 61). The distribution of the potassium channel Kir 4.1 and K+ conductivity is similar to that of the DDC and AQP4 (62-64). This codistribution seen in astrocytes and retinal Müller cells may enable these cells to respond to the potassium uptake with water influx (46, 60).

3.1.5. Transmitter Release One of the principal functions of astrocytes is uptake of neurotrans-

from Astrocytes mitters released from nerve terminals. However, astrocytes can also release neuroactive agents, including transmitters, eicosanoids, steroids, neuropeptides, and growth factors (65).

Neuropeptides expressed at the mRNA or protein level in astrocytes include angiotensin (66-68), atrial natriuretic peptide (69-71), enkephalin (72), neuropeptide Y (73, 74), nociceptin (75), somatostatin (76, 77), substance P (78), and vasoactive intestinal peptide (79, 80). The regional distribution of these peptides in basal conditions and postinjury and their possible role in neuron-astrocyte interactions have been reviewed previously (81). For many of these agents, it is still uncertain whether the expressed transcript translates into a peptide, whether all these agents are expressed in vivo, and whether the target of the released neuropeptides are receptors on glia or neurons, or both.

3.2. Astrocytes and the The proximity of astrocytes to brain capillaries suggested that

Blood-Brain Barrier these cells may have a role in maintaining the barrier properties of cerebral endothelium. The first study to demonstrate the inductive influence of astrocytes and the neural microenvironment on barrier features in brain vessels was performed by grafting neural tissue in the coelomic cavity of different bird species (82). The newly formed vessels originating from the host displayed BBB characteristics. Immediately after isolation, cerebral microvessels and rodent and human brain endothelial cells lose their functional BBB properties as indicated by their low electrical resistance (83). This suggests that the milieu surrounding the vasculature determines the characteristics of the blood vessels. Brain endothelial cells cocultured with astrocytes display a significant increase in tight junction formation, together with an increase in enzymatic systems such as g-glutamyl transpeptidase, Na+ IK ATPase, alkaline phosphatase, and transporters for neutral amino acids (84-89). Up-regulation of low-density lipoprotein receptors and P-glycoprotein was also reported (90, 91). Endothelial cells cultured with astrocyte-conditioned media also demonstrate an increase in barrier features including tight junction formation and increased electrical resistance, decrease in permeability and expression of g-GT, ATPase, HT7, and neurothelin, suggesting that soluble factors released by astrocytes are responsible for this effect (88, 92-95). It was further proposed that the basal lamina between capillary endothelial cells and astrocytic foot processes promotes interaction between the astroglia and endothelium, by increasing the local concentration of soluble factors secreted by astrocytes (89).

Over the years, several agents derived from astrocytes have been shown to increase the barrier properties in cultured endothe-lium. Treatment of cultured endothelial cells with src-suppressed C-kinase substrate-conditioned medium, a factor which stimulates the expression of angiopoietin-1 in astrocytes, results in an increase in tight junction proteins and decreased permeability to 3H-sucrose (96). Various molecules released by astrocytes such as transforming growth factor b1 (97), glial-derived neurotrophic factor (98), FGF-2 (99), and interleukin-6 (100) are able to induce some of the barrier properties in cultured brain endothelial cells. Human brain endothelial cells cultured in astrocyte-conditioned media show increased activity of protein kinase C, implicating a receptor-mediated action of an astrocyte-derived factor (101). Using angiotensinogen knockout mice with BBB disruption, it was shown that angiotensinogen production by reactive astrocytes was required to reinduce BBB function (102).

3.3. Astrocyte Astrocytes are electrically nonexcitable cells. Advances in Ca2+

Signaling Mechanisms imaging techniques led to the finding that astrocytes can com3 3 1 Ca++ SI nailn municate by Ca2+ signaling in two major ways. Firstly, signaling is

3.3.1. C SIgnalIng expressed as repetitive monophasic oscillations in cytosolic Ca2+

concentrations ([Ca2+];) limited to a single cell when activated by different transmitters, including glutamate, GABA, and ATP (adenosine 5'-triphosphate) (103, 104). They can also be evoked by changes in the extracellular environment including lowering of extracellular Ca2+, hypo-osmotic conditions, local application of potassium, or mechanical stress (105). Astrocytic Ca2+ oscillations are known to involve activation of phospholipase C, IP3 production, and release of Ca2+ from intracellular stores, rather than influx through membrane channels (106, 107).

The second type of Ca2+ signaling is in the form of propagating Ca2+ waves which can be stimulated by focal electric stimulation, mechanical stimulation, lowering extracellular Ca2+ levels, or by local application of transmitters such as glutamate or ATP (103). High-frequency neuronal spiking has been shown to induce astro-cytic Ca2+ waves in organotypic slices and in anesthetized mice following sensory stimulation (108, 109). In general, Ca2+ waves propagate with a velocity of about 8-20 |mm/s and engage as many as 50 neighboring astrocytes per wave (110). These signals are transmitted to other cell types in the brain including neurons, microglial cells, and oligodendrocytes (111, 112). Initially, these intracellular waves were thought to be propagated by diffusion of IP3 or calcium through intercellular gap junctions (103). Pharmacological approaches demonstrate that ATP is the diffusible messenger (113) and that connexin hemi-channels are the most significant mechanism of ATP release from astrocytes (110). Wave propagation is mediated by P2Y receptor subtypes including P2Y1, P2Y2, and P2Y4 (114). ATP, once released, can be broken down by ecto-ATPase and ecto-5-nucleosidase into adenosine, which has a dilating effect on cerebral vessels during functional hyperemia (115) and also has presynaptic and postsynaptic effects. Presynaptically, adenosine A1 receptors inhibit Ca2+ channel opening resulting in inhibition of excitatory synaptic transmission (116). Postsynaptically, A1 receptors open K+ channels resulting in hyper-polarization and decreased neuronal activity (117).

Both modalities of astrocytic signaling, Ca2+ oscillations and Ca2+ waves, are readily transmitted to surrounding neurons, which display prolonged increases in intracellular [Ca2+] (111, 118). Further details of astrocyte signaling can be obtained from reviews in the literature (41, 110, 119-121).

3.3.2. Astrocyte Signaling Neural activity is known to increase cerebral blood flow (CBF)

and Cerebral Blood Flow within seconds in the activated region, a process referred to as functional hyperemia (122-124). A variety of agents, including H+, K+, lactate, neurotransmitters, adenosine, arachidonic acid metabolites, and glutamate-induced activation of neuronal nitric oxide (NO) synthase are implicated in this increase in CBF (125, 126). The cellular source of these agents was not known and it was uncertain whether neurons were the source of the signals mediating vasodilatation or whether other cells were involved. The proximity of astrocytic processes with synapses and blood vessels suggested that astrocytes may have a role in CBF regulation. The first demonstration that astrocytic activity can influence vascular tone was observed in brain slices where electrical stimulation of neuronal processes increased the amount of intracellular Ca2+ in astrocytic end-feet leading to slow dilatation of local cerebral arterioles (127). Inhibitors of metabotropic glutamate (mGluR) receptors block the vascular response, while activation of these receptors by agonists increases the amount of intracellular Ca2+ and reproduces the vasodilatation observed by neuronal stimulation (127). Direct electrical and mechanical stimulation of individual astrocytes increases the amount of intracellular Ca2+ and induces vasodilatation (127). Several mediators are implicated in these vascular changes, including vasoactive metabolites of the cyclooxygenase (COX) or cytochrome P450 ©-hydroxylase pathways as reviewed previously (126).

The availability of two-photon laser scanning microscopy allowed the investigation of the relationships between neural activity, astrocytic Ca2+, and CBF in vivo in anesthetized mice (128). These authors reported that increase in astrocytic Ca2+ by photolysis of caged Ca2+ caused vasodilatation of cortical arterioles in less than a second. An 18% increase in arterial cross-sectional area corresponding to an almost 40% increase in local perfusion was observed. Pharmacological studies indicated that the vascular responses evoked from astrocytes are mediated by metabolites of the COX-1 pathway. The nonselective COX inhibitor indomethacin and the selective COX-1 inhibitor SC-560 attenuated the vascular response, whereas inhibitors of NO synthase, COX-2, p450 hepoxygenases, or adenosine receptors had no effect. Stimulation of neural activity by electrical stimulation resulted in an increase in astrocytic Ca2+ and vasodilatation of adjacent arterioles, which was attenuated by COX-1 inhibition suggesting that astrocytes induce vasodilatation predominantly through COX-1 reaction products. This is supported by a recent study demonstrating that COX-1 is the primary mediator of astrocyte-induced vasodilatation (129).

These studies provide strong evidence that during neural activity, changes in intracellular Ca2+ in astrocytic end-feet modulate vascular tone in adjacent arterioles. This is an exciting development in the field and reinforces the concept of a neurovascular unit in which neurons, astrocytes, and endothelial cells work together to maintain homeostasis of the brain microenvironment.

Ca2+ increases in astrocytic end-feet activates soluble PLA2, leading to the production of multiple vasoactive substances. PLA2 generates diffusible arachidonic acid (AA) from the plasma membrane, which can be converted into a number of compounds, some of which induce vasodilatation while others induce vasoconstriction. Dilating products include PGE2 from the action of COX enzymes (127, 128, 130) and several epoxyeicosatrienoic acids (EETs) (5,6-EET;8,9-EET; 11,12-EET and 14,15-EET) (131, 132) from the activity of a subtype of cytochrome P450 (CYP450) enzyme. Constricting molecules consist of PGF2 (133) and thromboxane A2 (134-136) from COX activity, endothelin peptide (137, 138), as well as 20-HETE (45, 139). The enzymes governing the production of these vasoactive products are sensitive to NO, suggesting that NO levels may dictate the direction of the vessel response (140).

Decrease in CBF occurs in vivo in response to norepinephrine (NE) (141), an effect that may help maintain CBF at a constant rate at higher blood pressures. In vitro work shows that NE triggers robust intracellular Ca2+ increases in astrocytes via activation of a1 and b adrenergic receptors (142). This precedes prominent vasoconstriction (45). When astrocytes are loaded with BAPTA-AM to chelate rises in intracellular Ca2+, NE-induced vascular constrictions are drastically reduced, suggesting Ca2+ is critical for the astrocyte-mediated effect. Thus, NE is another vasoactive transmitter affecting blood vessels besides glutamate.

Pial arteriolar dilatation is known to accompany increased neuronal activity in the cerebral cortex, despite the absence of direct neuronal connections from the cortex to these vessels. A recent study demonstrates that the vasodilating signal arising in the parenchyma is transmitted to pial arterioles via an astrocytic, rather than a vascular route (143). Selective injury to the glia limitans, but not endothelium, prevents neural activation-induced pial arteriolar dilatation, suggesting a key role for astrocytes in this process. This astrocytic signaling pathway is sensitive to con-nexin43 blockade, suggesting an important contribution from gap junctions and/or hemi-channels.

Certain findings question whether Ca2+ signaling is indeed a key factor in mediating functional hyperemia in vivo. Astrocyte Ca2+ increase often occurs independent of neuronal activity (144)

and spontaneous Ca2+ oscillations have been reported both in vivo (145) and in vitro (146). Secondly, neuronal activity does not consistently increase astrocyte Ca2+ with a time course that follows the time of activation (147). Also, the astrocyte Ca2+ waves that are considered to be important for relaying neuronal information through the astrocyte syncytium towards vessels are not always observed in vivo, suggesting that this signaling process may actually be attributable to aspects of the slice or culture condition, or that it manifests more easily in patho-physiological conditions such as epilepsy (148, 149), rather than being the "normal" method of signaling for neurovascular coupling. Further studies are required to understand the significance of Ca2+ signals in the control of cerebral vessels.

In summary, astrocytes are capable of eliciting changes in vessel diameter in both directions. Additional details of the role of astrocytes in modulation of CBF can be obtained from reviews in the literature (126, 140, 150, 151).

3.3.3. Signaling In Relation Astrocytes undergo swelling in response to a decrease in extracel-to Cell Volume lular osmolarity and this is followed by a corrective process lead ing to restoration of normal volume (152, 153). The latter is an active process resulting from extrusion of mainly K+, Cl- and organic molecules such as pyroles, and organic amines (153). Efflux of taurine, GABA, glutamate, and glycine has been documented in astrocyte cultures in response to hypo-osmotic stimuli. An increase in the cellular volume triggers swell-activated channels (153, 154). Volume-regulated anion channels (VRAC) tightly regulate cell volume homeostasis and act as release routes for transmitters including excitatory amino acids and ATP and chloride currents (155). VRACs contribute to neuronal damage via excitatory amino acid release in pathological conditions. For example, persistent swelling of astrocytes induced by lactacidosis appears to be secondary to inhibition of VRACs (156). For further details about ionic homeostasis in astrocytes, the reader is referred to reviews on this subject (119, 155, 157).

The presynaptic terminal and the postsynaptic neuron are well-known functionally important elements of the synapse. However, a third cellular component consisting of astrocytic processes is often associated with synapses in the cerebral (158, 159) (Fig. 3) and cerebellar cortex (160, 161), while synapses in the retina are contacted by Müller cells (astrocyte-like radial glia) (162). Thus, synapses should be considered a tripartite, rather than a bipartite, structure (163).

3.4. Astrocytes and Synaptic Transmission

3.4.1. The Tripartite Synapse

3.4.2. Neuron/Astrocyte Astrocytes express a wide variety of neurotransmitter receptors Interactions including metabotropic glutamate receptors (164, 165), GABAB

receptors (166), and muscarinic acetyl choline (Ach) receptors

(167). In cell culture systems and in brain slices, following electrical stimulation of neurons, a variety of neurotransmitters, including glutamate, GABA, adrenaline, ATP, serotonin, Ach, and several peptides, can activate astrocyte receptors leading to increases in intracellular [Ca2+] concentrations (164, 168). This in turn initiates gliotransmission and the release of gliotransmitters (to distinguish them from neurotransmitters released from neurons), including glutamate, ATP, adenosine, D-serine, eicosanoids such as prostaglandin and 20-hydroxyeicosatetraenoic acid, cytokines such as tumor necrosis factor-a, and proteins and peptides such as acetylcholine-binding protein and atrial natriuretic peptide (121, 169). The most significant of these agents is glutamate which plays a central bidirectional role in astrocyte-neuronal interactions. Release of glutamate from astrocytes in response to increases in [Ca2+) has been demonstrated in both culture (118, 170-172) and brain slice preparations (165, 166, 173, 174). Glutamate release from astrocytes may occur by numerous mechanisms including exocytosis (175-177), VRAC (178), hemi-channels (179), purinergic P2X receptors (180), and pannexins (181). The state of the brain dictates which mechanism is utilized to release transmitters

3.4.3. Synaptic Modulation In vitro studies demonstrate that activated astrocytes can regulate synaptic transmission by release of glutamate and ATP. Glutamate can have presynaptic effects that are mediated either by metabotro-pic glutamate receptors (182) or by kainate receptors that induce an enhancement of transmitter release (183). ATP can act through postsynaptic P2X receptors to induce an elevation of postsynaptic Ca2+ level, which is thought to drive a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors to mediate an increase of synaptic transmission (184). Extracellular hydrolysis of released ATP can cause reduction in synaptic transmission that is mediated by presynaptic adenosine A1 receptors (116, 185, 186) and modify neuronal excitability by activating K+ conductance, which hyperpolarizes the neuronal membrane potential (187). Experiments in brain slices of the hippocampus (165, 173) and thalamus (188) demonstrate that application of glutamate agonists and PGE2 in addition to neuronal stimulation evokes a Ca2+-dependent glutamate release from astrocytes which activates neighboring neurons and results in increases in neuronal Ca2+ levels (162). This astrocytic modulation of neurotransmitter release from the presynaptic terminal and stimulation of postsynaptic neurons constitutes direct modulation of synaptic regulation.

Astrocytes can also regulate synaptic transmission by "indirect" mechanisms, one of which is the uptake of glutamate via the high-affinity glutamate transporters, EAAT1 and EAAT2 subtypes, which are enriched in astrocytic processes (189, 190). Transmission at glutamatergic synapses is terminated by removal of glutamate from the synaptic cleft. Brain astrocytes and Müller cells in the retina account for the bulk of the glutamate uptake at synapses (36, 191, 192). Within the astrocyte, glutamate is converted to glutamine through an ATP-requiring reaction catalyzed by the astrocyte-specific enzyme glutamine synthetase (193-195). Glutamine is subsequently released to the extracellular space for uptake by neurons and recycled into glutamate for glutamatergic neurotransmission.

Astrocytes can also modulate synaptic transmission by releasing chemical cofactors. The best documented example of such modulation is activation of the NMDA receptor, which requires the presence of glutamate as well as the cofactor D-serine that binds to the glycine-binding site of the receptor (196, 197). Serine racemase is an enzyme highly expressed in astrocytes and is responsible for conversion of L- to D-serine (196, 198). Brain astrocytes and Müller cells are the sole source of D-serine in the CNS and most likely modulate synaptic transmission at the NMDA synapses by releasing D-serine (198, 199).

A third indirect mechanism of modulation is via glial regulation of extracellular ion levels. Neuronal activity leads to substantial variations in the concentrations of K+ and H+ in the extracellular space (200-202). These variations can alter synaptic transmission since increases in K+ levels depolarize synaptic terminals (203), while H+ blocks presynaptic Ca2+ channels (204, 205) and NMDA receptors (206). K+ and H+ taken up by astrocytes are dissipated through many cells via gap junction coupling, thus regulating the extracellular concentrations of these ions (207, 208).

Astrocytes can also modulate synaptic transmission by directly controlling synaptogenesis as discussed in the next section.

In summary, release of neurotransmitter from the presynaptic terminal not only stimulates the postsynaptic neuron, but also activates the perisynaptic astrocytic processes. The activated astro-cyte, in turn, releases gliotransmitters that can directly stimulate the postsynaptic neuron and can feed back onto the presynaptic terminal either to enhance or to depress further release of neu-rotransmitter. Thus, the perisynaptic astrocyte is an active partner in synaptic transmission. Further details of astrocytic modulation of synaptic transmission can be obtained from reviews in the literature (121, 162, 169, 209-211).

3.5. Astrocytes Astrocytes are known to enhance the formation of functional syn-

and CNS apses in the CNS. Retinal ganglion cells (RGCs) cultured in the

Synaptogenesis absence of astrocytes, even after many weeks in culture, exhibit very little spontaneous synaptic activity when excitatory postsyn-aptic currents are measured by patch clamp (212). However, when RGCs are cultured in the presence of a feeder layer of astro-cytes or in astrocyte-conditioned medium, they exhibit high levels of synaptic activity (212). Two subsequent studies show that a glial factor (or factors) enhances the number of synapses between RGCs sevenfold without changing neuronal survival or neurite growth (213, 214). Astrocytes are also required for synapse stability and maintenance. Coculture of purified RGCs with a feeder layer of astrocytes cultured on a removable insert results in synapse formation in 1 week. When the astrocyte insert is removed and the neurons are examined after 1 week by immunostaining and patch-clamp recording, the majority of synapses are no longer present (214).

The synaptogenic factor contained in the glia-conditioned medium was identified as cholesterol (215, 216). Other factors derived from astrocytes which are important for synapse development include tumor necrosis factor-a (217) and the thrombos-pondins (218). Astrocytes may also play a role in the sculpting of synaptic structure and function within the developing or even adult brain as reviewed previously (219, 220).

3.6. Astrocytes as Several studies have proposed that astrocytes might not only reg

Neural Progenitor ulate neurogenesis but also themselves be neuronal progenitor

Cells cells (221-223). Studies show that radial glia in development and specific subpopulations of astrocytes located in the subventricular zone (SVZ) (224), along the walls of the lateral ventricles and the subgranular zone within the dentate gyrus of the hippocampus

(225) of adults mammals, function as primary progenitors or neural stem cells (NSC) that give rise to differentiated neurons and glial cells during development and in the postnatal brain

(226). Antimitotic ablation of the SVZ kills off the neuroblasts, but the SVZ-astrocytes survive and can repopulate the entire SVZ including the neural precursors (227). This is a novel finding which contradicts the classic teaching that neurons and glia are derived from distinct pools of progenitor cells. During development and in the adult brain, neural progenitors are capable of giving rise to transit-amplifying, or intermediate, progenitor cells (IPCs) which can, in the subventicular zone, divide rapidly to expand the available pool of neural precursors (228). Within NSCs and IPCs, genetic programs unfold for generating the extraordinary diversity of cell types in the CNS. The timing in development and location of NSCs, a property tightly linked to their neuroepithelial origin, appear to be key determinants of the types of neurons generated. Further discussion of the role of astrocytes as neural progenitor cells can be obtained from published reviews (226, 228, 229).

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