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Astroglia and glutamate in physiology and pathology: aspects on glutamate transport, glutamate-induced cell swelling and gap-junction communication
Neurochemistry International 37 (2000) 317±329
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Astroglia and glutamate in physiology and pathology: aspects on glutamate transport, glutamate-induced cell swelling and gapjunction communication Elisabeth Hansson*, HaÊkan Muyderman, Julia Leonova, Louise Allansson, Jon Sinclair, Fredrik Blomstrand, Thorleif Thorlin, Michael Nilsson, Lars RoÈnnbaÈck Institute of Clinical Neuroscience, GoÈteborg University, Box 420, S-40530 GoÈteborg, Sweden Received 22 June 1999; accepted 31 August 1999
Abstract Astroglia have the capacity to monitor extracellular glutamate (Glu) and maintain it at low levels, metabolize Glu, or release it back into the extracellular space. Glu can induce an increase in astroglial cell volume with a resulting decrease of the extracellular space, and thereby alter the concentration of extracellular substances. Many lines of evidence show that K+ can be buered within the astroglial gap-junction-coupled network, and recent results show that gap junctions are permeable for Glu. All these events occur dynamically: the astroglial network has the capacity to interfere actively with neurotransmission, thereby contributing to a high signal-to-noise ratio for the Glu transmission. High-quality neuronal messages during normal physiology can then be maintained. With the same mechanisms, astroglia might exert a neuroprotective function in situations of moderately increased extracellular Glu concentrations, i.e., corresponding to conditions of pathological hyper-excitability, or corresponding to early stages of an acute brain injury. If the astroglial functions are failing, neuronal dysfunction can be reinforced. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Astroglia; Glutamate; Glutamate receptors; Glutamate transport; Cell volume; Glial network
1. Introduction Recent experimental data support the view that astroglia and neurons are intimate partners in glutamate (Glu) synaptic transmission (see Araque et al., 1999; Laming et al., 1998; Hansson and RoÈnnbaÈck, 1995; Hansson et al., 1997). Astroglia express functional ionotropic and metabotropic Glu receptors (iGluRs and mGluRs; SteinhaÈuser and Gallo, 1996), and thereby have the ability to monitor neuronally released Glu. With the aid of the Glu transporters, GLAST and GLT-1, the astroglia clear the extracellu* Corresponding author. Tel.: +46-31-7733363; fax: +46-317733330. E-mail address: [email protected] (E. Hansson).
lar space from Glu, in fact, they do so more eectively than the neurons do. Alterations in astroglial cell volume can in¯uence extracellular volume and thereby the concentrations of neuroactive substances, with eects on neuronal excitability. Astroglia respond to neuronal signals by intracellular Ca2+ increases with complex spatio-temporal patterns. Astroglia metabolize Glu and release glutamine back to the neurons to be transformed and recycled as a transmitter. Furthermore, astroglia can release Glu and probably signal back to neurons (Parpura et al., 1994; Nedergaard, 1994). Notably, it has been suggested that the functional status of the gap-junction-coupled astroglial network is important for the supportive and protective functions of astroglia. It is interesting that the functional status of the network has also been shown to be
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important in the development of acute brain damage (Cotrina et al., 1998). Astroglia can react dynamically by exerting supportive and protective functions in the handling of Glu in the brain, even if astroglial reactions during pathophysiological processes appear to be destructive. In this paper, we focus on those aspects of astroglial interactions with Glu that are important for the astroglial support of normal neurotransmission, as well as on astroglial reactions in pathological states of overexcitability and during the development of a brain injury. 2. Astroglia monitor extracellular glutamate levels 2.1. Glutamate receptors Glu is the major excitatory transmitter in the CNS and is released at the neuronal presynaptic terminal, and activates dierent receptor types at the postsynaptic terminal. Consequently, GluRs are regarded as one of the most important receptor groups, and they have been studied extensively throughout the years (SteinhaÈuser and Gallo, 1996). Classically, GluRs have been subdivided into two groups, iGluRs and mGluRs, on the basis of their pharmacological and electrophysiological properties (see Hollmann and Heinemann, 1994; Pin and Duvoisin, 1995). Metabotropic GluRs are G protein coupled and their activation elicits both electrical and metabolic events in glial cells. Today, eight cloned mGluRs are subdivided into three groups according to their amino-acid similarity, pharmacological pro®le, and signal-transduction mechanism (Nakanishi, 1992, 1994). In short, mGluR1 and 5 (group I) are positively coupled to phospholipase C (PLC) as well as to various K+ channels by a Go or Gq protein. Their activation results in massive phosphoinositide (PI) hydrolysis and membrane depolarisation, while group II (mGluR2±3) and III (mGluR4,6±8) are associated with ion channels and the inhibition of adenylate cyclase. The all iGluRs contain integrated, ligandgated, cationic ion channels assembled from several subunits. They are further subdivided into N-methyl-Daspartate (NMDA) receptors and non-NMDA [aamino-3-hydroxy-5-methyl-isoxazole-4-proprionic acid(AMPA) or kainate- (KA) preferring] receptors. Both the NMDA receptors and the AMPA or KA receptors open transmembrane cation channels that generate Na in¯ow, K+ eux, membrane depolarisation and/ or calcium in¯ow, depending on the subunit composition. Activation of the AMPA-preferring subunits results in a rapid, fast-desensitising depolarisation, while KA-receptor activation produces long-lasting, non-desensitisation responses (Jabs et al., 1994). Expression of GluRs have been shown in several glial-cell
Fig. 1. Astrocytes, acutely isolated and from primary cultures, have been shown to express functional glutamate receptors. A. Acutely isolated astrocyte from rat cerebral cortex at p14 shows increases in [Ca2+]i when stimulated with (1S,3R )-1-aminocyclopentane-1,3dicarboxylic acid (ACPD; 10ÿ4 M), a metabotropic glutamate receptor (mGluR) agonist. The response is blocked by the selective group 1 mGluR antagonist (RS)-1-aminoindan-1,5 dicarboxylic acid (AIDA; 10ÿ3 M). B. Corresponding curves from a cultured cortical astrocyte. C. Stimulation with the kainate receptor agonist kainic acid (KA; 10ÿ5 M) and blockade of the receptor with the antagonist CNQX (10ÿ4 M). Drugs were applied as indicated by the horizontal bars, and the cells were allowed to rest for 10±15 min between the stimulations. Changes in [Ca2+]i were determined by microspectro¯uorometry, by using the calciumsensitive probes ¯uo-3 (A) or fura2 (B,C). Data are presented as change in ¯uorescence (F ) from the resting value (F0), (F ÿ F0)/F0%, or as changes in [Ca2+]i.
Cationic channel opening Ca2+ in¯ow Membrane depolarisation Fast desensitising Cationic channel opening Ca2+ in¯ow Non-desensitising Membrane depolarisation. Cationic channel opening Ca2+ in¯ow
AMPA
Activating PLC, IP3-mediated Ca2+ release
Inhibitory action on adenylyl cyclase. Decreased cAMP production
Inhibitory action on adenylyl cyclase. Decreased cAMP production
MGluR I (1/5)
MGluR II (2±3)
MGluR III (4/6±8)
NMDA
Kainate
Mechanism
Receptor
Table 1 Characteristics of glutamate receptor subtypes
Gi/o-protein coupled
Gi/o-protein coupled
Gq-protein coupled
Ligand-gated ion channel
Ligand-gated ion channel
Ligand-gated ion channel
Coupling
(1S,3R )ACPD QA (1S,3R )ACPD DCG-IV (S )-3C4HPG L-CCG-I L-AP4 L-SOP
CHPG (R,S )-3,5-DHPG
NMDA Cis-ACBD
Kainic acid Domoic acid
AMPA Quisqualate (QA)
Agonists
CPPG MSOP MAP4
EGLU MCCG PCCG-IV)
D-AP5 MK-801 CNQX AIDA L-AP3 (S)-4-CPG
CNQX DNQX
CNQX DNQX
Antagonists
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preparations both in vitro and in situ (Seifert and SteinhaÈuser, 1995; Porter and McCarthy, 1997; Kimelberg et al., 1997; Thorlin et al., 1998). Many of the ion channels, i.e., voltage-gated K+, Na+, Clÿ, and Ca2+ channels, previously described in cell cultures, have lately been reported in acutely isolated astrocytes (Barres et al., 1990; Tse et al., 1992; Sontheimer and Waxman, 1993; Duy and Mac Vicar, 1994). Although both iGluRs and mGluRs are commonly found on astrocytes, the presence of NMDA receptors is still unclear. NMDA-mediated currents have been observed in a small population of astrocytes in the hippocampus, and in neocortical astrocytes, while the NMDA-receptor group has been found on astrocytes in the visual cortex by using subunit-speci®c antibodies (Aoki et al., 1994). Recently, Ziak et al. (1998) showed NMDA-evoked membrane currents in glial cells in rat spinal cord, but the physiological implications of functional NMDA receptors in glia remain unknown. In astrocytes, activation of GluRs elicits complex changes in intracellular calcium ([Ca2+]i) (Fig. 1), which is often composed of dierent spatio-temporal patterns in the form of intracellular waves and oscillations. Recently, both neuroprotective action (e.g., Bruno et al., 1998) and the modulation of synaptic activity (Araque et al., 1998) as mediated through glial mGluRs have been shown, indicating an extensive complexity in Glu-induced neuroglial interactions. It has also been shown that both iGluRs and mGluRs mediate the induction of immediate early genes (Condorelli et al., 1993). The ability of Glu to elicit cell-tocell signalling (Cornell-Bell et al., 1990), to induce volume changes (see below and Hansson, 1994), and to decrease Glu release from astrocytes (Ye and Sontheimer, 1999) indicates that glial GluRs might play a major role as participant in both physiological and pathophysiological events within the CNS. (For agonists and antagonists for the dierent Glu receptors, refer to Table 1).
3. Astroglia clear glutamate from the extracellular space 3.1. High-anity glutamate uptake and glutamatemediated astroglial cell swelling The intracellular concentrations of Glu are rather high: 50 mM in astrocytes, and as much as 10 mM in Glu-ergic neurons. However, the extracellular concentration has to be maintained at approximately 1±3 m M to assure a high signal-to-noise ratio at normal Glu-ergic neurotransmission and to avoid excitotoxic actions of Glu on neurons. The removal of Glu from the extracellular space is achieved by a high-anity uptake into the cells surrounding the synaptic cleft.
Astrocytes have been shown to play a dominant role in this process. Given that Glu clearance is of paramount importance for normal transmission and for preventing neuronal injury, the biochemical and pharmacological properties of the Glu uptake have been scrutinized by neuroscientists since a high-anity transport system for this amino acid was initially observed in the early 1970s (Logan and Snyder, 1971). Increases in extracellular Glu levels have also been implicated as an important factor in astroglial cell swelling (Schneider et al., 1992; Hansson and RoÈnnbaÈck, 1995). After a circulatory disturbance in the brain, the astrocytes in the aected area respond within a few minutes by increasing the cell volume. Because of the relatively small volume of the extracellular space, astroglial swelling leads to changes in the concentration of neuroactive substances therein, which, in turn, aects neuronal function. Depolarization of the astrocytes causes both a release of Glu and de®cits in the ability to take up Glu from the extracellular space, which may lead to abnormal Gluergic neurotransmission and to neurotoxicity (Norenberg, 1998). Astrocytic swelling could also limit the diusion of Glu from the lesion site (Maxwell et al., 1994). The increase in volume may, however, result in metabolic disarray in the injured tissue, which might be harmful for neurons and might lead to destruction of the astrocytes (Kimelberg et al., 1990). The swelling of astrocytes by Glu results from an active intracellular accumulation of the amino acid (Barbour et al., 1988; Kimelberg et al., 1989). The mechanisms underlying Glu-induced astroglial swelling have been studied by using dierent in vitro model systems, but are far from fully explored. To better understand the mechanisms regulating the cell volume in various cell types, it is important to develop technical instrumentation and analysis methods that are capable of detecting and characterising dynamic cell-shape changes in a quantitative way. We have developed a new method, on the basis of ¯uorescence microscopy in conjunction with advanced image analysis, for evaluating cell-volume changes in three-dimensions (3D) (Allansson et al., 1999). Fig. 2 demonstrates the principle with this method. It is suggested that the astroglial swelling is caused by an osmotically driven transmembrane transport of Glu coupled to the uptake of Na+ ions. The uptake of Glu occurs against a steep intracellular to extracellular concentration dierence in the astrocytes, which is energy consuming, and the transport is driven by the electrochemical gradient for Na+ (Barbour et al., 1988; Drejer et al., 1983). Thus, there is a relation between Glu transport over the cell membrane and cell volume changes. We will discuss the aspects of the mechanisms behind each of
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these two processes and also suggest probable connections between them. Recent evidence suggests that one Glu is co-transported with three Na+ ions and one H+, while one K+ ion is counter-transported to the exterior (Zerangue and Kavanaugh, 1996). Five distinct transporters have been cloned so far: GLAST, GLT-1, EAAC1, EAAT4 and EAAT5. The cloned transporters exhibit distinct patterns of distribution in dierent brain regions and cell types. GLAST and GLT-1 are considered to be glia-speci®c Glu transporters (for details on tissue and cell distribution of Glu transporters, see Gegelashvili and Schousboe, 1998; Danbolt et al., 1998). Measuring the accumulation of a radio-labelled amino acid in the cells sheds light on the kinetics of Glu-transport activity. Depending on the kind of cellular preparation that is studied, and the brain region, the Vmax varies between 2 and 60 nmol/min/mg protein at 37;8C, and the Km varies between 3 and 80 mM (for a summary of the pharmacological and kinetic properties of Glu uptake, see Erecinska and Silver, 1990; Robinson and Dowd, 1997). Though both GLT-1 and GLAST are almost exclusively found in astrocytes, their patterns of expression are dierentially regulated and regionally and developmentally segregated. GLT-1 is expressed at the highest concentrations in the hippocampus and cerebral cortex, while GLAST is preferentially expressed in the molecular layer of the cerebellum (Lehre et al., 1995). It is noteworthy that, in adult brain, astrocytic membranes that face nerve terminals, axons, and spines have a denser population of Glu transporters than
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those facing capillaries, pia, or stem dendrites (Chaudhry et al., 1995), which is consistent with the importance of astrocytic carriers for removal of Glu from the extracellular space after synaptic transmission. The Glu transporters are probably related to the regulation of the cell volume, as the Glu-induced astroglial cell swelling can be at least partially blocked by the Glu-carrier blocker PDC (Hansson, 1994); it is probable that these two processes are linked. The major role of astrocytic transporters in Glu clearance and in preventing neurodegeneration was unequivocally demonstrated by the in vivo application of the antisense oligonucleotid technique, a method that permits selective inhibition of synthesis of dierent Glu carriers. The loss of the glial transporters GLT-1 and GLAST in rat produced elevated extracellular Glu levels, neurodegeneration characteristic of excitotoxicity, and a progressive paralysis. Failure of Glu uptake has also been demonstrated in ischemia, ALS and traumatic brain injury. The resulting increase in extracellular Glu levels has been considered an important factor in neuronal damage in these conditions (Raghavendra Rao et al., 1998), whereas the loss of the neuronal Glu transporter EAAC1 did not elevate the extracellular levels of Glu (Rothstein et al., 1996). Possible mechanisms for aecting Glu transporters in the regulation of extracellular Glu levels and thereby also regulating the cell volume and extracellular space volume are important. A large number of factors have been shown to aect the activity and expression of Glu transporters (Table 2). Importantly, the activity of dierent Glu transporters may be regulated dierentially by the same eectors. For example,
Fig. 2. The ®gure to the left demonstrates the principle for optical sectioning of ¯uorescent labelled astroglial cells (a). Data is collected from dierent focus planes (b), resulting in a stack of 2D images (c). The 2D images are reconstructed, and an edge detection algorithm is applied for volume estimation. The right ®gure presents the total cell volume, as well as the slice area for each 2D image in one astroglial cell. 1 = control, 2 = swollen cell.
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Table 2 Eects on astroglial glutamate (Glu) uptake capacity and gap junction permeability (GJ) caused by dierent substances or changed conditions Substance/condition
Eects on Glu uptake
Reference
Eects on GJ
Reference
Glutamate Kainate Phenylephrine or methoxamine (a1) Isoproterenol (b) 5-HT Arachidonic acid Endothelin-1 Oxygen free radicals Depolarization pH (acidosis) Energy status (ATP levels q)
Q Q Q q n.e q q q q q q
Gegelashvili et al. (1996) Gegelashvili et al. (1996) Hansson and RoÈnnbaÈck (1991) Hansson and RoÈnnbaÈck (1991) Hansson and RoÈnnbaÈck (1991) Barbour et al. (1989) unpublished results Volterra et al. (1994) Barbour et al. (1988) Swanson et al. (1995) Swanson (1992)
Q Q q Q Q or q q q q Q q q
Enkvist and McCarthy (1994) Enkvist and McCarthy (1994) Giaume et al. (1991) Giaume et al. (1991) Blomstrand et al. (1999) Giaume et al. (1991) Giaume et al. (1992) Bolanos and Medina (1996) Enkvist and McCarthy (1994) Pappas et al. (1996) Vera et al. (1996)
GLT-1 is stimulated by phosphorylation by PKC, while GLAST is inhibited by PKC at a non-PKC consensus site (for reviews see Gegelashvili and Schousboe, 1997; Danbolt et al., 1998). We have shown that stimulation of various membrane receptors aects high-anity uptake kinetics. Glu uptake increased in the presence of the a1-adrenergic agonist phenylephrine, while b-adrenoreceptor activation slightly inhibited it (Hansson and RoÈnnbaÈck, 1991). Stimulation of receptors for endothelin, a highly potent vasoconstrictor with neuromodulatory properties, robustly decreases Glu uptake in cultured rat astrocytes (Fig. 3). This interaction between endothelin receptors and Glu transporters in astrocytes is of particular interest, as endothelin is known to be released at subarachnoid hemorrhage, a condition which entails raised extracellular Glu levels caused by a decrease in Glu uptake (Pluta et al., 1997). It should be kept in mind, however, that mechanisms other than the Glu transporters are important in cell-volume regulation. The net increase of intracellular
osmolarity caused by the in¯ux of Na+ and Glu has been proposed as one mechanism that could be responsible for glial swelling (Schneider et al., 1992). Inhibition of the Na+±K+-ATPase by ouabain, which leads to failure of the energy supply or of Na+ transport, prevents Glu-induced astroglial swelling (Bender et al., 1998). This relationship suggests that the Na+± K+-pump is also involved in the process of astroglial swelling (Kempski et al., 1988). This might also be the case with the Na+±K+ÿ2Clÿ co-transporter, or the opening of Clÿ channels, as well as with activation of the mGluRs (Staub et al., 1993; Hansson, 1994). Both extracellular and intracellular Ca2+ have been shown to in¯uence cell-volume regulation in dierent cell types (McCarthy and O'Neil, 1992; Koyoama et al., 1991). In addition to avoiding Glu excitotoxicity, astrocytic Glu transporters and cell-volume regulation might also play a role in the modulation of Glu-ergic synaptic transmission (see below). This is suggested by the complex interplay between the two subtypes of astroglial Glu transporters and the activation of iGluRs and mGluRs, and a multitude of factors that aect their activity and expression both spatially and temporally.
4. The astroglial gap-junction-coupled network and intercellular Ca2+ signalling
Fig. 3. Endothelin-induced decrease of glutamate uptake in astrocytes. [ET ÿ 1] = 10ÿ7 M, n = 4.
A major feature of astrocytes in vitro and in vivo is their extensive coupling by small pores, the so-called gap-junction channels. Astrocytic gap-junction channels are mainly composed of connexin-43, and they couple the cells into three-dimensional networks, the astroglial syncytia. The degree of gap-junction coupling depends on the number of channels expressed and on their probability of opening. The expression and distribution of connexin-43 is regulated developmentally and regionally and is dynamically remodelled by short- and long-term mechanisms (see Giaume and
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McCarthy, 1996). Various neurotransmitters and neuromodulators regulate the permeability of astrocytic gap-junction channels (Table 2). The physiological role for the astroglial syncytium and its plasticity is to a large extent unknown. The coupling permits inter-cytoplasmic exchanges of ions and small molecules (MW up to 1±1.2 kDa). Noteworthy here is the possibility for a spatial buering of K+ (Gardner-Medwin, 1983), and for a spatial distribution of neurotransmitters and metabolites such as Glu, glutamine, lactate and glucose (Tabernero et al., 1996; Giaume et al., 1997). Thus, controlled by various neuroactive substances (Table 2), astrocytes might act cooperatively to transfer metabolic substrates within the brain and between the circulation and brain cells. Also, with the aid of the uptake capacity and gap-junction permeability for Glu, the astroglial network may serve as a sink for neuronally released Glu. Furthermore, direct inter-astrocytic couplings, with the aid of gap-junction channels, seem to be one pathway for the propagation of intercellular Ca2+ waves (see Giaume and Venance, 1998). Such intercellular waves of increased [Ca2+]i can be elicited from a single astrocyte by electrical, mechanical, or chemical stimulation (e.g., by focal application of Glu). The roles of these Ca2+ waves have not yet been elucidated, but they might be a mechanism by which functional domains of astrocytes could modulate the extracellular homeostasis in a co-ordinated manner and thereby aect neuronal physiology and excitability (see Smith, 1992). In support of these ideas, recent experimental data from several independent groups show that astrocytes can respond to neuronal activity and signal back by means of Ca2+-dependent Glu release, thereby causing feedback regulation of neuronal activity (see Araque et al., 1999). Experimental data indicate that gap-junction communication is aected after an ischemic period. Increased immunoreactivity for connexin-43 is seen 2 days after mild ischemic injury in rat hippocampus and striatum, while decreased connexin-43 levels are seen after severe neuronal damage (Hossain et al., 1994a). Similarly, in kainic-acid lesioned brain tissue, astrocytic connexin-43 immunostaining is decreased at sites depleted of neurons (Hossain et al., 1994b). Metabolic inhibition of acutely isolated slices reduces but does not block the permeability of astrocytic gap junctions (Cotrina et al., 1998). Enhanced gap-junction coupling in astrocytes is seen in vitro by short-term incubations in 10, 23, or 55 mM K+ or 400 mM Glu (Table 2). Furthermore, inter-glial gap-junction permeability is increased by nerve impulses in the frog optic nerve (Marrero and Orkand, 1996). Gap-junction communication and intercellular Ca2+ signalling are increased in cells isolated and cultured from hyperexcitable human tissue (Lee et al., 1995) and there are in-
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dications of an increase in the level of connexin-43 mRNA in the temporal cortex of patients exhibiting seizure disorders (Naus et al., 1991). Such changes might re¯ect mechanisms for increased astroglial capacity of spatial buering in regions of high neurotransmission and in lesioned and vulnerable tissue. Decreased communication in irreversibly damaged regions may re¯ect a mechanism to limit the ¯ow of harmful metabolites through the network. In fact, it was recently shown in vitro that gap junctions can mediate propagation of cell injury (Lin et al., 1998), and that blockage of astroglial gap-junction communication reduced the infarct volume in a rat stroke model (Rawanduzy et al., 1997). 5. Astrocytes and glutamate in neurodysfunction After traumatic or ischemic injury to the CNS there is a pathological release of Glu from neurons and astrocytes, which plays a central role in mediating more extensive excitotoxic neuronal degeneration (Benveniste et al., 1984; Katayama et al., 1990). The increased extracellular Glu concentration activates an excitotoxic cascade as a consequence of uncontrolled activation of both iGluRs and mGluRs. The molecular mechanisms behind these toxic eects are not fully known, but involve intracellular Ca2+ ``overloading'' and a massive in¯ux of Na+. Ca2+ entry through both Glu-operated and voltage-operated Ca2+ channels, together with the release from intracellular stores, results in uncontrolled activation of neuronal protein kinases, phospholipases, proteases and nitric oxide synthase (NOS) (Hudspith, 1997). The consequent proteolysis, lipid peroxidation, and free-radical formation results in degeneration of central neurons. Although Glu-induced neuronal death may occur within minutes and a proportion of the neurons die in the acute phase of injury, a large number of neurons instead suer delayed death (Lynch and Dawson, 1994). Much interest has therefore arisen from the possibility of ameliorating excitotoxic neuronal damage by modi®cation of Glu release, GluR antagonism, or inhibition of subsequent proteolysis and lipid peroxidation (McIntosh et al., 1996; Berg-Johnsen et al., 1998). How do the astroglial cells interfere with or contribute to these mechanisms? Astrocytes are not merely static components of brain tissue; they are greatly responsive to external stimuli, and are capable of communicating with neurons and other glial cells through the extracellular space and intercellularly through gap junctions by modulating ion concentrations, Glu levels, substrates, and fuel for energy. These properties mean that astrocytes can respond to a wide variety of insults to the nervous system. The initial changes are closely related to their normal role, but subsequently astro-
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Fig. 4. Schematic drawing showing aspects on normal astroglial responses, preferentially GluRs, ion channels and intracellular Ca2+ responses to extracellular Glu. Possible events during cerebral glutamate overexcitation is shown to the right.
cytes may lose their ability to restore homeostasis and may trigger changes that can exacerbate the original injury and contribute to a decline of neurological function. This change means that potentially protective functions like Glu uptake, K+ buering, substrate production, and elimination of free radicals will be eventually reduced or reversed and might instead contribute to the development of neural damage. This reduction or reversal applies in particular to the
following functions involving Glu (1±4; see also Fig. 4): (1) Reduced uptake of Glu and reversed operation of Glu transporters, when intracellular ATP levels fall. The fall of brain ATP levels in anoxia or ischemia leads to slowing of the Na+/K+ pump and a rundown of the transmembrane gradients for [Na+], [K+] and membrane potential. This change leads to Glu transporters running backwards and releasing Glu into the extracellular space, where it activates GluRs and may
Fig. 5. Three preparations showing GFAP stained rat astrocytes from cerebral cortex: A. astrocytes in primary culture, B. an acute isolated astrocyte and C. astrocytes in a cortical brain slice.
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Fig. 6. Schematic drawing which indicates alterations in astroglial capacities for Glu uptake, gap-junction permeability and cell volume at two conditions involving increased extracellular glutamate levels ([Glu]e).
contribute to neuronal death (Takahashi et al., 1997). (2) Astroglial swelling is an early response to an acute injury (see above). Swelling is due to intracellular accumulation of osmoles such as Glu and K+ with a secondary in¯ux of water, and re¯ects the attempts of the astrocytes to remove toxic compounds from the extracellular space (Landis, 1994). If the injury persists, astrocytes may be unable to cope with the excessive production of metabolites and several potentially neurotoxic events may occur. First, the neural tissue swells at the site of injury, which leads to an increase of intra-cerebral pressure and reduction of vascular perfusion. Second, this swelling may increase the release of Glu in the extracellular space, which contributes to excitotoxicity by the activation of NMDA receptors
(Kimelberg et al., 1990). The reduction of extracellular space can also alter the ion concentration, especially K+ and Ca2+, which can eect neuronal excitability. (3) Several results show that astroglial Ca2+ waves may play a role in spreading depression (SD) (Nedergaard, 1994). Ischemic insult can induce SD waves and the extent of the resultant damage increases with each incidence of SD. Furthermore, it has been demonstrated that elevations in astroglial Ca2+ induce elevation of [Ca2+]i in neighbouring neurons (e.g., Parpura et al., 1994; Nedergaard, 1994). One possible explanation for this eect is that Glu is released from the astrocytes as a consequence of the elevated [Ca2+]i. Glu then depolarises adjacent neurons and activates neuronal NMDA receptors. Such a mechan-
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ism is consistent with the ability of a propagating astroglial Ca2+ wave to induce propagating neuronal depolarisation, similar to that which is observed in SD. (4) Astrocytes have the ability to generate nitric oxide (NO) and peroxynitrite (ONOOÿ) without cell death occurring (Heales et al., 1999). For that reason it is possible that in certain neurological disorders, astroglial-derived NO/ONOOÿ may cause mitochondrial damage to susceptible target cells, such as neurons. Exposure to Glu and various cytokines leads to an induction of NOS and marked generation of NO and ONOOÿ (formed from the reaction between NO and superoxide). Mitochondrial damage may occur in the astrocytes, but ATP levels are maintained as a result of the increase in glycolytic ¯ux. Further cellular damage is minimised due to relatively high glutathione (GSH) concentrations. Diusion of NO and/or ONOOÿ into neighbouring neurons may, if the cellular concentration of GSH is low, lead to mitochondrial damage. Since neurons are unable to compensate by increasing glycolysis, a cellular energy-de®ciency state ensues that may lead ultimately to cell death. Thus, non-neuronal cells, in particular, astrocytes, may contribute to neuronal damage and also modify neuronal responses to Glu excitotoxicity. This might lead to a new conception of how neural dysfunction evolves during and after dierent kinds of injury in the CNS. 6. Concluding remarks: do astroglia play an active role in glutamate handling even in vivo? As described in this paper, many studies have proven the eects of GluR activation on astrocytes. However, it is important to emphasize that most of the data on astrocyte properties that have accumulated during the last two decades have been obtained in cellculture studies. It can be questioned whether the ®ndings on membrane receptors, ion channel expression, and uptake mechanisms are artifacts of an arti®cial system or are representative of the in vivo cells. For example, it has been proposed that the cultures re¯ect reactive astrocytes that have been selected during isolation and cultivation more than they express properties of astrocytes in vivo. To progress in these aspects of astrocyte research, eorts have been made during the last few years to study astrocytes in model systems, such as acutely isolated cells and brain slices, which are preparations that can be considered to have a high level of concordance with the in vivo cells (Fig. 5). With the anatomical location of the astrocytes between the blood vessels and the neurons and their expression, both in vitro and in vivo, of functional membrane receptors, ion channels and aminoacid transporters (see above), it seems possible that
the cells sense synaptic activities from many synapses simultaneously. Furthermore, the cells have the ability to register the composition of the extracellular milieu and also the serum composition concerning neuroactive substances. These conditions may aect the functional status of the cell network for membrane potential, gap-junction status, and Ca2+ signalling. In this way, the Glu-uptake capacity can be regulated not only at the single-cell level, but it can also be synchronized within multicellular systems. The eciency of the K+ uptake and K+ buering abilities may be regulated in similar ways, as may the cell volume and concentrations of substances in the extracellular space. In this way, astroglia can interact with and support the neurons by achieving a high signal-to-noise ratio for the Glu transmission. To complete the picture of astroglial functions in neuronal circuits and in neuronal information processing, the roles of astroglia as providers of neurotrophic factors and energy to the neurons, and as partners to the neurons for the regulation of the local micro-circulation, should be taken into consideration. Recently, more direct experimental evidence was presented for functional Glu-ergic interactions between astrocytes and neurons in vivo. Astrocytes in brain slices were found to potentiate inhibitory synaptic transmission in a process involving activation of iGluRs (Kang et al., 1998), and synaptic activity was shown to rapidly evoke Glu-transporter currents, which indicates an uptake of considerable amounts of synaptically released neurotransmitter into astrocytes (Bergles and Craig, 1997). From the information available, it is thus tempting to postulate that the astroglial network could be a cell syncytium that interlinks the neuronal networks, with the capacity, depending on the requirements of neuronal activity, not only to support passively, but to act as a ®ne tuner to optimize the quality of the neuronal message in normal transmission. In fact, all data obtained during the recent years indicate that astrocytes have active roles in the Glu-ergic neurotransmission. In the human nervous system, one might expect to ®nd that the astroglial network interacts with neuronal circuits in higher brain functions in normal physiological situations. In pathology, knowledge of astroglial cell properties might be used to develop new pharmacological strategies to exert neuroprotective treatment (Fig. 6).
Acknowledgements The skilful work by Ulrika Johansson and Barbro Eriksson is greatly appreciated. The work was supported by the Swedish Medical Research Council (pro-
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Astroglia and glutamate in physiology and pathology: aspects on glutamate transport, glutamate-induced cell swelling and gapjunction communication Elisabeth Hansson*, HaÊkan Muyderman, Julia Leonova, Louise Allansson, Jon Sinclair, Fredrik Blomstrand, Thorleif Thorlin, Michael Nilsson, Lars RoÈnnbaÈck Institute of Clinical Neuroscience, GoÈteborg University, Box 420, S-40530 GoÈteborg, Sweden Received 22 June 1999; accepted 31 August 1999
Abstract Astroglia have the capacity to monitor extracellular glutamate (Glu) and maintain it at low levels, metabolize Glu, or release it back into the extracellular space. Glu can induce an increase in astroglial cell volume with a resulting decrease of the extracellular space, and thereby alter the concentration of extracellular substances. Many lines of evidence show that K+ can be buered within the astroglial gap-junction-coupled network, and recent results show that gap junctions are permeable for Glu. All these events occur dynamically: the astroglial network has the capacity to interfere actively with neurotransmission, thereby contributing to a high signal-to-noise ratio for the Glu transmission. High-quality neuronal messages during normal physiology can then be maintained. With the same mechanisms, astroglia might exert a neuroprotective function in situations of moderately increased extracellular Glu concentrations, i.e., corresponding to conditions of pathological hyper-excitability, or corresponding to early stages of an acute brain injury. If the astroglial functions are failing, neuronal dysfunction can be reinforced. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Astroglia; Glutamate; Glutamate receptors; Glutamate transport; Cell volume; Glial network
1. Introduction Recent experimental data support the view that astroglia and neurons are intimate partners in glutamate (Glu) synaptic transmission (see Araque et al., 1999; Laming et al., 1998; Hansson and RoÈnnbaÈck, 1995; Hansson et al., 1997). Astroglia express functional ionotropic and metabotropic Glu receptors (iGluRs and mGluRs; SteinhaÈuser and Gallo, 1996), and thereby have the ability to monitor neuronally released Glu. With the aid of the Glu transporters, GLAST and GLT-1, the astroglia clear the extracellu* Corresponding author. Tel.: +46-31-7733363; fax: +46-317733330. E-mail address: [email protected] (E. Hansson).
lar space from Glu, in fact, they do so more eectively than the neurons do. Alterations in astroglial cell volume can in¯uence extracellular volume and thereby the concentrations of neuroactive substances, with eects on neuronal excitability. Astroglia respond to neuronal signals by intracellular Ca2+ increases with complex spatio-temporal patterns. Astroglia metabolize Glu and release glutamine back to the neurons to be transformed and recycled as a transmitter. Furthermore, astroglia can release Glu and probably signal back to neurons (Parpura et al., 1994; Nedergaard, 1994). Notably, it has been suggested that the functional status of the gap-junction-coupled astroglial network is important for the supportive and protective functions of astroglia. It is interesting that the functional status of the network has also been shown to be
0197-0186/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 1 9 7 - 0 1 8 6 ( 0 0 ) 0 0 0 3 3 - 4
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important in the development of acute brain damage (Cotrina et al., 1998). Astroglia can react dynamically by exerting supportive and protective functions in the handling of Glu in the brain, even if astroglial reactions during pathophysiological processes appear to be destructive. In this paper, we focus on those aspects of astroglial interactions with Glu that are important for the astroglial support of normal neurotransmission, as well as on astroglial reactions in pathological states of overexcitability and during the development of a brain injury. 2. Astroglia monitor extracellular glutamate levels 2.1. Glutamate receptors Glu is the major excitatory transmitter in the CNS and is released at the neuronal presynaptic terminal, and activates dierent receptor types at the postsynaptic terminal. Consequently, GluRs are regarded as one of the most important receptor groups, and they have been studied extensively throughout the years (SteinhaÈuser and Gallo, 1996). Classically, GluRs have been subdivided into two groups, iGluRs and mGluRs, on the basis of their pharmacological and electrophysiological properties (see Hollmann and Heinemann, 1994; Pin and Duvoisin, 1995). Metabotropic GluRs are G protein coupled and their activation elicits both electrical and metabolic events in glial cells. Today, eight cloned mGluRs are subdivided into three groups according to their amino-acid similarity, pharmacological pro®le, and signal-transduction mechanism (Nakanishi, 1992, 1994). In short, mGluR1 and 5 (group I) are positively coupled to phospholipase C (PLC) as well as to various K+ channels by a Go or Gq protein. Their activation results in massive phosphoinositide (PI) hydrolysis and membrane depolarisation, while group II (mGluR2±3) and III (mGluR4,6±8) are associated with ion channels and the inhibition of adenylate cyclase. The all iGluRs contain integrated, ligandgated, cationic ion channels assembled from several subunits. They are further subdivided into N-methyl-Daspartate (NMDA) receptors and non-NMDA [aamino-3-hydroxy-5-methyl-isoxazole-4-proprionic acid(AMPA) or kainate- (KA) preferring] receptors. Both the NMDA receptors and the AMPA or KA receptors open transmembrane cation channels that generate Na in¯ow, K+ eux, membrane depolarisation and/ or calcium in¯ow, depending on the subunit composition. Activation of the AMPA-preferring subunits results in a rapid, fast-desensitising depolarisation, while KA-receptor activation produces long-lasting, non-desensitisation responses (Jabs et al., 1994). Expression of GluRs have been shown in several glial-cell
Fig. 1. Astrocytes, acutely isolated and from primary cultures, have been shown to express functional glutamate receptors. A. Acutely isolated astrocyte from rat cerebral cortex at p14 shows increases in [Ca2+]i when stimulated with (1S,3R )-1-aminocyclopentane-1,3dicarboxylic acid (ACPD; 10ÿ4 M), a metabotropic glutamate receptor (mGluR) agonist. The response is blocked by the selective group 1 mGluR antagonist (RS)-1-aminoindan-1,5 dicarboxylic acid (AIDA; 10ÿ3 M). B. Corresponding curves from a cultured cortical astrocyte. C. Stimulation with the kainate receptor agonist kainic acid (KA; 10ÿ5 M) and blockade of the receptor with the antagonist CNQX (10ÿ4 M). Drugs were applied as indicated by the horizontal bars, and the cells were allowed to rest for 10±15 min between the stimulations. Changes in [Ca2+]i were determined by microspectro¯uorometry, by using the calciumsensitive probes ¯uo-3 (A) or fura2 (B,C). Data are presented as change in ¯uorescence (F ) from the resting value (F0), (F ÿ F0)/F0%, or as changes in [Ca2+]i.
Cationic channel opening Ca2+ in¯ow Membrane depolarisation Fast desensitising Cationic channel opening Ca2+ in¯ow Non-desensitising Membrane depolarisation. Cationic channel opening Ca2+ in¯ow
AMPA
Activating PLC, IP3-mediated Ca2+ release
Inhibitory action on adenylyl cyclase. Decreased cAMP production
Inhibitory action on adenylyl cyclase. Decreased cAMP production
MGluR I (1/5)
MGluR II (2±3)
MGluR III (4/6±8)
NMDA
Kainate
Mechanism
Receptor
Table 1 Characteristics of glutamate receptor subtypes
Gi/o-protein coupled
Gi/o-protein coupled
Gq-protein coupled
Ligand-gated ion channel
Ligand-gated ion channel
Ligand-gated ion channel
Coupling
(1S,3R )ACPD QA (1S,3R )ACPD DCG-IV (S )-3C4HPG L-CCG-I L-AP4 L-SOP
CHPG (R,S )-3,5-DHPG
NMDA Cis-ACBD
Kainic acid Domoic acid
AMPA Quisqualate (QA)
Agonists
CPPG MSOP MAP4
EGLU MCCG PCCG-IV)
D-AP5 MK-801 CNQX AIDA L-AP3 (S)-4-CPG
CNQX DNQX
CNQX DNQX
Antagonists
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preparations both in vitro and in situ (Seifert and SteinhaÈuser, 1995; Porter and McCarthy, 1997; Kimelberg et al., 1997; Thorlin et al., 1998). Many of the ion channels, i.e., voltage-gated K+, Na+, Clÿ, and Ca2+ channels, previously described in cell cultures, have lately been reported in acutely isolated astrocytes (Barres et al., 1990; Tse et al., 1992; Sontheimer and Waxman, 1993; Duy and Mac Vicar, 1994). Although both iGluRs and mGluRs are commonly found on astrocytes, the presence of NMDA receptors is still unclear. NMDA-mediated currents have been observed in a small population of astrocytes in the hippocampus, and in neocortical astrocytes, while the NMDA-receptor group has been found on astrocytes in the visual cortex by using subunit-speci®c antibodies (Aoki et al., 1994). Recently, Ziak et al. (1998) showed NMDA-evoked membrane currents in glial cells in rat spinal cord, but the physiological implications of functional NMDA receptors in glia remain unknown. In astrocytes, activation of GluRs elicits complex changes in intracellular calcium ([Ca2+]i) (Fig. 1), which is often composed of dierent spatio-temporal patterns in the form of intracellular waves and oscillations. Recently, both neuroprotective action (e.g., Bruno et al., 1998) and the modulation of synaptic activity (Araque et al., 1998) as mediated through glial mGluRs have been shown, indicating an extensive complexity in Glu-induced neuroglial interactions. It has also been shown that both iGluRs and mGluRs mediate the induction of immediate early genes (Condorelli et al., 1993). The ability of Glu to elicit cell-tocell signalling (Cornell-Bell et al., 1990), to induce volume changes (see below and Hansson, 1994), and to decrease Glu release from astrocytes (Ye and Sontheimer, 1999) indicates that glial GluRs might play a major role as participant in both physiological and pathophysiological events within the CNS. (For agonists and antagonists for the dierent Glu receptors, refer to Table 1).
3. Astroglia clear glutamate from the extracellular space 3.1. High-anity glutamate uptake and glutamatemediated astroglial cell swelling The intracellular concentrations of Glu are rather high: 50 mM in astrocytes, and as much as 10 mM in Glu-ergic neurons. However, the extracellular concentration has to be maintained at approximately 1±3 m M to assure a high signal-to-noise ratio at normal Glu-ergic neurotransmission and to avoid excitotoxic actions of Glu on neurons. The removal of Glu from the extracellular space is achieved by a high-anity uptake into the cells surrounding the synaptic cleft.
Astrocytes have been shown to play a dominant role in this process. Given that Glu clearance is of paramount importance for normal transmission and for preventing neuronal injury, the biochemical and pharmacological properties of the Glu uptake have been scrutinized by neuroscientists since a high-anity transport system for this amino acid was initially observed in the early 1970s (Logan and Snyder, 1971). Increases in extracellular Glu levels have also been implicated as an important factor in astroglial cell swelling (Schneider et al., 1992; Hansson and RoÈnnbaÈck, 1995). After a circulatory disturbance in the brain, the astrocytes in the aected area respond within a few minutes by increasing the cell volume. Because of the relatively small volume of the extracellular space, astroglial swelling leads to changes in the concentration of neuroactive substances therein, which, in turn, aects neuronal function. Depolarization of the astrocytes causes both a release of Glu and de®cits in the ability to take up Glu from the extracellular space, which may lead to abnormal Gluergic neurotransmission and to neurotoxicity (Norenberg, 1998). Astrocytic swelling could also limit the diusion of Glu from the lesion site (Maxwell et al., 1994). The increase in volume may, however, result in metabolic disarray in the injured tissue, which might be harmful for neurons and might lead to destruction of the astrocytes (Kimelberg et al., 1990). The swelling of astrocytes by Glu results from an active intracellular accumulation of the amino acid (Barbour et al., 1988; Kimelberg et al., 1989). The mechanisms underlying Glu-induced astroglial swelling have been studied by using dierent in vitro model systems, but are far from fully explored. To better understand the mechanisms regulating the cell volume in various cell types, it is important to develop technical instrumentation and analysis methods that are capable of detecting and characterising dynamic cell-shape changes in a quantitative way. We have developed a new method, on the basis of ¯uorescence microscopy in conjunction with advanced image analysis, for evaluating cell-volume changes in three-dimensions (3D) (Allansson et al., 1999). Fig. 2 demonstrates the principle with this method. It is suggested that the astroglial swelling is caused by an osmotically driven transmembrane transport of Glu coupled to the uptake of Na+ ions. The uptake of Glu occurs against a steep intracellular to extracellular concentration dierence in the astrocytes, which is energy consuming, and the transport is driven by the electrochemical gradient for Na+ (Barbour et al., 1988; Drejer et al., 1983). Thus, there is a relation between Glu transport over the cell membrane and cell volume changes. We will discuss the aspects of the mechanisms behind each of
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these two processes and also suggest probable connections between them. Recent evidence suggests that one Glu is co-transported with three Na+ ions and one H+, while one K+ ion is counter-transported to the exterior (Zerangue and Kavanaugh, 1996). Five distinct transporters have been cloned so far: GLAST, GLT-1, EAAC1, EAAT4 and EAAT5. The cloned transporters exhibit distinct patterns of distribution in dierent brain regions and cell types. GLAST and GLT-1 are considered to be glia-speci®c Glu transporters (for details on tissue and cell distribution of Glu transporters, see Gegelashvili and Schousboe, 1998; Danbolt et al., 1998). Measuring the accumulation of a radio-labelled amino acid in the cells sheds light on the kinetics of Glu-transport activity. Depending on the kind of cellular preparation that is studied, and the brain region, the Vmax varies between 2 and 60 nmol/min/mg protein at 37;8C, and the Km varies between 3 and 80 mM (for a summary of the pharmacological and kinetic properties of Glu uptake, see Erecinska and Silver, 1990; Robinson and Dowd, 1997). Though both GLT-1 and GLAST are almost exclusively found in astrocytes, their patterns of expression are dierentially regulated and regionally and developmentally segregated. GLT-1 is expressed at the highest concentrations in the hippocampus and cerebral cortex, while GLAST is preferentially expressed in the molecular layer of the cerebellum (Lehre et al., 1995). It is noteworthy that, in adult brain, astrocytic membranes that face nerve terminals, axons, and spines have a denser population of Glu transporters than
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those facing capillaries, pia, or stem dendrites (Chaudhry et al., 1995), which is consistent with the importance of astrocytic carriers for removal of Glu from the extracellular space after synaptic transmission. The Glu transporters are probably related to the regulation of the cell volume, as the Glu-induced astroglial cell swelling can be at least partially blocked by the Glu-carrier blocker PDC (Hansson, 1994); it is probable that these two processes are linked. The major role of astrocytic transporters in Glu clearance and in preventing neurodegeneration was unequivocally demonstrated by the in vivo application of the antisense oligonucleotid technique, a method that permits selective inhibition of synthesis of dierent Glu carriers. The loss of the glial transporters GLT-1 and GLAST in rat produced elevated extracellular Glu levels, neurodegeneration characteristic of excitotoxicity, and a progressive paralysis. Failure of Glu uptake has also been demonstrated in ischemia, ALS and traumatic brain injury. The resulting increase in extracellular Glu levels has been considered an important factor in neuronal damage in these conditions (Raghavendra Rao et al., 1998), whereas the loss of the neuronal Glu transporter EAAC1 did not elevate the extracellular levels of Glu (Rothstein et al., 1996). Possible mechanisms for aecting Glu transporters in the regulation of extracellular Glu levels and thereby also regulating the cell volume and extracellular space volume are important. A large number of factors have been shown to aect the activity and expression of Glu transporters (Table 2). Importantly, the activity of dierent Glu transporters may be regulated dierentially by the same eectors. For example,
Fig. 2. The ®gure to the left demonstrates the principle for optical sectioning of ¯uorescent labelled astroglial cells (a). Data is collected from dierent focus planes (b), resulting in a stack of 2D images (c). The 2D images are reconstructed, and an edge detection algorithm is applied for volume estimation. The right ®gure presents the total cell volume, as well as the slice area for each 2D image in one astroglial cell. 1 = control, 2 = swollen cell.
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Table 2 Eects on astroglial glutamate (Glu) uptake capacity and gap junction permeability (GJ) caused by dierent substances or changed conditions Substance/condition
Eects on Glu uptake
Reference
Eects on GJ
Reference
Glutamate Kainate Phenylephrine or methoxamine (a1) Isoproterenol (b) 5-HT Arachidonic acid Endothelin-1 Oxygen free radicals Depolarization pH (acidosis) Energy status (ATP levels q)
Q Q Q q n.e q q q q q q
Gegelashvili et al. (1996) Gegelashvili et al. (1996) Hansson and RoÈnnbaÈck (1991) Hansson and RoÈnnbaÈck (1991) Hansson and RoÈnnbaÈck (1991) Barbour et al. (1989) unpublished results Volterra et al. (1994) Barbour et al. (1988) Swanson et al. (1995) Swanson (1992)
Q Q q Q Q or q q q q Q q q
Enkvist and McCarthy (1994) Enkvist and McCarthy (1994) Giaume et al. (1991) Giaume et al. (1991) Blomstrand et al. (1999) Giaume et al. (1991) Giaume et al. (1992) Bolanos and Medina (1996) Enkvist and McCarthy (1994) Pappas et al. (1996) Vera et al. (1996)
GLT-1 is stimulated by phosphorylation by PKC, while GLAST is inhibited by PKC at a non-PKC consensus site (for reviews see Gegelashvili and Schousboe, 1997; Danbolt et al., 1998). We have shown that stimulation of various membrane receptors aects high-anity uptake kinetics. Glu uptake increased in the presence of the a1-adrenergic agonist phenylephrine, while b-adrenoreceptor activation slightly inhibited it (Hansson and RoÈnnbaÈck, 1991). Stimulation of receptors for endothelin, a highly potent vasoconstrictor with neuromodulatory properties, robustly decreases Glu uptake in cultured rat astrocytes (Fig. 3). This interaction between endothelin receptors and Glu transporters in astrocytes is of particular interest, as endothelin is known to be released at subarachnoid hemorrhage, a condition which entails raised extracellular Glu levels caused by a decrease in Glu uptake (Pluta et al., 1997). It should be kept in mind, however, that mechanisms other than the Glu transporters are important in cell-volume regulation. The net increase of intracellular
osmolarity caused by the in¯ux of Na+ and Glu has been proposed as one mechanism that could be responsible for glial swelling (Schneider et al., 1992). Inhibition of the Na+±K+-ATPase by ouabain, which leads to failure of the energy supply or of Na+ transport, prevents Glu-induced astroglial swelling (Bender et al., 1998). This relationship suggests that the Na+± K+-pump is also involved in the process of astroglial swelling (Kempski et al., 1988). This might also be the case with the Na+±K+ÿ2Clÿ co-transporter, or the opening of Clÿ channels, as well as with activation of the mGluRs (Staub et al., 1993; Hansson, 1994). Both extracellular and intracellular Ca2+ have been shown to in¯uence cell-volume regulation in dierent cell types (McCarthy and O'Neil, 1992; Koyoama et al., 1991). In addition to avoiding Glu excitotoxicity, astrocytic Glu transporters and cell-volume regulation might also play a role in the modulation of Glu-ergic synaptic transmission (see below). This is suggested by the complex interplay between the two subtypes of astroglial Glu transporters and the activation of iGluRs and mGluRs, and a multitude of factors that aect their activity and expression both spatially and temporally.
4. The astroglial gap-junction-coupled network and intercellular Ca2+ signalling
Fig. 3. Endothelin-induced decrease of glutamate uptake in astrocytes. [ET ÿ 1] = 10ÿ7 M, n = 4.
A major feature of astrocytes in vitro and in vivo is their extensive coupling by small pores, the so-called gap-junction channels. Astrocytic gap-junction channels are mainly composed of connexin-43, and they couple the cells into three-dimensional networks, the astroglial syncytia. The degree of gap-junction coupling depends on the number of channels expressed and on their probability of opening. The expression and distribution of connexin-43 is regulated developmentally and regionally and is dynamically remodelled by short- and long-term mechanisms (see Giaume and
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McCarthy, 1996). Various neurotransmitters and neuromodulators regulate the permeability of astrocytic gap-junction channels (Table 2). The physiological role for the astroglial syncytium and its plasticity is to a large extent unknown. The coupling permits inter-cytoplasmic exchanges of ions and small molecules (MW up to 1±1.2 kDa). Noteworthy here is the possibility for a spatial buering of K+ (Gardner-Medwin, 1983), and for a spatial distribution of neurotransmitters and metabolites such as Glu, glutamine, lactate and glucose (Tabernero et al., 1996; Giaume et al., 1997). Thus, controlled by various neuroactive substances (Table 2), astrocytes might act cooperatively to transfer metabolic substrates within the brain and between the circulation and brain cells. Also, with the aid of the uptake capacity and gap-junction permeability for Glu, the astroglial network may serve as a sink for neuronally released Glu. Furthermore, direct inter-astrocytic couplings, with the aid of gap-junction channels, seem to be one pathway for the propagation of intercellular Ca2+ waves (see Giaume and Venance, 1998). Such intercellular waves of increased [Ca2+]i can be elicited from a single astrocyte by electrical, mechanical, or chemical stimulation (e.g., by focal application of Glu). The roles of these Ca2+ waves have not yet been elucidated, but they might be a mechanism by which functional domains of astrocytes could modulate the extracellular homeostasis in a co-ordinated manner and thereby aect neuronal physiology and excitability (see Smith, 1992). In support of these ideas, recent experimental data from several independent groups show that astrocytes can respond to neuronal activity and signal back by means of Ca2+-dependent Glu release, thereby causing feedback regulation of neuronal activity (see Araque et al., 1999). Experimental data indicate that gap-junction communication is aected after an ischemic period. Increased immunoreactivity for connexin-43 is seen 2 days after mild ischemic injury in rat hippocampus and striatum, while decreased connexin-43 levels are seen after severe neuronal damage (Hossain et al., 1994a). Similarly, in kainic-acid lesioned brain tissue, astrocytic connexin-43 immunostaining is decreased at sites depleted of neurons (Hossain et al., 1994b). Metabolic inhibition of acutely isolated slices reduces but does not block the permeability of astrocytic gap junctions (Cotrina et al., 1998). Enhanced gap-junction coupling in astrocytes is seen in vitro by short-term incubations in 10, 23, or 55 mM K+ or 400 mM Glu (Table 2). Furthermore, inter-glial gap-junction permeability is increased by nerve impulses in the frog optic nerve (Marrero and Orkand, 1996). Gap-junction communication and intercellular Ca2+ signalling are increased in cells isolated and cultured from hyperexcitable human tissue (Lee et al., 1995) and there are in-
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dications of an increase in the level of connexin-43 mRNA in the temporal cortex of patients exhibiting seizure disorders (Naus et al., 1991). Such changes might re¯ect mechanisms for increased astroglial capacity of spatial buering in regions of high neurotransmission and in lesioned and vulnerable tissue. Decreased communication in irreversibly damaged regions may re¯ect a mechanism to limit the ¯ow of harmful metabolites through the network. In fact, it was recently shown in vitro that gap junctions can mediate propagation of cell injury (Lin et al., 1998), and that blockage of astroglial gap-junction communication reduced the infarct volume in a rat stroke model (Rawanduzy et al., 1997). 5. Astrocytes and glutamate in neurodysfunction After traumatic or ischemic injury to the CNS there is a pathological release of Glu from neurons and astrocytes, which plays a central role in mediating more extensive excitotoxic neuronal degeneration (Benveniste et al., 1984; Katayama et al., 1990). The increased extracellular Glu concentration activates an excitotoxic cascade as a consequence of uncontrolled activation of both iGluRs and mGluRs. The molecular mechanisms behind these toxic eects are not fully known, but involve intracellular Ca2+ ``overloading'' and a massive in¯ux of Na+. Ca2+ entry through both Glu-operated and voltage-operated Ca2+ channels, together with the release from intracellular stores, results in uncontrolled activation of neuronal protein kinases, phospholipases, proteases and nitric oxide synthase (NOS) (Hudspith, 1997). The consequent proteolysis, lipid peroxidation, and free-radical formation results in degeneration of central neurons. Although Glu-induced neuronal death may occur within minutes and a proportion of the neurons die in the acute phase of injury, a large number of neurons instead suer delayed death (Lynch and Dawson, 1994). Much interest has therefore arisen from the possibility of ameliorating excitotoxic neuronal damage by modi®cation of Glu release, GluR antagonism, or inhibition of subsequent proteolysis and lipid peroxidation (McIntosh et al., 1996; Berg-Johnsen et al., 1998). How do the astroglial cells interfere with or contribute to these mechanisms? Astrocytes are not merely static components of brain tissue; they are greatly responsive to external stimuli, and are capable of communicating with neurons and other glial cells through the extracellular space and intercellularly through gap junctions by modulating ion concentrations, Glu levels, substrates, and fuel for energy. These properties mean that astrocytes can respond to a wide variety of insults to the nervous system. The initial changes are closely related to their normal role, but subsequently astro-
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Fig. 4. Schematic drawing showing aspects on normal astroglial responses, preferentially GluRs, ion channels and intracellular Ca2+ responses to extracellular Glu. Possible events during cerebral glutamate overexcitation is shown to the right.
cytes may lose their ability to restore homeostasis and may trigger changes that can exacerbate the original injury and contribute to a decline of neurological function. This change means that potentially protective functions like Glu uptake, K+ buering, substrate production, and elimination of free radicals will be eventually reduced or reversed and might instead contribute to the development of neural damage. This reduction or reversal applies in particular to the
following functions involving Glu (1±4; see also Fig. 4): (1) Reduced uptake of Glu and reversed operation of Glu transporters, when intracellular ATP levels fall. The fall of brain ATP levels in anoxia or ischemia leads to slowing of the Na+/K+ pump and a rundown of the transmembrane gradients for [Na+], [K+] and membrane potential. This change leads to Glu transporters running backwards and releasing Glu into the extracellular space, where it activates GluRs and may
Fig. 5. Three preparations showing GFAP stained rat astrocytes from cerebral cortex: A. astrocytes in primary culture, B. an acute isolated astrocyte and C. astrocytes in a cortical brain slice.
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Fig. 6. Schematic drawing which indicates alterations in astroglial capacities for Glu uptake, gap-junction permeability and cell volume at two conditions involving increased extracellular glutamate levels ([Glu]e).
contribute to neuronal death (Takahashi et al., 1997). (2) Astroglial swelling is an early response to an acute injury (see above). Swelling is due to intracellular accumulation of osmoles such as Glu and K+ with a secondary in¯ux of water, and re¯ects the attempts of the astrocytes to remove toxic compounds from the extracellular space (Landis, 1994). If the injury persists, astrocytes may be unable to cope with the excessive production of metabolites and several potentially neurotoxic events may occur. First, the neural tissue swells at the site of injury, which leads to an increase of intra-cerebral pressure and reduction of vascular perfusion. Second, this swelling may increase the release of Glu in the extracellular space, which contributes to excitotoxicity by the activation of NMDA receptors
(Kimelberg et al., 1990). The reduction of extracellular space can also alter the ion concentration, especially K+ and Ca2+, which can eect neuronal excitability. (3) Several results show that astroglial Ca2+ waves may play a role in spreading depression (SD) (Nedergaard, 1994). Ischemic insult can induce SD waves and the extent of the resultant damage increases with each incidence of SD. Furthermore, it has been demonstrated that elevations in astroglial Ca2+ induce elevation of [Ca2+]i in neighbouring neurons (e.g., Parpura et al., 1994; Nedergaard, 1994). One possible explanation for this eect is that Glu is released from the astrocytes as a consequence of the elevated [Ca2+]i. Glu then depolarises adjacent neurons and activates neuronal NMDA receptors. Such a mechan-
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ism is consistent with the ability of a propagating astroglial Ca2+ wave to induce propagating neuronal depolarisation, similar to that which is observed in SD. (4) Astrocytes have the ability to generate nitric oxide (NO) and peroxynitrite (ONOOÿ) without cell death occurring (Heales et al., 1999). For that reason it is possible that in certain neurological disorders, astroglial-derived NO/ONOOÿ may cause mitochondrial damage to susceptible target cells, such as neurons. Exposure to Glu and various cytokines leads to an induction of NOS and marked generation of NO and ONOOÿ (formed from the reaction between NO and superoxide). Mitochondrial damage may occur in the astrocytes, but ATP levels are maintained as a result of the increase in glycolytic ¯ux. Further cellular damage is minimised due to relatively high glutathione (GSH) concentrations. Diusion of NO and/or ONOOÿ into neighbouring neurons may, if the cellular concentration of GSH is low, lead to mitochondrial damage. Since neurons are unable to compensate by increasing glycolysis, a cellular energy-de®ciency state ensues that may lead ultimately to cell death. Thus, non-neuronal cells, in particular, astrocytes, may contribute to neuronal damage and also modify neuronal responses to Glu excitotoxicity. This might lead to a new conception of how neural dysfunction evolves during and after dierent kinds of injury in the CNS. 6. Concluding remarks: do astroglia play an active role in glutamate handling even in vivo? As described in this paper, many studies have proven the eects of GluR activation on astrocytes. However, it is important to emphasize that most of the data on astrocyte properties that have accumulated during the last two decades have been obtained in cellculture studies. It can be questioned whether the ®ndings on membrane receptors, ion channel expression, and uptake mechanisms are artifacts of an arti®cial system or are representative of the in vivo cells. For example, it has been proposed that the cultures re¯ect reactive astrocytes that have been selected during isolation and cultivation more than they express properties of astrocytes in vivo. To progress in these aspects of astrocyte research, eorts have been made during the last few years to study astrocytes in model systems, such as acutely isolated cells and brain slices, which are preparations that can be considered to have a high level of concordance with the in vivo cells (Fig. 5). With the anatomical location of the astrocytes between the blood vessels and the neurons and their expression, both in vitro and in vivo, of functional membrane receptors, ion channels and aminoacid transporters (see above), it seems possible that
the cells sense synaptic activities from many synapses simultaneously. Furthermore, the cells have the ability to register the composition of the extracellular milieu and also the serum composition concerning neuroactive substances. These conditions may aect the functional status of the cell network for membrane potential, gap-junction status, and Ca2+ signalling. In this way, the Glu-uptake capacity can be regulated not only at the single-cell level, but it can also be synchronized within multicellular systems. The eciency of the K+ uptake and K+ buering abilities may be regulated in similar ways, as may the cell volume and concentrations of substances in the extracellular space. In this way, astroglia can interact with and support the neurons by achieving a high signal-to-noise ratio for the Glu transmission. To complete the picture of astroglial functions in neuronal circuits and in neuronal information processing, the roles of astroglia as providers of neurotrophic factors and energy to the neurons, and as partners to the neurons for the regulation of the local micro-circulation, should be taken into consideration. Recently, more direct experimental evidence was presented for functional Glu-ergic interactions between astrocytes and neurons in vivo. Astrocytes in brain slices were found to potentiate inhibitory synaptic transmission in a process involving activation of iGluRs (Kang et al., 1998), and synaptic activity was shown to rapidly evoke Glu-transporter currents, which indicates an uptake of considerable amounts of synaptically released neurotransmitter into astrocytes (Bergles and Craig, 1997). From the information available, it is thus tempting to postulate that the astroglial network could be a cell syncytium that interlinks the neuronal networks, with the capacity, depending on the requirements of neuronal activity, not only to support passively, but to act as a ®ne tuner to optimize the quality of the neuronal message in normal transmission. In fact, all data obtained during the recent years indicate that astrocytes have active roles in the Glu-ergic neurotransmission. In the human nervous system, one might expect to ®nd that the astroglial network interacts with neuronal circuits in higher brain functions in normal physiological situations. In pathology, knowledge of astroglial cell properties might be used to develop new pharmacological strategies to exert neuroprotective treatment (Fig. 6).
Acknowledgements The skilful work by Ulrika Johansson and Barbro Eriksson is greatly appreciated. The work was supported by the Swedish Medical Research Council (pro-
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