Neuron-astrocyte cross-talk during synaptic transmission: physiological and neuropathological implications.
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Animals↗
Biomedical subjects
A Volterra
Publications and source records attributed to A Volterra.
A neuron-glia signalling network in the active brain.
Glial cells are active partners of neurons in processing information and synaptic integration. They receive coded signals from synapses and elaborate modulatory responses. The active properties of glia, including long-range signalling and regulated transmitter release, are beginning to be elucidated. Recent insights suggest that the active brain should no longer be regarded as a circuitry of neuronal contacts, but as an integrated network of interactive neurons and glia.
Action Potentials↗
CXCR4-activated astrocyte glutamate release via TNFalpha: amplification by microglia triggers neurotoxicity.
Astrocytes actively participate in synaptic integration by releasing transmitter (glutamate) via a calcium-regulated, exocytosis-like process. Here we show that this process follows activation of the receptor CXCR4 by the chemokine stromal cell-derived factor 1 (SDF-1). An extraordinary feature of the ensuing signaling cascade is the rapid extracellular release of tumor necrosis factor-alpha (TNFalpha). Autocrine/paracrine TNFalpha-dependent signaling leading to prostaglandin (PG) formation not only controls glutamate release and astrocyte communication, but also causes their derangement when activated microglia cooperate to dramatically enhance release of the cytokine in response to CXCR4 stimulation. We demonstrate that altered glial communication has direct neuropathological consequences and that agents interfering with CXCR4-dependent astrocyte-microglia signaling prevent neuronal apoptosis induced by the HIV-1 coat glycoprotein, gp120IIIB. Our results identify a new pathway for glia-glia and glia-neuron communication that is relevant to both normal brain function and neurodegenerative diseases.
Animals↗
Synaptic transmission with the glia.
For decades, scientists thought that all of the missing secrets of brain function resided in neurons. However, a wave of new findings indicates that glial cells, formerly considered mere supporters and subordinate to neurons, participate actively in synaptic integration and processing of information in the brain.
Calcium Signaling↗
Inhibitory effect of the neuroprotective agent idebenone on arachidonic acid metabolism in astrocytes.
Idebenone, a compound with protective efficacy against neurotoxicity both in in vitro and in in vivo models, exists in two different oxidative states: the ubiquinol-derivative (reduced idebenone) and the ubiquinone-derivative (oxidised idebenone). In the present study, we have observed that both the redox forms of idebenone have a dose-dependent inhibitory effect on the enzymatic metabolism of arachidonic acid in astroglial homogenates (IC50 reduced idebenone: 1.76 +/- 0.86 microM; IC50 oxidised idebenone: 16.65 +/- 3.48 microM), while in platelets, they are apparently less effective (IC50 reduced idebenone: 18.28 +/- 4.70 microM; IC50 oxidised idebenone: > 1 mM). We have also observed that the oxidised form preferentially inhibited cyclooxygenase vs. lipoxygenase metabolism (IC50 ratio lipoxygenase/cyclooxygenase: 3.22), while the reduced form did not discriminate between the two pathways (IC50 ratio lipoxygenase/cyclooxygenase: 1.38). In this respect, the inhibitory action of reduced idebenone resembled that of the antioxidant nordihydroguaiaretic acid, while oxidised idebenone behaved similarly as indomethacin and piroxicam--two typical anti-inflammatory agents. Our results suggest the existence of two distinct mechanisms of action for the two redox forms of idebenone and a preferential action of the drug on arachidonic acid metabolism in the central nervous system.
Animals↗
Astrocytes as active participants of glutamatergic function and regulators of its homeostasis.
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ATP-Binding Cassette Transporters↗
The active role of astrocytes in synaptic transmission.
In the central nervous system, astrocytes form an intimately connected network with neurons, and their processes closely enwrap synapses. The critical role of these cells in metabolic and trophic support to neurons, ion buffering and clearance of neurotransmitters is well established. However, recent accumulating evidence suggests that astrocytes are active partners of neurons in additional and more complex functions. In particular, astrocytes express a repertoire of neurotransmitter receptors mirroring that of neighbouring synapses. Such receptors are stimulated during synaptic activity and start calcium signalling into the astrocyte network. Intracellular oscillations and intercellular calcium waves represent the astrocyte's own form of excitability, as they trigger release of transmitter (i.e. glutamate) via a novel process sensitive to blockers of exocytosis and involving cyclooxygenase eicosanoids. Astrocyte-released glutamate activates receptors on the surrounding neurons and modifies their electrical and intracellular calcium ([Ca2+]i) state. These exciting new findings reveal an active participation of astrocytes in synaptic transmission and the involvement of neuronastrocyte circuits in the processing of information in the brain.
Animals↗
The highly integrated dialogue between neurons and astrocytes in brain function.
For decades neurons have been regarded as the only cells involved in the generation and control of brain signalling, while the surrounding glia was supposed to provide structural and metabolic support to neuronal function. However, based on a number of recent findings, a new view is emerging: astrocytes, the glial cells ensheathing synaptic specializations, are active and integrated participants of neurotransmission. Not only do astrocytes take up and remove synaptically released glutamate (the major excitatory neurotransmitter), thus ending its stimulatory action and preventing neuronal damage, but also and outstandingly, they are able to undergo rapid bidirectional communication with neurons, based on reciprocal glutamatergic signalling. Thus, release of glutamate from synaptic terminals, in addition to postsynaptic neurons, turns on the astrocytes nearby which respond by liberating the same neurotransmitter via a novel Ca(2+)-dependent mechanism and thereby signal back to neurons. The present review discusses the above findings and their important implications as well as additional evidence supporting the new concept of an integrated neuron-astrocyte communication in brain function.
Animals↗
Prostaglandins stimulate calcium-dependent glutamate release in astrocytes.
Astrocytes in the brain form an intimately associated network with neurons. They respond to neuronal activity and synaptically released glutamate by raising intracellular calcium concentration ([Ca2+]i), which could represent the start of back-signalling to neurons. Here we show that coactivation of the AMPA/kainate and metabotropic glutamate receptors (mGluRs) on astrocytes stimulates these cells to release glutamate through a Ca2+-dependent process mediated by prostaglandins. Pharmacological inhibition of prostaglandin synthesis prevents glutamate release, whereas application of prostaglandins (in particular PGE2) mimics and occludes the releasing action of GluR agonists. PGE2 promotes Ca2+-dependent glutamate release from cultured astrocytes and also from acute brain slices under conditions that suppress neuronal exocytotic release. When applied to the CA1 hippocampal region, PGE2 induces increases in [Ca2+]i both in astrocytes and in neurons. The [Ca2+]i increase in neurons is mediated by glutamate released from astrocytes, because it is abolished by GluR antagonists. Our results reveal a new pathway of regulated transmitter release from astrocytes and outline the existence of an integrated glutamatergic cross-talk between neurons and astrocytes in situ that may play critical roles in synaptic plasticity and in neurotoxicity.
Animals↗
Glutamate transporters are oxidant-vulnerable: a molecular link between oxidative and excitotoxic neurodegeneration?
Increasing evidence indicates that glutamate transporters are vulnerable to the action of biological oxidants, resulting in reduced uptake function. This effect could contribute to the build-up of neurotoxic extracellular glutamate levels, with major pathological consequences. Specific 'redox-sensing' elements, consisting of cysteine residues, have been identified in the structures of at least three transporter subtypes (GLT1, GLAST and EAAC1) and shown to regulate transport rate via thiol-disulphide redox interconversion. In this article, Davide Trotti, Niels Danbolt and Andrea Volterra discuss these findings in relation to the emerging view that in brain diseases oxidative and excitotoxic mechanisms might often operate in tight conjunction to induce neuronal damage. In particular, they review evidence suggesting a possible involvement of oxidative alterations of glutamate transporters in specific pathologies, including amyotrophic lateral sclerosis, Alzheimer's disease, brain trauma and ischaemia.
Animals↗
Intracellular calcium oscillations in astrocytes: a highly plastic, bidirectional form of communication between neurons and astrocytes in situ.
The spatial-temporal characteristics of intracellular calcium ([Ca2+]i) changes elicited in neurons and astrocytes by various types of stimuli were investigated by means of confocal fluorescent microscopy in acute rat brain slices loaded with the Ca2+ indicator indo-1. Neurons and astrocytes from the visual cortex and CA1 hippocampal region were identified in situ on the basis of their morphological, electrophysiological, and pharmacological features. We show here that stimulation of neuronal afferents triggered periodic [Ca2+]i oscillations in astrocytes. The frequency of these oscillations was under a dynamic control by neuronal activity as it changed according to the pattern of stimulation. After repetitive episodes of neuronal stimulation as well as repetitive stimulation with a metabotropic glutamate receptor agonist, astrocytes displayed a long-lasting increase in [Ca2+]i oscillation frequency. Oscillating astrocytes were accompanied by repetitive [Ca2+]i elevations in adjacent neurons, most likely because of the release of glutamate via a tetanus toxin-resistant process. These results reveal that [Ca2+]i oscillations in astrocytes represent a highly plastic signaling system that underlies the reciprocal communication between neurons and astrocytes.
Animals↗
HIV-1 gp120 glycoprotein affects the astrocyte control of extracellular glutamate by both inhibiting the uptake and stimulating the release of the amino acid.
The mechanisms of HIV-1 neurotoxicity remain still undefined although the induction of signalling events and a modest inhibition of glutamate uptake induced by the envelope glycoprotein, gp120, have called attention to astrocytes. Here we demonstrate that the levels at which the viral glycoprotein affects glutamate homeostasis of astrocyte cultures are at least two: not only the inhibition of uptake, due to an effect at site(s) away from the transporters of the amino acid but also a slow stimulation of release. The combination of these two events accounts for a considerable steady increase of the extracellular concentration of the excitatory amino acid which could play an important role in the neurotoxicity often observed in AIDS patients.
Animals↗
Non-synaptic localization of the glutamate transporter EAAC1 in cultured hippocampal neurons.
It has been postulated for several years that the high affinity neuronal glutamate uptake system plays a role in clearing glutamate from the synaptic cleft. Four different glutamate transporter subtypes are now identified, the major neuronal one being EAAC1. To be a good candidate for the reuptake of glutamate at the synaptic cleft, EAAC1 should be properly located at synapses, either at pre- or postsynaptic sites. We have investigated the distribution of EAAC1 in primary cultures of hippocampal neurons, which represent an advantageous model for the study of synaptogenesis and synaptic specializations. We have demonstrated that EAAC1 immunoreactivity is segregated in the somatodendritic compartment of fully differentiated hippocampal neurons, where it is localized in the dendritic shaft and in the spine neck, outside the area facing the active zone. No co-localization of EAAC1 immunoreactivity with the stainings produced by typical presynaptic and postsynaptic markers was ever observed, indicating that EAAC1 is not to be considered a synaptic protein. Accordingly, the developmental pattern of expression of EAAC1 was found to be different from that of typical synaptic markers. Moreover, EAAC1 was expressed in the somatodendritic compartment of hippocampal neurons already at stages preceding the formation of synaptic contacts, and was also expressed in GABAergic interneurons with identical subcellular distribution. Taken together, these data rule against a possible role for EAAC1 in the clearance of glutamate from within the cleft and in the regulation of its time in the synapse. They suggest an unconventional non-synaptic function of this high-affinity glutamate carrier, not restricted to glutamatergic fibres.
Amino Acid Sequence↗
Differential modulation of the uptake currents by redox interconversion of cysteine residues in the human neuronal glutamate transporter EAAC1.
Control of extrasynaptic glutamate concentration in the central nervous system is an important determinant of neurotransmission and excitotoxicity. Mechanisms that modulate glutamate transporter function are therefore critical factors in these processes. The redox modulation of glutamate uptake was examined by measuring transporter-mediated electrical currents and radiolabelled amino acid influx in voltage-clamped Xenopus oocytes expressing the human neuronal glutamate transporter EAAC1. Up and down changes of the glutamate uptake currents in response to treatment with dithiothreitol and 5,5'-dithio-bis-(2-nitrobenzoic) acid (DTNB) were observed in oocytes clamped at -60 mV. The redox interconversion of cysteines induced by dithiothreitol/DTNB influenced the Vmax (Imax) of transport, while the apparent affinity for glutamate was not affected. Formation or breakdown of disulphide groups did not affect the pre-steady-state currents, suggesting that these manipulations do not interfere with the Na+ binding/unbinding and/or the charge distribution on the transporter molecule. The glutamate-evoked net uptake current of EAAC1 was composed of the inward current from electrogenic glutamate transport and the current arising from the glutamate-activated Cl- conductance. The structural rearrangement produced by the formation or breakdown of disulphide groups only affected the current from electrogenic glutamate transport. The electrogenic currents of EAAC1 were significantly reduced by peroxynitrite, an endogenously occurring oxidant formed in certain pathological brain processes, and the mechanism of inhibition partially depended on the formation of disulphide groups.
Amino Acid Transport System X-AG↗
Neuronal and glial glutamate transporters possess an SH-based redox regulatory mechanism.
Glutamate uptake into nerve cells and astrocytes via high-affinity transporters controls the extracellular glutamate concentration in the brain, with major implications for physiological excitatory neurotransmission and the prevention of excitotoxicity. We report here that three recently cloned rat glutamate transporter subtypes, viz. EAAC1 (neuronal), GLT1 and GLAST (glial), possess a redox-sensing property, undergoing opposite functional changes in response to oxidation or reduction of reactive sulphydryls present in their structure. In particular, thiol oxidation with 5,5'-dithio-bis(2-nitrobenzoic) acid (DTNB) and disulphide reduction with dithiothreitol (DTT) result, respectively, in reduced and increased uptake capacity by a preparation of partially purified brain transporters as well as by the three recombinant proteins reconstituted into liposomes. In this model system, EAAC1, GLT1 and GLAST react similarly to DTT/DTNB exposures despite their different contents of cysteines, suggesting that only the conserved residues might be involved in redox modulation. Redox sensitivity is a property of the glutamate transporters also when present in their native cell environment. Thus, by using cultured cortical astrocytes and the whole-cell patch-clamp technique we were able to observe dynamic increase and decrease of the glutamate uptake current in response to application of DTT and DTNB in sequence. Moreover, in the same paradigm, DDT-reversible current inhibition was observed with hydrogen peroxide instead of DTNB, indicating that the SH-based redox modulatory site is targeted by endogenous oxidants and might constitute an important physiological or pathophysiological regulatory mechanism of glutamate uptake in vivo.
ATP-Binding Cassette Transporters↗
Peroxynitrite inhibits glutamate transporter subtypes.
The reuptake of glutamate in neurons and astrocytes terminates excitatory signals and prevents the persistence of excitotoxic levels of glutamate in the synaptic cleft. This process is inhibited by oxygen radicals and hydrogen peroxide (H2O2). Here we show that another biological oxidant, peroxynitrite (ONOO-), formed by combination of superoxide (O2-) and nitric oxide (NO), potently inhibits glutamate uptake by purified or recombinant high affinity glutamate transporters reconstituted in liposomes. ONOO- reduces selectively the Vmax of transport; its action is fast (reaching > or = 90% within 20 s), dose-dependent (50% inhibition at 50 microM), persistent upon ONOO- (or by product) removal, and insensitive to the presence of the lipid antioxidant vitamin E in the liposomal membranes. Therefore, it likely depends on direct interaction of ONOO- with the glutamate transporters. Three distinct recombinant glutamate transporters from the rat brain, GLT1, GLAST, and EAAC1, exhibit identical sensitivity to ONOO . H2O2 also inhibits reconstituted transport, and its action matches that of ONOO- on all respects; however, this is observed only with 5-10 mM H202 and after prolonged exposure (10 min) in highly oxygenated buffer. NO, released from NO donors (up to 10 mM), does not modify reconstituted glutamate uptake, although in parallel conditions it promotes cGMP formation in synaptosomal cytosolic fraction. Overall, our results suggest that the glutamate transporters contain conserved sites in their structures conferring vulnerability to ONOO- and other oxidants.
ATP-Binding Cassette Transporters↗
The competitive transport inhibitor L-trans-pyrrolidine-2, 4-dicarboxylate triggers excitotoxicity in rat cortical neuron-astrocyte co-cultures via glutamate release rather than uptake inhibition.
We studied the early and late effects of L-trans-pyrrolidine-2,4-dicarboxylate (PDC), a competitive inhibitor of glutamate uptake with low affinity for glutamate receptors, in co-cultures of rat cortical neurons and glia expressing spontaneous excitatory amino acid (EAA) neurotransmission. At 100 or 200 microM, PDC induced different patterns of electrical changes: 100 microM prolonged tetrodotoxin-sensitive excitation triggered by synaptic glutamate release; 200 microM produced sustained, tetrodotoxin-insensitive and EAA-mediated neuronal depolarization, overwhelming synaptic activity. At 200 microM, but not at 100 microM, PDC caused rapid elevation of the glutamate concentration ([Glu]o) in the culture medium, resulting in NMDA receptor-mediated excitotoxic death of neurons 24 h later. The increase in [Glu]o was largely insensitive to tetrodotoxin, independent of extracellular Ca2+, and present also in astrocyte-pure cultures. By the use of glutamate transporters functionally reconstituted in liposomes, we showed directly that PDC activates carrier-mediated release of glutamate via heteroexchange. Glutamate release and delayed neurotoxicity in our cultures were suppressed if PDC was applied in a Na(+)-free medium containing Li+. However, replacement of Na+ with choline instead of Li+ did not result in an identical effect, suggesting that Li+ does not act simply as an external Na+ substitute. In conclusion, our data indicate that alteration of glutamate transport by PDC has excitotoxic consequences and that active release of glutamate rather than just uptake inhibition is responsible for the generation of neuronal injury.
Animals↗
Arachidonic acid inhibits a purified and reconstituted glutamate transporter directly from the water phase and not via the phospholipid membrane.
Glutamate is believed to be the major excitatory transmitter in the mammalian central nervous system. Keeping the extracellular concentration of glutamate low, the glutamate transporters are required for normal brain function. Arachidonic acid (AA) inhibits glutamate uptake in relatively intact preparations (cells, tissue slices, and synaptosomes (Rhoads, D.E., Ockner, R. K., Peterson, N. A., and Raghupathy, E. (1983) Biochemistry 22, 1965-1970 and Volterra, A., Trotti, D., Cassutti, P., Tromba, C., Salvaggio, A., Melcangi, R. C., and Racagni, G. (1992b) J. Neurochem. 59, 600-606). The present study demonstrates that the effect of AA occurs also in a reconstituted system, consisting of a purified glutamate transporter protein incorporated into artificial cell membranes (liposomes). The characteristics of the AA effect in this system and in intact cells are similar with regard to specificity, sensitivity, time course, changes in Vmax, and affinity. AA-ethyl ester is inactive, suggesting that the free carboxylic group is required for inhibitory activity. When incubated with proteoliposomes, AA (300 microM, 15 min) mostly partitions to the lipid phase (lipid/water about 95:5). However, uptake inhibition is abolished by rapid dilution (6.5-fold) of the incubation medium (water phase), a procedure that does not modify the amount of AA associated with lipids. On the contrary, inhibition remains sustained if the same dilution volume contains as little as 5 microM AA, a concentration inactive before saturation of liposome lipids with 300 microM AA. The same degree of inhibition (60%) is obtained by 5 microM AA following preincubation with the inactive AA-ethyl ester (300 microM) instead of AA. The lipids apparently inactivate AA by extracting it from the water phase. The results suggest that AA acts on the transporter from the water phase rather than via the membrane. This could be true for other proteins as well since gamma-aminobutyric acid uptake is similarly affected by AA.
ATP-Binding Cassette Transporters↗