Astrocyte sodium signaling and neuro-metabolic coupling in the brain.

At tripartite synapses, astrocytes undergo calcium signaling in response to release of neurotransmitters and this calcium signaling has been proposed to play a critical role in neuron-glia interaction. Recent work has now firmly established that, in addition, neuronal activity also evokes sodium transients in astrocytes, which can be local or global depending on the number of activated synapses and the duration of activity. Furthermore, astrocyte sodium signals can be transmitted to adjacent cells through gap junctions and following release of gliotransmitters. A main pathway for activity-related sodium influx into astrocytes is via high-affinity sodium-dependent glutamate transporters. Astrocyte sodium signals differ in many respects from the well-described glial calcium signals both in terms of their temporal as well as spatial distribution. There are no known buffering systems for sodium ions, nor is there store-mediated release of sodium. Sodium signals thus seem to represent rather direct and unbiased indicators of the site and strength of neuronal inputs. As such they have an immediate influence on the activity of sodium-dependent transporters which may even reverse in response to sodium signaling, as has been shown for GABA transporters for example. Furthermore, recovery from sodium transients through Na(+)/K(+)-ATPase requires a measurable amount of ATP, resulting in an activation of glial metabolism. In this review, we present basic principles of sodium regulation and the current state of knowledge concerning the occurrence and properties of activity-related sodium transients in astrocytes. We then discuss different aspects of the relationship between sodium changes in astrocytes and neuro-metabolic coupling, putting forward the idea that indeed sodium might serve as a new type of intracellular ion signal playing an important role in neuron-glia interaction and neuro-metabolic coupling in the healthy and diseased brain.

Na+ signals at central synapses.

A basic characteristic of animal cells is the maintenance of a steep inwardly directed electrochemical gradient for sodium ions. In vertebrate neurons, this Na+ gradient energizes intracellular ion regulation and enables influx of Na+ during action potentials and excitatory postsynaptic currents. Several studies suggested that Na+ ions could also play a role in activity-dependent synaptic plasticity. This review focuses on recent studies that demonstrated the presence of substantial intracellular Na+ transients during action potential firing or excitatory synaptic transmission in postsynaptic dendrites and dendritic spines. The large amplitudes of these activity-induced Na+ transients suggest that this signal will significantly alter electrical and biochemical properties of spines and dendrites and might influence the properties of synaptic transmission.

Stores not just for storage. intracellular calcium release and synaptic plasticity.

Activation of most excitatory synapses of central neurons produces calcium release signals from intracellular stores. Synaptically evoked calcium release from stores is frequently triggered by the binding of glutamate to metabotropic receptors and the subsequent activation of IP(3) receptors in spines and dendrites. There is increasing evidence for the presence of local calcium signals caused by calcium-induced calcium release (CICR) through activation of ryanodine or IP(3) receptors. Recent work on mutant mice indicates that store signaling determines activity-dependent synaptic plasticity.

NMDA receptor-mediated Na+ signals in spines and dendrites.

Spines and dendrites of central neurons represent an important site of synaptic signaling and integration. Here we identify a new, synaptically mediated spine signal with unique properties. Using two-photon Na(+) imaging, we show that suprathreshold synaptic stimulation leads to transient increases in Na(+) concentration in postsynaptic spines and their adjacent dendrites. This local signal is restricted to a dendritic domain near the site of synaptic input. In presumed active spines within this domain, the Na(+) level increases by 30-40 mm even during short bursts of synaptic stimulation. During a long-term potentiation induction protocol (100 Hz, 1 sec), the Na(+) level in the active spines reaches peak amplitudes of approximately 100 mm. We find that the Na(+) transients are mainly mediated by Na(+) entry through NMDA receptor channels and are detected during the coincident occurrence of synaptic potentials and backpropagating action potentials. The large amplitudes of the Na(+) transients and their location on dendritic spines suggest that this signal is an important determinant of electrical and biochemical spine characteristics.

Neurotrophin-evoked rapid excitation through TrkB receptors.

Neurotrophins are a family of structurally related proteins that regulate the survival, differentiation and maintenance of function of different populations of peripheral and central neurons. They are also essential for modulating activity-dependent neuronal plasticity. Here we show that neurotrophins elicit action potentials in central neurons. Even at low concentrations, brain-derived neurotrophic factor (BDNF) excited neurons in the hippocampus, cortex and cerebellum. We found that BDNF and neurotrophin-4/5 depolarized neurons just as rapidly as the neurotransmitter glutamate, even at a more than thousand-fold lower concentration. Neurotrophin-3 produced much smaller responses, and nerve growth factor was ineffective. The neurotrophin-induced depolarization resulted from the activation of a sodium ion conductance which was reversibly blocked by K-252a, a protein kinase blocker which prefers tyrosine kinase Trk receptors. Our results demonstrate a very rapid excitatory action of neurotrophins, placing them among the most potent endogenous neuro-excitants in the mammalian central nervous system described so far.

Leech giant glial cell: functional role in a simple nervous system.

The giant glial cell in the central nervous system of the leech Hirudo medicinalis has been the subject of a series of studies trying to link its physiological properties with its role in neuron-glia interactions. Isolated ventral cord ganglia of this annelid offer several advantages for these studies. First, single giant glial cells can easily be identified and are quite accessible to electrophysiological and microfluorometric studies. Second, only two giant macroglial cells are located in the neuropil of each ganglion, rendering them well suited for studying neuron-glia interactions. Third, many neurons can be identified and are well known with respect to their physiology and their roles in controlling simple behaviors in the leech. This review briefly outlines the major recent findings gained by studying this preparation and its contributions to our knowledge of the functional role of glia in nervous systems. Emphasis is directed to glial responses during neuronal activity and to the analysis of intracellular Ca(2+) and H(+) transients mediated by neurotransmitter receptors and ion-driven carriers. Among its numerous properties, the leech giant glial cell prominently expresses a large K(+) conductance, voltage-dependent Ca(2+) channels, ionotropic non-NMDA glutamate receptors, and an electrogenic, reversible Na(+)-HCO(3)(-) cotransporter.

K(+)-induced reversal of astrocyte glutamate uptake is limited by compensatory changes in intracellular Na+.

Glutamate uptake is coupled to counter-transport of K+, and high external K+ concentrations can induce reversal of glutamate uptake in whole-cell patch-clamp and isolated membrane preparations. However, high external K+ causes little or no reversal of glutamate uptake in intact astrocytes, suggesting a regulatory mechanism not evident in membrane preparations. One mechanism by which intact cells could limit the effects of altered extracellular ion concentrations on glutamate transport is by compensatory changes in intracellular Na+ concentrations. This possibility was examined using astrocyte cultures treated in two ways to reduce the driving force for glutamate uptake: incubation in high K+ (with reciprocal reduction in Na+), and incubation with metabolic inhibitors to induce ATP depletion. ATP depletion produced a rise in intracellular Na+, a collapse of the membrane sodium gradient and a massive reversal of glutamate uptake. By contrast, incubation in high K+/low Na+ medium did not significantly alter the sodium gradient and did not induce glutamate uptake reversal. The sodium gradient was shown to be maintained under these conditions by compensatory reductions in intracellular Na+ that approximately matched the reductions in extracellular Na+. These findings suggest a mechanism by which astrocytes may limit reversal of glutamate uptake under high K+/low Na+ conditions, and further suggest a general mechanism by which Na(+)-dependent transport processes could be shielded from fluctuating extracellular ion concentrations.

Two-photon Na+ imaging in spines and fine dendrites of central neurons.

Dendritic spines are assumed to be the smallest units of neuronal integration. Because of their miniature size, however, many of their functional properties are still unclear. New insights in spine physiology have been provided by two-photon laser-scanning microscopy which allows fluorescence imaging with high spatial resolution and minimal photodamage. For example, two-photon imaging has been employed successfully for the measurement of activity-induced calcium transients in individual spines. Here, we describe the first application of two-photon imaging to measure Na+ transients in spines and dendrites of CA1 pyramidal neurons in hippocampal slices. Whole-cell patch-clamped neurons were loaded with the Na(+)-indicator dye SBFI (sodium-binding benzofuran-isophthalate). In situ calibration of SBFI fluorescence with ionophores enabled the determination of the actual magnitude of the [Na+]i changes. We found that back-propagating action potentials (APs) evoked Na+ transients throughout the proximal part of the dendritic tree and adjacent spines. The action-potential-induced [Na+]i transients reached values of 4 mM for a train of 20 APs and monotonically decayed with a time constant of several seconds. These results represent the first demonstration of activity-induced Na+ accumulation in spines. Our results demonstrate that two-photon Na+ imaging represents a powerful tool for extending our knowledge on Na+ signaling in fine cellular subcompartments.

Effects of glucose deprivation, chemical hypoxia, and simulated ischemia on Na+ homeostasis in rat spinal cord astrocytes.

A steep inwardly directed Na+ gradient is essential for glial functions such as glutamate reuptake and regulation of intracellular ion concentrations. We investigated the effects of glucose deprivation, chemical hypoxia, and simulated ischemia on intracellular Na+ concentration ([Na+]i) in cultured spinal cord astrocytes using fluorescence ratio imaging with sodium-binding benzofuran isophthalate (SBFI) AM. Glucose removal or chemical hypoxia (induced by 10 mM NaN3) for 60 min increased [Na+]i from a baseline of 8.3 to 11 mM. Combined glycolytic and respiratory blockage by NaN3 and 0 glucose saline caused [Na+]i to increase by 20 mM, similar to the [Na+]i increases elicited by blocking the Na+/K+-ATPase with ouabain. Recovery from large [Na+]i increases (>15 mM) induced by the glutamatergic agonist kainate was attenuated during glucose deprivation or NaN3 application and was blocked in NaN3 and 0 glucose. To mimic in vivo ischemia, we exposed astrocytes to NaN3 and 0 glucose saline containing L-lactate and glutamate with increased [K+] and decreased [Na+], [Ca2+], and pH. This induced an [Na+]i decrease followed by an [Na+]i rise and a further [Na+]i increase after reperfusion with standard saline. Similar multiphasic [Na+]i changes were observed after NaN3 and 0 glucose saline with only reduced [Na+]e. Our results suggest that the ability to maintain a low [Na+]i enables spinal cord astrocytes to continue uptake of K+ and/or glutamate at the onset of energy failure. With prolonged energy failure, however, astrocytic [Na+]i rises; with loss of their steep transmembrane Na+ gradient, astrocytes may aggravate metabolic insults by carrier reversal and release of acid, K+, and/or glutamate into the extracellular space.

Gap junctions equalize intracellular Na+ concentration in astrocytes.

Gap junctions between glial cells allow intercellular exchange of ions and small molecules. We have investigated the influence of gap junction coupling on regulation of intracellular Na+ concentration ([Na+]i) in cultured rat hippocampal astrocytes, using fluorescence ratio imaging with the Na+ indicator dye SBFI (sodium-binding benzofuran isophthalate). The [Na+]i in neighboring astrocytes was very similar (12.0 +/- 3.3 mM) and did not fluctuate under resting conditions. During uncoupling of gap junctions with octanol (0.5 mM), baseline [Na+]i was unaltered in 24%, increased in 54%, and decreased in 22% of cells. Qualitatively similar results were obtained with two other uncoupling agents, heptanol and alpha-glycyrrhetinic acid (AGA). Octanol did not alter the recovery from intracellular Na+ load induced by removal of extracellular K+, indicating that octanol's effects on baseline [Na+]i were not due to inhibition of Na+, K+-ATPase activity. Under control conditions, increasing [K+]o from 3 to 8 mM caused similar decreases in [Na+]i in groups of astrocytes, presumably by stimulating Na+, K+-ATPase. During octanol application, [K+]o-induced [Na+]i decreases were amplified in cells with increased baseline [Na+]i, and reduced in cells with decreased baseline [Na+]i. This suggests that baseline [Na+]i in astrocytes "sets" the responsiveness of Na+, K+-ATPase to increases in [K]o. Our results indicate that individual hippocampal astrocytes in culture rapidly develop different levels of baseline [Na+]i when they are isolated from one another by uncoupling agents. In astrocytes, therefore, an apparent function of coupling is the intercellular exchange of Na+ ions to equalize baseline [Na+]i, which serves to coordinate physiological responses that depend on the intracellular concentration of this ion.

Regulation of intracellular sodium in cultured rat hippocampal neurones.

1. We studied regulation of intracellular Na+ concentration ([Na+]i) in cultured rat hippocampal neurones using fluorescence ratio imaging of the Na+ indicator dye SBFI (sodium-binding benzofuran isophthalate). 2. In standard CO2/HCO3(-)-buffered saline with 3 mM K+, neurones had a baseline [Na+]i of 8.9 +/- 3.8 mM (mean +/- S.D.). Spontaneous, transient [Na+]i increases of 5 mM were observed in neurones on 27% of the coverslips studied. These [Na+]i increases were often synchronized among nearby neurones and were blocked reversibly by 1 microM tetrodotoxin (TTX) or by saline containing 10 mM Mg2+, suggesting that they were caused by periodic bursting activity of synaptically coupled cells. Opening of voltage-gated Na+ channels by application of 50 microM veratridine caused a TTX-sensitive [Na+]i increase of 25 mM. 3. Removing extracellular Na+ caused an exponential decline in [Na+]i to values close to zero within 10 min. Inhibition of Na+,K(+)-ATPase by removal of extracellular K+ or ouabain application evoked a [Na+]i increase of 5 mM min-1. Baseline [Na+]i was similar in the presence or absence of CO2/HCO3-; switching from CO2/HCO3(-)-free to CO2/HCO3(-)-buffered saline, however, increased [Na+]i transiently by 3 mM, indicating activation of Na(+)-dependent Cl(-)-HCO3- exchange. Inhibition of Na(+)-K(+)-2Cl- cotransport by bumetanide had no effect on [Na+]i. 4. Brief, small changes in extracellular K+ concentration ([K+]o) influenced neuronal [Na+]i only weakly. Virtually no change in [Na+]i was observed with elevation or reduction of [K+]o by 1 mM. Only 30% of cells reacted to 3 min [K+]o elevations of up to 5 mM. In contrast, long [K+]o alterations (> or = 10 min) to 6 mM or greater slowly changed steady-state [Na+]i in the majority of cells. 5. Our results indicate several differences between [Na+]i regulation in cultured hippocampal neurones and astrocytes. Baseline [Na+]i is lower in neurones compared with astrocytes and is mainly determined by Na+,K(+)-ATPase, whereas Na(+)-dependent Cl(-)-HCO3- exchange, Na(+)-HCO3- cotransport or Na(+)-K(+)-2Cl- cotransport do not play a significant role. In contrast to glial cells, [Na+]i of neurones changes only weakly with small alterations in bath [K+]o, suggesting that activity-induced [K+]o changes in the brain might not significantly influence neuronal Na+,K(+)-ATPase activity.

Pharmacological characterization of Na+ influx via voltage-gated Na+ channels in spinal cord astrocytes.

Spinal cord astrocytes display a high density of voltage-gated Na+ channels. To study the contribution of Na+ influx via these channels to Na+ homeostasis in cultured spinal cord astrocytes, we measured intracellular Na+ concentration ([Na+]i) with the fluorescent dye sodium-binding benzofuran isophthalate. Stellate and nonstellate astrocytes, which display Na+ currents with different properties, were differentiated. Baseline [Na+]i was 8.5 mM in these cells and was not altered by 100 microM tetrodotoxin (TTX). Inhibition of Na+ channel inactivation by veratridine (100 microM) evoked a [Na+]i increase of 47.1 mM in 44% of stellate and 9.7 mM in 64% of nonstellate astrocytes. About 30% of cells reacted to veratridine with a [Na+]i decrease of approximately 2 mM. Qualitatively similar [Na+]i changes were caused by aconitine. The effects of veratridine were blocked by TTX, amplified by (alpha-)scorpion toxin and usually were readily reversible. Veratridine-induced [Na+]i increases were reduced upon membrane depolarization with elevated extracellular [K+]. Recovery to baseline [Na+]i was unaltered during blocking of K+ channels with 4-aminopyridine. [Na+]i increases evoked by the ionotropic non-N-methyl--aspartate receptor agonist kainate were not altered by TTX. Our results indicate that influx of Na+ via voltage- gated Na+ channels is not a prerequisite for glial Na+,K+-ATPase activity in spinal cord astrocytes at rest nor does it seem to be involved in [Na+]i increases evoked by kainate. During pharmacological inhibition of Na+ channel inactivation, however, Na+ channels can serve as prominent pathways of Na+ influx and mediate large perturbations in [Na+]i, suggesting that Na+ channel inactivation plays an important functional role in these cells.

Mechanisms of H+ and Na+ changes induced by glutamate, kainate, and D-aspartate in rat hippocampal astrocytes.

The excitatory transmitter glutamate (Glu), and its analogs kainate (KA), and D-aspartate (D-Asp) produce significant pH changes in glial cells. Transmitter-induced pH changes in glial cells, generating changes in extracellular pH, may represent a special form of neuronal-glial interaction. We investigated the mechanisms underlying these changes in intracellular H+ concentration ([H+]i) in cultured rat hippocampal astrocytes and studied their correlation with increases in intracellular Na+ concentration ([Na+]i), using fluorescence ratio imaging with 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) or sodium-binding benzofuran isophthalate (SBFI). Glu, KA, or D-Asp evoked increases in [Na+]i; Glu or D-Asp produced parallel acidifications. KA, in contrast, evoked biphasic changes in [H+]i, alkaline followed by acid shifts, which were unaltered after Ca2+ removal and persisted in 0 CI(-)-saline, but were greatly reduced in CO2/HCO3(-)-free or Na(+)-free saline, or during 4,4'-diisothiocyanato-stilbene-2,2'-disulphonic acid (DIDS) application. The non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) blocked KA-evoked changes in [H+]i and [Na+]i, indicating that they were receptor-ionophore mediated. In contrast, CNQX increased the [H+]i change and decreased the [Na+]i change induced by Glu. D-Asp, which is transported but does not act at Glu receptors, induced [H+]i and [Na+]i changes that were virtually unaltered by CNQX. Our study indicates that [Na+]i increases are not primarily responsible for Glu- or KA-induced acidifications in astrocytes. Instead, intracellular acidifications evoked by Glu or D-Asp are mainly caused by transmembrane movement of acid equivalents associated with Glu/Asp-uptake into astrocytes. KA-evoked biphasic [H+]i changes, in contrast, are probably attributable to transmembrane ion movements mediated by inward, followed by outward, electrogenic Na+/HCO3- cotransport, reflecting KA-induced biphasic membrane potential changes.

Intracellular sodium homeostasis in rat hippocampal astrocytes.

1. We determined the intracellular Na+ concentration ([Na+]i) and mechanisms of its regulation in cultured rat hippocampal astrocytes using fluorescence ratio imaging of the Na+ indicator SBFI-AM (acetoxymethylester of sodium-binding benzofuran isophthalate, 10 microM). Dye signal calibration within the astrocytes showed that the ratiometric dye signal changed monotonically with changes in [Na+]i from 0 to 140 nM. The K+ sensitivity of the dye was negligible; intracellular pH changes, however, slightly affected the 'Na+' signal. 2. Baseline [Na+]i was 14.6 +/- 4.9 mM (mean +/- S.D.) in CO2/HCO3(-)-containing saline with 3 mM K+. Removal of extracellular Na+ decreased [Na+]i in two phases: a rapid phase of [Na+]i reduction (0.58 +/- 0.32 mM min-1) followed by a slower phase (0.15 +/- 0.09 mM min-1). 3. Changing from CO2/HCO3(-)-free to CO2/HCO3(-)-buffered saline resulted in a transient increase in [Na+]i of approximately 5 mM, suggesting activation of inward Na(+)-HCO3- cotransport by CO2/HCO3-. During furosemide (frusemide, 1 mM) or bumetanide (50 microM) application, a slow decrease in [Na+]i of approximately 2 mM was observed, indicating a steady inward transport of Na+ via Na(+)-K(+)-2Cl- cotransport under control conditions. Tetrodotoxin (100 microM) did not influence [Na+]i in the majority of cells (85%), suggesting that influx of Na+ through voltage-gated Na+ channels contributed to baseline [Na+]i in only a small subpopulation of hippocampal astrocytes. 4. Blocking Na+, K(+)-ATPase activity with cardiac glycosides (ouabain or strophanthidin, 1 mM) or removal of extracellular K+ led to an increase in [Na+]i of about 2 and 4 mM min-1, respectively. This indicated that Na+, K(+)-ATPase activity was critical in maintaining low [Na+]i in the face of a steep electrochemical gradient, which would favour a much higher [Na+]i. 5. Elevation of extracellular K+ concentration ([K+]o) by as little as 1 mM (from 3 to 4 mM) resulted in a rapid and reversible decrease in [Na+]i. Both the slope and the amplitude of the [K+]o-induced reductions in [Na+]i were sensitive to bumetanide. A reduction of [K+]o by 1 mM increased [Na+]i by 3.0 +/- 2.3 mM. In contrast, changing extracellular Na+ concentration by 20 mM resulted in changes in [Na+]i of less than 3 mM. 6. These results implied that in hippocampal astrocytes low baseline [Na+]i is determined by the action of Na(+)-HCO3- cotransport, Na(+)-K(+)-2Cl- cotransport and Na+, K(+)-ATPase, and that both Na+, K(+)-ATPase and inward Na(+)-K(+)-2Cl cotransport are activated by small, physiologically relevant increases in [K+]o. These mechanisms are well suited to help buffer increases in [K+]o associated with neural activity.

Extracellular Ca2+ changes during transmitter application in the leech central nervous system.

Changes in extracellular Ca2+ concentration ([Ca2+]e) evoked by transmitters and transmitter agonists, respectively, and by elevation of bath K+ concentration were recorded in isolated segmental ganglia of the leech Hirudo medicinalis using Ca(2+)-selective microelectrodes. A 1-min bath application of kainate (10 microM), glutamate (1 mM), aspartate (1 mM), or carbachol (200 microM) decreased [Ca2+]e by up to 1 mM, whereas the inhibitory transmitters gamma-amino butyric acid (GABA, 100 microM) and serotonin (5-HT, 100 microM) did not change [Ca2+]e. The amplitude of the kainate-induced changes in [Ca2+]e increased with repetitive applications, and changes were blocked by 6-cyano-7-dinitroquinozaline-2,3-dione (CNQX). Elevation of bath K+ concentration from 4 to 40 mM led to a Ni(2+)-sensitive decrease in [Ca2+]e by 0.9 mM. Our results suggest that excitatory transmission in the leech central nervous system might be accompanied by substantial decreases in [Ca2+]e.