Metabolic substrates other than glucose support axon function in central white matter.

We tested the hypothesis that non-glucose energy sources can support axon function in the rat optic nerve. Axon function was assessed by monitoring the stimulus-evoked compound action potential (CAP). CAP was maintained at full amplitude for 2 hr in 10 mM glucose. 20 mM lactate, 20 mM pyruvate, 10 mM fructose, or 10 mM mannose supported axon function as effectively as did glucose, and 10 mM glutamine provided partial support, but beta-hydroxybutyrate, octanoate, sorbitol, alanine, aspartate, and glutamate failed to support axon function. Our results indicated that a variety of compounds can sustain function in CNS myelinated axons. Axons probably use lactate, pyruvate, and glutamine directly as energy substrates, whereas mannose and fructose could be shuttled through astrocytes to lactate, which is then exported to axons.

(1R,3S)-1-Aminocyclopentane-1,3-dicarboxylic acid (RS-ACPD) reduces intracellular glutamate levels in astrocytes.

(+/-)-1-Aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD) is an equimolar mixture of two enantiomers: (1S,3R)-1-Aminocyclopentane-1,3-dicarboxylic acid (SR-ACPD) and 1R,3S-1-Aminocyclopentane-1,3-dicarboxylic acid (RS-ACPD). t-ACPD and SR-ACPD have been commonly used as agonists for metabotropic glutamate receptors (mGluR). Here we demonstrated that RS-ACPD, but not SR-ACPD, is transported into astrocytes with a K(m) of 6.51 +/- 2.38 mM and V(max) of 22.8 +/- 3.4 nmol/mg/min. This low-affinity transport is Na(+)-dependent and is competitively blocked by glutamate or other substrates for the glutamate transporter. RS-ACPD therefore is probably taken up by the glutamate transporter. Prolonged incubation with high levels of RS-ACPD (> 500 microM) induced significant swelling of astrocytes. At lower concentrations (100 microM), RS-ACPD reduced intracellular glutamate content ([Glu](i)) by > 50% without obvious morphological changes. The reduction in [Glu](i) was accompanied by an increase in [glutamine](i). The RS-ACPD's effect on [Glu](i) required glutamine and high levels of phosphate, suggesting that RS-ACPD inhibited phosphate-activated glutaminase (PAG). These data suggest that astrocytic PAG is actively involved in determining the equilibrium between intracellular glutamate and glutamine. By reducing [Glu](i), RS-ACPD reduces the amount of glutamate available for release.

Expression and function of lysophosphatidic acid receptors in cultured rodent microglial cells.

Microglia are the resident tissue macrophages of the central nervous system. They are rapidly activated by a variety of insults; and recently, receptors linked to cytoplasmic Ca(2+) signals have been implicated in such events. One potential class of receptors are those recognizing lysophosphatidic acid (LPA). LPA is a phospholipid signaling molecule that has been shown to cause multiple cellular responses, including increases in cytoplasmic calcium. We examined whether any of the known LPA receptor genes (lp(A1)/Edg2, lp(A2)/Edg4, and lp(A3)/Edg7) are expressed by cultured mouse or rat microglia. Reverse transcriptase-polymerase chain reaction indicated that mouse microglia predominantly expressed the lp(A1) gene, whereas rat microglia predominantly expressed lp(A3). Although LPA induced increases in the cytoplasmic Ca(2+) concentration in both microglial preparations, the responses differed substantially. The Ca(2+) signal in rat microglia occurred primarily through Ca(2+) influx via the plasma membrane, whereas the Ca(2+) signal in mouse microglia was due to release from intracellular stores. Only at high concentrations was an additional influx component recruited. Additionally, LPA induced increased metabolic activity in mouse (but not rat) microglial cells. Our findings provide evidence for functional LPA receptors on microglia. Thus, LPA might play an important role as a mediator of microglial activation in response to central nervous system injury.

Ionic mechanisms of aglycemic axon injury in mammalian central white matter.

The authors investigated ionic mechanisms underlying aglycemic axon injury in adult rat optic nerve, a central white matter tract. Axon function was assessed using evoked compound action potentials (CAPs). Glucose withdrawal led to delayed CAP failure, an alkaline extracellular pH shift, and an increase in extracellular [K(+)]. Sixty minutes of glucose withdrawal led to irreversible axon injury. Aglycemic axon injury required extracellular calcium; the extent of injury progressively declined as bath [Ca(2+)] was decreased. To evaluate Ca(2+) movements during aglycemia, the authors recorded extracellular [Ca(2+)] ([Ca(2+)](o)) using Ca(2+)-sensitive microelectrodes. Under control conditions, [Ca(2+)](o) fell with a similar time course to CAP failure, indicating extracellular Ca(2+) moved to an intracellular position during aglycemia. The authors quantified the magnitude of [Ca(2+)]o decrease as the area below baseline [Ca(2+)]o during aglycemia and used this as a qualitative measure of Ca(2+) influx. The authors studied the mechanisms of Ca(2+) influx. Blockade of Na(+) influx reduced Ca(2+) influx and improved CAP recovery, suggesting Na(+)-Ca(2+) exchanger involvement. Consistent with this hypothesis, bepridil reduced axon injury. In addition, diltiazem or nifedipine decreased Ca(2+) influx and increased CAP recovery. The authors conclude aglycemic central white matter injury is caused by Ca(2+) influx into intracellular compartments through reverse Na(+)-Ca(2+) exchange and L-type Ca(2+) channels.

Axonal L-type Ca2+ channels and anoxic injury in rat CNS white matter.

We studied the magnitude and route(s) of Ca2+ flux from extra- to intracellular compartments during anoxia in adult rat optic nerve (RON), a central white matter tract, using Ca2+ sensitive microelectrodes to monitor extracellular [Ca2+] ([Ca2+]o). One hour of anoxia caused a rapid loss of the stimulus-evoked compound action potential (CAP), which partially recovered following re-oxygenation, indicating that irreversible injury had occurred. After an initial increase caused by extracellular space shrinkage, anoxia produced a sustained decrease of 0.42 mM (29%) in [Ca2+]o. We quantified the [Ca2+]o decrease as the area below baseline [Ca2+]o during anoxia and used this as a qualitative index of suspected Ca2+ influx. The degree of RON injury was predicted by the amount of Ca2+ leaving the extracellular space. Bepridil, 0 Na+ artificial cerebrospinal fluid or tetrodotoxin reduced suspected Ca2+ influx during anoxia implicating reversal of the Na+/Ca2+ exchanger as a route of Ca2+ influx. Diltiazem reduced suspected Ca2+ influx during anoxia, suggesting that Ca2+ influx via L-type Ca2+ channels is a route of toxic Ca2+ influx into axons during anoxia. Immunocytochemical staining was used to demonstrate and localize high-threshold Ca2+ channels. Only alpha1(C) and alpha1(D) subunits were detected, indicating that only L-type Ca2+ channels were present. Double labeling with anti-neurofilament antibodies or anti-glial fibrillary acidic protein antibodies localized L-type Ca2+ channels to axons and astrocytes.

Thrombin-induced activation of cultured rodent microglia.

Microglia are the resident immune cells of the CNS. Upon brain damage, these cells are rapidly activated and function as tissue macrophages. The first steps in this activation still remain unclear, but it is widely believed that substances released from damaged brain tissue trigger this process. In this article, we describe the effects of the blood coagulation factor thrombin on cultured rodent microglial cells. Thrombin induced a transient Ca(2+) increase in microglial cells, which persisted in Ca(2+)-free media. It was blocked by thapsigargin, indicating that thrombin caused a Ca(2+) release from internal stores. Preincubation with pertussis toxin did not alter the thrombin-induced [Ca(2+)](i) signal, whereas it was blocked by hirudin, a blocker of thrombin's proteolytic activity. Incubation with thrombin led to the production of nitric oxide and the release of the cytokines tumor necrosis factor-alpha, interleukin-6, interleukin-12, the chemokine KC, and the soluble tumor necrosis factor-alpha receptor II and had a significant proliferative effect. Our findings indicate that thrombin, a molecule that enters the brain at sites of injury, rapidly triggered microglial activation.

Astrocytic glycogen influences axon function and survival during glucose deprivation in central white matter.

We tested the hypothesis that astrocytic glycogen sustains axon function during and enhances axon survival after 60 min of glucose deprivation. Axon function in the rat optic nerve (RON), a CNS white matter tract, was monitored by measuring the area of the stimulus-evoked compound action potential (CAP). Switching to glucose-free artificial CSF (aCSF) had no effect on the CAP area for approximately 30 min, after which the CAP rapidly failed. Exposure to glucose-free aCSF for 60 min caused irreversible injury, which was measured as incomplete recovery of the CAP. Glycogen content of the RON fell to a low stable level 30 min after glucose withdrawal, compatible with rapid use in the absence of glucose. An increase of glycogen content induced by high-glucose pretreatment increased the latency to CAP failure and improved CAP recovery. Conversely, a decrease of glycogen content induced by norepinephrine pretreatment decreased the latency to CAP failure and reduced CAP recovery. To determine whether lactate represented the fuel derived from glycogen and shuttled to axons, we used the lactate transport blockers quercetin, alpha-cyano-4-hydroxycinnamic acid (4-CIN), and p-chloromercuribenzene sulfonic acid (pCMBS). All transport blockers, when applied during glucose withdrawal, decreased latency to CAP failure and decreased CAP recovery. The inhibitors 4-CIN and pCMBS, but not quercetin, blocked lactate uptake by axons. These results indicated that, in the absence of glucose, astrocytic glycogen was broken down to lactate, which was transferred to axons for fuel.

Activity-dependent extracellular K+ accumulation in rat optic nerve: the role of glial and axonal Na+ pumps.

1. We measured activity-dependent changes in [K+]o with K(+)-selective microelectrodes in adult rat optic nerve, a CNS white matter tract, to investigate the factors responsible for post-stimulus recovery of [K+]o. 2. Post-stimulus recovery of [K+]o followed a double-exponential time course with an initial, fast time constant, tau fast, of 0.9 +/- 0.2 s (mean +/- S.D.) and a later, slow time constant, tau slow, of 4.2 +/- 1 s following a 1 s, 100 Hz stimulus. tau fast, but not tau slow, decreased with increasing activity-dependent rises in [K+]o. tau slow, but not tau fast, increased with increasing stimulus duration. 3. Post-stimulus recovery of [K+]o was temperature sensitive. The apparent temperature coefficients (Q10, 27-37 degrees C) for the fast and slow components following a 1 s, 100 Hz stimulus were 1.7 and 2.6, respectively. 4. Post-stimulus recovery of [K+]o was sensitive to Na+ pump inhibition with 50 microM strophanthidin. Following a 1 s, 100 Hz stimulus, 50 microM strophanthidin increased tau fast and tau slow by 81 and 464%, respectively. Strophanthidin reduced the temperature sensitivity of post-stimulus recovery of [K+]o. 5. Post-stimulus recovery of [K+]o was minimally affected by the K+ channel blocker Ba2+ (0.2 mM). Following a 10 s, 100 Hz stimulus, 0.2 mM Ba2+ increased tau fast and tau slow by 24 and 18%, respectively. 6. Stimulated increases in [K+]o were followed by undershoots of [K+]o. Post-stimulus undershoot amplitude increased with stimulus duration but was independent of the peak preceding [K+]o increase. 7. These observations imply that two distinct processes contribute to post-stimulus recovery of [K+]o in central white matter. The results are compatible with a model of K+ removal that attributes the fast, initial phase of K+ removal to K+ uptake by glial Na+ pumps and the slower, sustained decline to K+ uptake via axonal Na+ pumps.

Lysophosphatidic acid-induced calcium signals in cultured rat oligodendrocytes.

Oligodendrocytes are the myelin forming glial cells of the CNS and are known to express receptors linked to ion channels and intracellular second messenger cascades. In this paper, we describe the intracellular calcium responses of cells from the oligodendrocyte lineage to application of lysophosphatidic acid (LPA), a naturally occurring, growth factor-like phospholipid. Oligodendrocyte precursors did not respond to application of LPA (1 microM). In mature oligodendrocytes, however, LPA (1 microM) induced an increase in the intracellular calcium concentration ([Ca2+]i). In the majority of cells this increase was followed by a persistent plateau phase. The LPA-induced [Ca2+]i signal vanished in Ca2+-free medium, implying that it arose due to a Ca2+ influx across the plasma membrane. Preincubation of the cells with Pertussis-toxin prevented the generation of LPA-induced [Ca2+]i signals. We conclude that cultured rat oligodendrocytes express functional LPA receptors, which mediate a transmembrane Ca2+ influx via a Pertussis-toxin-sensitive G-protein.

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.

Changes in [Ca2+]0 during anoxia in CNS white matter.

Irreversible anoxic injury of axons in the rat optic nerve requires the presence of extracellular Ca2+. To test the hypothesis that Ca2+ enters an intracellular compartment during anoxia we monitored [Ca2+]0 in this CNS white matter tract using ion-sensitive microelectrodes. Periods of anoxia lasting 15 min resulted in a rapid, reversible increase in [Ca2+]0 accompanied by transient loss of nerve conduction. This increase in [Ca2+]0 was apparently the result of extracellular space shrinkage. Anoxic periods lasting 60 min resulted in an initial rise followed by a sustained fall in [Ca2+]0, indicative of net influx of Ca2+ into an intracellular compartment. Following reoxygenation after 60 min of anoxia, [Ca2+]0 slowly returned toward control levels but nerve conduction recovered incompletely, indicating irreversible loss of function. Removal of bath Ca2+ lowered [Ca2+]0 to about 100 microM, prevented the anoxia-induced fall in [Ca2+]0, and protected against irreversible loss of the compound action potential.

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.

Axon conduction and survival in CNS white matter during energy deprivation: a developmental study.

We investigated the postnatal development of axon sensitivity to the withdrawal of oxygen, glucose, or the combined withdrawal of oxygen + glucose in the isolated rat optic nerve (a CNS white matter tract). Removal of either oxygen or glucose for 60 min resulted in irreversible injury in optic nerves from adult rats, assessed by loss of the evoked compound action potential (CAP). Optic nerves at ages 45 min caused the selective loss of late CAP components; this was not seen with oxygen deprivation. The amplitude of the early component recovered to 94.8% of control after 60 min of glucose withdrawal, although total CAP area recovered to only 42.3%. Combined oxygen + glucose withdrawal for 60 min produced a greater degree of permanent CAP loss than 60 min of glucose or oxygen withdrawal individually in optic nerves from rats older than P4. Younger than P4 optic nerves showed no permanent loss of function from 60 min of combined oxygen + glucose withdrawal. Unexpectedly, optic nerves from P21-P49 rats recovered significantly less after all three conditions than adult opticnerves (>P50). It is probable that this period of final myelination corresponds to a time of heightened metabolic activity in white matter. The tolerance of CNS white matter to energy deprivation can be categorized into four stages that are correlated with specific developmental features: premyelination (P0-P4), highly tolerant to anoxia, aglycemia and combined anoxia/aglycemia; early myelination (P5-P20), partially tolerant of anoxia and aglycemia but not to combined anoxia/aglycemia; late myelination (P21-P49), very low tolerance of anoxia, aglycemia and combined anoxia/aglycemia; and mature (>P50), low tolerance of anoxia, aglycemia and combined anoxia/aglycemia. The relative resistance of optic nerve function to glucose withdrawal in the presence of oxygen, compared with glucose withdrawal in the absence of oxygen, is presumably due to the presence of oxygen-dependent energy reserves such as astrocytic glycogen, amino acids. and phospholipids.

Does astrocytic glycogen benefit axon function and survival in CNS white matter during glucose deprivation?

Axons, the functional elements in CNS white matter, are frequently injured by ischemia, especially in the context of stroke. The pathophysiology of axonal injury induced by energy deprivation has been analyzed in the rat optic nerve and involves excessive calcium influx by way of reverse Na+/Ca2+ exchange and Ca2+ channels. Evidence is presented that CNS axonal function can be supported in the absence of glucose by intrinsic energy reserves provided through the breakdown of astrocytic glycogen. It is argued that energy is transferred from astrocytes to axons in the form of lactate, which is able to maintain axonal function when substituted for glucose. These observations complement the increasingly convincing hypothesis that astrocytes and neurons interact metabolically, both in the course of normal activity and under pathological conditions such as ischemia. The emerging picture would be no surprise to Camillo Golgi, who predicted a close facsimile of this glial-neuronal interaction more than a century ago.

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.

Ischemic injury of optic nerve axons: the nuts and bolts.

Anterior ischemic optic neuropathy is the most common cause of persistent monocular visual loss in persons over the age of 50. At the heart of this form of optic neuropathy is a sequence of cytoplasmic and membrane events that culminate in axonal destruction. Early depletion of ATP is followed by membrane depolarization, influx of Na+ and Ca2+ via specific voltage-gated channels and reverse operation of the Na+/Ca2+ exchange protein. Toxic Ca2+ overload is the ultimate consequence of these events. Preventing or modulating any of these well-defined steps mitigates against the development of anoxic injury. Translating these molecular insights about how optic nerve axons are damaged by ischemia-like conditions into clinical gains remains the challenge for the future.

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.