Astrocyte membrane responses and potassium accumulation during neuronal activity

Older studies suggest that astrocytes act as potassium electrodes and depolarize with the potassium efflux accompanying neuronal activity. Newer studies suggest that astrocytes depolarize in response to neuronal glutamate release and the activity of electrogenic glial glutamate transporters, thus casting doubt on the fidelity with which astrocytes might sense extracellular potassium rises. Any K(+)-induced astrocyte depolarization might reflect a spatial buffering effect of astrocytes during neuronal activity. For these reasons, we studied stimulus-evoked currents in hippocampal CA1 astrocytes. Hippocampal astrocytes exhibited stimulus-evoked transient glutamate transporter currents and slower Ba(2+)-sensitive inward rectifier potassium (K(ir)) currents. In whole-cell astrocyte recordings, Ba(2+) blocked a very weakly rectifying component of the astrocyte membrane conductance. The slow stimulus-elicited current, like measurements from K(+)-sensitive electrodes under the same conditions, predicted small bulk [K(+)](o) increases (<0.5 mM) following the termination of short-stimulus trains. These currents indicate the potential for astrocyte spatial K(+) buffering. However, Ba(2+) did not significantly affect resting [K(+)](o) or the [K(+)](o) rises detected by the K(+)-sensitive electrode. To test whether local K(+) rises may be significantly higher than those detected by glial recordings or by K(+) electrodes, we assayed EPSCs and fiber volleys, two measures very sensitive to K(+) increases. We found that Ba(2+) had little effect on neuronal axonal or synaptic function during short-stimulus trains, indicating that K(ir)s do not influence local [K(+)](o) rises enough, under these conditions to affect synaptic transmission. In conclusion, our results indicate that hippocampal astrocytes are faithful sensors of [K(+)](o) rises, but we find little evidence for physiologically relevant spatial K(+) buffering during brief bursts of presynaptic activity.     

Regulation of extracellular calcium in the hippocampus in vivo during epileptiform activity--role of astrocytes

Astrocytes have been suggested to regulate the extracellular calcium concentration ([Ca(2+)](o)), but this has not been thoroughly investigated. Adult, male Sprague-Dawley rats were used to record changes in [Ca(2+)](o) in the hippocampus during epileptiform activity. Maximal decreases in [Ca(2+)](o) in CA1 were measured in the pyramidal cell layer during 20 Hz, 20s stimulus trains to the contralateral CA3 region. Maximal decreases in [Ca(2+)](o) in the dentate gyrus were measured when maximal dentate activation had appeared-irrespective of the location, frequency or duration of the stimulation. Maximal decreases were 36% greater in the dentate gyrus than in CA1. During prolonged discharges, [Ca(2+)](o) recovered partially towards the baseline in both hippocampal regions. To investigate the role of astrocytes, local injections of fluorocitrate (FC), a metabolic toxin selectively taken up by astrocytes, were used. FC (0.1, 0.25 or 0.5mM FC), but not vehicle (2 microl), caused a small, but significant decrease in the maximal changes in CA1, but an increase in the dentate gyrus. The results suggest that maximal decreases in [Ca(2+)](o) occur in the hippocampus in response to burst firing of neurons and that astrocytes play a minimal role in the regulation of [Ca(2+)](o) during epileptiform activity.     

Background potassium concentrations and epileptiform discharges. I. Electrophysiological characteristics of neuronal activity

Intra- and extracellular recording techniques were used to study the epileptiform activity generated by guinea pig hippocampal slices perfused with free-magnesium artificial cerebrospinal fluid in the presence of physiologic (4 mM), reduced (2 mM) or elevated (8 mM) extracellular potassium concentrations ([K(+)](o)). Extracellular field potentials along with intracellular recordings were recorded in CA1 or CA3 region. Reduction of [K(+)](o) significantly increased the latency of epileptiform field potential (EFP) appearance as well as burst discharge duration and decreased EFP repetition rate. Depending on different background [K(+)](o), epileptiform burst discharges appeared in different patterns including varied types of paroxysmal depolarisation shifts and burst activity in CA1 and CA3 subfields. Comparison with physiological and increased [K(+)](o,) reduction of [K(+)](o) significantly increased the mean duration of bursts, mean amplitude of depolarisation, mean after-hyperpolarisation duration, and inter-spike intervals in both CA1 and CA3 areas. Three distinct patterns were distinguished on the basis of their evoked firing pattern in response to application of depolarising current pulses in the interval of epileptiform burst discharges. Neurons superfused with 2 mM [K(+)](o) presented fast adapting pattern while cells washed with 4 or 8 mM [K(+)](o) exhibited intrinsically bursting or slow adapting patterns. Comparing the groups with different background [K(+)](o), there is a more severe form of discharges in low K(+) and a subtle difference between 4 and 8 mM K(+). The data indicate the importance of background [K(+)](o) on epileptiform burst discharge pattern and characteristics.     

Relationship between calcium-dependent potassium channel and ectopic spontaneous discharges of injured dorsal root ganglion neurons in the rat.

The ectopic spontaneous discharges (ESD) of single myelinated dorsal root fiber originated from the injured dorsal root ganglion (DRG) neurons were recorded in vivo. When [Ca(2+)](o) perfusing the injured DRG had been enhanced or caffeine been used, the ESD was inhibited in dose-dependent manner, while using Ni(2+) or EGTA, the ESD facilitated. The increment of [K(+)](o) and the use of TEA could both facilitate the ESD in dose-dependent manner. Apamin, a special antagonist of calcium-dependent potassium channel (K(Ca)), had markedly increased the number of ESD. These results suggest that the generation of ESD has close relation to the activity of K(Ca) channel.     

Shunting and hyperpolarizing GABAergic inhibition in the high-potassium model of ictogenesis in the developing rat hippocampus

Ontogenesis of GABAergic signaling may play an important role in developmental changes in seizure susceptibility in the high-potassium model of ictogenesis in vitro. The age-dependent effects of [K(+)](o) on the reversal potential of the GABA(A)-mediated responses and membrane potential in hippocampal slices in vitro were compared with the effect of GABA(A)-receptors antagonists and GABA(A) modulators on high-potassium induced seizures in the CA3 pyramidal layer of rat hippocampus in vivo. GABA(A) responses were depolarizing at P8-12 and hyperpolarizing at P17-21. In P8-12 rats, GABA(A) responses switch their polarity from depolarizing to hyperpolarizing upon elevation of extracellular potassium. At approximately 10 mM [K(+)](o), activation of GABA(A) receptors produced an isoelectric, purely shunting response characterized by no changes in the membrane potential but an increase in the membrane conductance. In P17-21 rats, the hyperpolarizing GABA(A) driving force progressively increased with elevation of [K(+)](o). In P8-12 rats in vivo, GABA(A)-receptor antagonists did not affect the occurrence of ictal discharges induced by intrahippocampal injection of 10 mM [K(+)](o), but significantly increased seizure duration. Diazepam and isoguvacine completely prevented seizures induced by 10 mM [K(+)](o). In P17-21 rats, GABA(A)-receptor antagonists strongly increased the occurrence of ictal activity induced both by 10 mM [K(+)](o). Taken together, these results suggest that anticonvulsive effects of GABA are because of the combination of shunting and hyperpolarizing actions of GABA. Although shunting GABA is already efficient in the young age group, a developmental increase in the hyperpolarizing GABA(A) driving force likely contributes to the increase in the GABAergic control of seizures upon maturation.     

Intracellular calcium handling in rat olfactory ensheathing cells and its role in axonal regeneration

Intracellular calcium handling by rat olfactory ensheathing cells (OECs) is implicated in their support for regrowth of adult CNS neurites in a coculture model of axonal regeneration. Pretreatment of OECs with BAPTA-AM to sequester glial intracellular calcium ([Ca(2+)](i)) reduces significantly the numbers of cocultured neurons regrowing neurites. The mean resting [Ca(2+)](i) of OECs cultured alone or with neurons was 300 nM in an external solution containing 2.5 mM calcium ([Ca(2+)](o)). In high [K(+)](o) or zero [Ca(2+)](o), resting [Ca(2+)](i) significantly decreased. [Ca(2+)](i) significantly increased when [Ca(2+)](o) was increased to 20 mM, lonomycin, thapsigargin, and thimerosal increased [Ca(2+)](i), and caffeine, ryanodine, and cyclopiazonic acid were without effect. Of the receptor agonists tested, none induced a change in [Ca(2+)](i). The calcium influx induced by high [Ca(2+)](o) was blocked by La(3+) and SKF96365, partially inhibited by Cd(2+), and insensitive to Ni(2+) and nifedipine. Pretreatment of OECs with La(3+) reduced neurite regrowth in cocultures in a concentration-dependent manner over the range that blocked the non-voltage-gated calcium flux through a putative TRP-like channel, which, we propose, is activated in OEC-mediated axonal regeneration.     

Alterations in the microenvironment during spreading depression associated with epileptiform activity in the immature neocortex

Local changes in extracellular ion concentrations were measured with ion-sensitive microelectrodes in slices of mature (greater than 40 days of age) or immature (16-30 days of age) rat neocortex maintained in vitro. Repetitive stimulation resulted in increases in extracellular potassium ([K+]o) to levels of 8.85 +/- 2.1 mM in slices from adult animals and 12.77 +/- 1.8 mM in slices from immature animals. During exposure to picrotoxin, maximum levels were 11.3 +/- 2.6 and 14.8 +/- 2.5 mM in the mature and immature groups, respectively. Picrotoxin (50 microM) induced spontaneous bursts of repetitive spiking, followed by a slow, negative field potential, associated with spreading depression (SD), in slices from immature animals. [K+]o levels increased to 10.2 +/- 3.9 mM during repetitive spike discharges and reached 30.3 +/- 18.5 mM during SDs. Variations in the size of the extracellular space (ES) were examined during SD. The ES was found to reversibly decrease by 39 +/- 4.5%. Clusters of repetitive spikes were associated with 0.1-0.2 mM decreases in [Ca2+]o, whereas 1.12 +/- 0.06 mM decreases were observed during SDs. Decreases in [Na+]o and [Cl-]o of 56 +/- 10 mM and 41 +/- 9 mM, respectively, were observed during SDs suggesting that a net transmembrane movement of water occurred during SDs. These results indicate that changes in [K+]o associated with epileptiform activity in the immature nervous system are quantitatively different from those observed in the mature brain. These large increases in [K+]o may contribute to the prolonged nature of epileptiform discharges in the developing nervous system.     

Calcium dependence of rapid astrocyte death induced by transient hypoxia, acidosis, and extracellular ion shifts

Exposure to hypoxic, acidic, ion-shifted Ringer (HAIR) for 15-40 min has been shown to cause rapid astrocyte death upon reperfusion with normal media. The ion shifts of the HAIR solution included a rise in extracellular K(+) (e.g., [K(+)](o)) and a fall in [Na(+)](o), [Cl(-)](o), and [Ca(2+)](o), characteristic of ischemic-traumatic brain insults. We investigated the ionic basis of the HAIR-induced injury. After HAIR exposure, reperfusion in 0 Ca(2+)/EGTA media completely protected astrocytes. Preincubation of cells in BAPTA-AM ester was also protective, indicating that the injury was triggered by Ca(2+) influx during reperfusion. Neither nimodipine, CNQX, APV, nor TTX reduced injury. Astrocyte death could be blocked by 100 microM Ni(2+) or 100 microM benzamil, suggesting involvement of Na(+)-Ca(2+) exchange. KB-R7943, which preferentially inhibits reverse Na(+)-Ca(2+) exchange, also protected astrocytes. Elevation of [K(+)](o) was not necessary for astrocyte death. However, when [Na(+)](o) was maintained at 151 mM throughout the HAIR protocol, cell death was markedly reduced. We postulate that [Na(+)](o) shifts aid reversal of Na(+)-Ca(2+) exchange by favoring cytosolic Na(+) loading. Possible means of astrocytic Na(+) accumulation are discussed.     

Extracellular K+ and Ca2+ changes during epileptiform discharges in the immature rat neocortex

Picrotoxin-induced epileptiform activity was examined in neocortical slices prepared from 8- to 15-day-old rats. This activity consisted of spontaneous bursts of 3-5 discharges that resembled interictal spikes and were interspersed with ictal-like paroxysms lasting 10-30 s. Measurements of extracellular potassium ([K+]o) and calcium ([Ca2+]o) were made during these spontaneous epileptiform events, using ion-sensitive electrodes. Individual interictal spikes were associated with [Ca2+]o decreases of 0.1-0.2 mM, whereas sustained ictal-like discharges were accompanied by decreases of 0.3-0.4 mM. Measurement of [K+]o showed that individual interictal spikes were associated with increases in [K+]o up to 12 mM, whereas increases to more than 20 mM accompanied long-lasting ictal-like discharges. Maximum increases in [K+]o were observed ca. 600 microns below the pial surface. [K+]o increases were followed by undershoots of the resting [K+]o level. The unusually high [K+]o levels associated with epileptiform discharges in the immature neocortex suggest that disturbances in [K+]o regulation may contribute to the generation of the picrotoxin-induced, spontaneous, prolonged ictal-like discharges observed in the 8- to 15-day age group.     

Epileptiform discharges induced by altering extracellular potassium and calcium in the rat hippocampal slice

During and after intense neuronal activity the concentration of extracellular potassium ([K+]o) increases while the concentration of calcium ([Ca2+]o) decreases. The present study examined the effect of increased [K+]o alone, and with a parallel decrease in [Ca2+]o, on overall excitability, long-term potentiation (LTP), and the appearance of epileptiform discharges. [K+]o and [Ca2+]o were varied over the range in which they fluctuate in vivo. Hippocampal slices were first equilibrated in a control artificial CSF containing 3.1 mM K+ and 1.5 mM Ca2+ and then reequilibrated in an identical solution except that the K+ was increased to 3.55, 4, 5, 6, or 8 mM with and without a decrease in Ca2+ to 1.0 mM. Raising [K+]o caused a leftward shift of input-output curves. Lowering [Ca2+]o to 1.0 mM had no effect on the ability of [K+]o to shift the input-output curve to the left. LTP was not changed by increasing [K+]o. Lowering [Ca2+]o to 1.0 mM blocked LTP and increasing the [K+]o did not overcome this blockade. When [K+]o alone was altered, the [K+]oS at which epileptiform bursts occurred 50% of the time were 5.6 and 7.6 mM for stimulus-locked and spontaneous bursting, respectively. The combination of decreased [Ca2+]o and increased [K+]o made slices considerably more prone to epileptiform activity. In 1.0 mM [Ca2+]o, the [K+]o at which 50% of the slices showed stimulus-locked bursting was decreased to 3.6 mM while that for spontaneous discharges was 5.4 mM. The sensitivity of hippocampal slices to [K+]o and [Ca2+]o, and the synergistic actions of alterations of these ions, indicates that even small changes in the aggregate extracellular ionic milieu may be important in epileptogenesis.     

Background potassium concentrations and epileptiform discharges. II. Involvement of calcium channels

Potassium- and calcium conductances regulate neuronal excitability and epileptiform activity. In this study, the effects of different extracellular potassium concentrations ([K(+)](o)) were investigated on the modulatory effect of the L-type transmembranous calcium currents on epileptiform discharges. The in vitro brain slice technique was used to examine the effects of calcium channel blockers, verapamil and nifedipine, on the repetition rate, amplitude, and duration of epileptiform field potentials (EFP) in the presence of low, physiological, and high background [K(+)](o) in guinea pig hippocampal slices. Epileptiform activity was induced by omission of Mg(2+) from artificial cerebrospinal fluid contained 2, 4, and 8 mM [K(+)](o). Both verapamil and nifedipine suppressed EFP after a transient increase in repetition rate. The extent of EFP frequency rate acceleration significantly increased with reduction of [K(+)](o). The increase in EFP frequency rate induced by application of verapamil and nifedipine was accompanied by a reduction in the EFP amplitude and a reversible increase in the burst discharge duration. The extent of burst discharge prolongation was also significantly higher with decreasing [K(+)](o). Further application of verapamil and nifedipine suppressed the epileptiform burst activity in the presence of different [K(+)](o). The latency of EFP depression was significantly diminished both with increased and decreased background potassium concentrations. The data indicate the importance of the effect of the L-type transmembranous calcium currents on the regulatory effect of background [K(+)](o) on epileptiform burst discharge frequency and duration.     

Antioxidants prevent elevation in [Ca(2+)](i) induced by low extracellular magnesium in cultured canine cerebral vascular smooth muscle cells: possible relationship to Mg(2+) deficiency-induced vasospasm and stroke

Low serum concentrations of Mg(2+) ions have been reported, recently, in patients with coronary disease, atherosclerosis and stroke as well as in patients with cerebral hemorrhage. The aim of the present study was to determine whether potent antioxidants [alpha-tocopherol and pyrrolidine dithiocarbamate (PDTC)] can prevent or ameliorate intracellular Ca(2+) ([Ca(2+)](i)) overload associated with cerebral vascular injury induced by low extracellular free Mg(2+) ([Mg(2+)](o)). Exposure of cultured canine cerebral vascular smooth muscle cells to low [Mg(2+)](o) (0.15-0.6 mM) vs. normal [Mg(2+)](o) (1.2 mM) for either 10 min or 2 h induced concentration-dependent rises in [Ca(2+)](i). Treatment of the cultured cells with either PDTC (0.1 microM) or alpha-tocopherol (15 microM) for 24 h, alone, failed to interfere with basal [Ca(2+)](i) levels. However, preincubation of the cells with either alpha-tocopherol or PDTC for 24 h completely inhibited the elevation of [Ca(2+)](i) induced by exposure to low [Mg(2+)](o), not only for 10 min, but also for 2 h. These results indicate that alpha-tocopherol and PDTC prevent rises in [Ca(2+)](i) produced by low [Mg(2+)](o), which probably result from low [Mg(2+)](o)-induced lipid peroxidation of cerebral vascular smooth muscle cell membranes. Moreover, these new results suggest that such protective effects of alpha-tocopherol and PDTC on cerebral vascular cells might be useful therapeutic tools in cerebral vascular injury associated with low [Mg(2+)](o) and accumulation of [Ca(2+)](i).     

Astrocytes contribute to regulation of extracellular calcium and potassium in the rat cerebral cortex during spreading depression

This study used spreading depression (SD), which is characterized by redistribution of ions, to examine the role of astrocytes in the regulation of extracellular potassium ([K+]o) and calcium ([Ca2+]o) levels. Recurrent spreading depression episodes were induced by application of 3 M potassium chloride to the cortex of adult anesthetized rats while monitoring the extracellular direct current (DC) potential shifts and changes in [K+]o or [Ca2+]o 6-7 mm away. The reversible glial toxins, fluorocitrate (FC) and fluoroacetate (FA), were injected locally into the cortex at doses that are selective for reducing glial function. The peak changes and area under the curve for [K+]o and [Ca2+]o, recovery rate for [K+]o, and interval between spreading depression episodes were measured before and at various times after administration of the toxins. Both fluorocitrate and fluroacetate slowed the recovery of the [K+]o and altered the recovery of the [Ca2+]o. Local injection of glutamate uptake inhibitors or barium had no effect on the peak changes in [K+]o or the rate of recovery of the [K+]o. The slowing of the recovery rate is consistent with the hypothesis that glial cells play a role in the return of [K+]o to baseline after spreading depression in the cortex in vivo. The change in movement of calcium after administration of FC suggests that astrocytes normally extrude calcium during spreading depression, resulting in rapid recovery of the levels of [Ca2+]o with an overshoot. These findings demonstrate that astrocytes contribute to the regulation of both potassium and calcium during and after a stress to the ionic homeostatic mechanisms.     

Effects of barium, furosemide, ouabaine and 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) on ionophoretically-induced changes in extracellular potassium concentration in hippocampal slices from rats and from patients with epilepsy

Glial cells limit local K(+)-accumulation by K(+)-uptake through different mechanisms, sensitive to Ba(2+), ouabaine, furosemide, or DIDS. Since the relative contribution of these mechanisms has not yet been determined, we studied the effects of bath-applied barium (2 mM), ouabaine (9 microM), furosemide (2 mM), and DIDS (1 mM) on ionophoretically-induced rises in [K(+)](o) in the pyramidal layer of area CA1 from normal rat slices, in the presence of glutamate receptor (Glu-R) antagonists. We also investigated the effect of barium on ionophoretically-induced tetrapropylammonium (TPA(+))-signals in order to test for barium-induced changes of the extracellular space. Finally, we repeated the barium experiment on slices from human non-sclerotic and sclerotic hippocampal specimens to assess a reduced glial capability for barium-sensitive K(+)-uptake in sclerotic tissue from epilepsy patients. In normal rat slices barium augmented ionophoretically-induced rises in [K(+)](o) by approximately 120%, also in the presence of tetrodotoxin (TTX) (by approximately 150%), but did not significantly affect the TPA(+)-signal. Ouabaine also augmented the K(+)-signal, but only by 27%. Furosemide and DIDS had negligible effects. In slices from sclerotic human hippocampus an augmentation of the K(+)-signal by barium was absent. Thus barium augments ionophoretically-induced K(+)-signals to a similar extent as previously shown for stimulus-induced signals. We suggest that glial barium-sensitive K(+)-buffer mechanisms reduce fast local rises of [K(+)](o) by at least 50%. This capability of glial cells is extremely reduced in area CA1 of slices from human sclerotic hippocampal specimens.     

Effect of halothane on neuronal excitation in the superficial dorsal horn of rat spinal cord slices: evidence for a presynaptic action

The action of the volatile anaesthetic halothane on optically recorded neuronal excitation in juvenile rat spinal cord slices was investigated. Prolonged neuronal excitation lasting approximately 100 ms was evoked in the superficial dorsal horn after single-pulse dorsal root stimulation that activated both A- and C-fibres. Halothane depressed the neuronal excitation in a concentration-dependent manner (IC(50) 0.21 mm, I(max) 28%). In Ca(2+)-free solution, dorsal root stimulation induced excitation with a short duration of several tens of milliseconds, in which the excitation of the postsynaptic component was largely eliminated. Under these conditions, halothane also depressed the excitation concentration-dependently (IC(50) 0.46 mm, I(max) 60%). Most of the suppression occurred within 5 min of halothane application, and the effect of halothane was fully reversible upon washout of the anaesthetic. Application of bicuculline and strychnine or picrotoxin, or reduction of extracellular Cl(-) concentration ([Cl(-)](o)), had no effect on halothane inhibition. Applications of K(+) channel blockers tetraethyl ammonium, 4-aminopyridine, Cs(+) or Ba(2+) either had no effect or augmented the inhibitory effect of halothane. On the other hand, the degree of inhibition by halothane was found to be dependent on [K(+)](o); the higher [K(+)](o), the larger the depression. In addition, decreases in [Na+]o and [Mg(2+)](o) reduced the excitation similar to that of halothane treatment, and the degree of halothane inhibition became larger with lower [Mg(2+)](o). These results lead to a hypothesis that halothane suppresses the excitation of presynaptic elements by inhibiting presynaptic Na(+) channels by shifting the steady-state inactivation curve in the hyperpolarizing direction.     

Glutamate, NMDA, and AMPA induced changes in extracellular space volume and tortuosity in the rat spinal cord.

Glutamate release, particularly in pathologic conditions, may result in cellular swelling. The authors studied the effects of glutamate, N-methyl-D-aspartate (NMDA), and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) on extracellular pH (pH(e)), extracellular potassium concentration ([K(+)](e)), and changes in extracellular space (ECS) diffusion parameters (volume fraction alpha, tortuosity lambda) resulting from cellular swelling. In the isolated spinal cord of 4-to 12-day-old rats, the application of glutamate receptor agonists induced an increase in [K(+)](e), alkaline-acid shifts, a substantial decrease in alpha, and an increase in lambda. After washout of the glutamate receptor agonists, alpha either returned to or overshot normal values, whereas lambda remained elevated. Pretreatment with 20 mmol/L Mg(++), MK801, or CNQX blocked the changes in diffusion parameters, [K(+)](e) and pH(e) evoked by NMDA or AMPA. However, the changes in diffusion parameters also were blocked in Ca(2+)-free solution, which had no effect on the [K(+)](e) increase or acid shift. The authors conclude that increased glutamate release may produce a large, sustained and [Ca(2+)](e)-dependent decrease in alpha and increase in lambda. Repetitive stimulation and pathologic states resulting in glutamate release therefore may lead to changes in ECS volume and tortuosity, affecting volume transmission and enhancing glutamate neurotoxicity and neuronal damage.     

Neocortical microenvironment in patients with intractable epilepsy: potassium and chloride concentrations

PURPOSE: The regulation of extracellular ion concentrations plays an important role in neuronal function and epileptogenesis. Despite the many studies into the mechanisms of epileptogenesis in human experimental models, no data are available regarding the fluctuations of extracellular potassium ([K(+)](o)) and chloride ([Cl(-)](o)) concentrations, which could underlie seizure susceptibility in human chronically epileptic tissues in vivo. METHODS: By using cerebral microdialysis during surgical resection of epileptic foci, the basic [K(+)](o) and [Cl(-)](o) as well as their changes after epicortical electric stimulation were studied in samples of dialysates obtained from 11 patients by ion-selective microelectrodes. RESULTS: The mean basal values of [K(+)](o) and [Cl(-)](o) in all patients were 3.83 +/- 0.08 mM and 122.9 +/- 2.6 mM, respectively. However, significant differences were observed in the basal levels of both [K(+)](o) and [Cl(-)](o) between different patients. Statistically, no correlation was found between basal [K(+)](o) or [Cl(-)](o) and electrocorticogram (ECoG) spike activity, but in one patient, dramatically lowered baseline [Cl(-)](o) was accompanied by enhanced ECoG spike activity. Application of epicortical electrical stimulation increased [K(+)](o) but not [Cl(-)](o) in all cases. According to the velocity as well as spatial distribution of [K(+)](o) reduction to the prestimulation levels, three different types of responses were observed: slow decline, fast decline, and slow and fast declines at adjacent sites. CONCLUSIONS: These data may represent abnormalities in ion homeostasis of the epileptic brain.     

Excitotoxic death induced by released glutamate in depolarized primary cultures of mouse cerebellar granule cells is dependent on GABAA receptors and niflumic acid-sensitive chloride channels

Excitotoxic neuronal death has been linked to neurological and neurodegenerative diseases. Several studies have sought to clarify the involvement of Cl(-) channels in neuronal excitotoxicity using either N-methyl-D-aspartic acid (NMDA) or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainic acid agonists. In this work we induced excitotoxic death in primary cultures of cerebellar granule cells by means of endogenously released glutamate. Excitotoxicity was provoked by exposure to high extracellular K(+) concentrations ([K(+)](o)) for 5 min. Under these conditions, a Ca(2+)-dependent release of glutamate was evoked. When extracellular glutamate concentration rose to between 2 and 4 microM, cell viability was significantly reduced by 30-40%. The NMDA receptor antagonists (MK-801 and D-2-amino-5-phosphonopentanoic acid) prevented cell death. Exposure to high [K(+)](o) produced a (36)Cl(-) influx which was significantly reduced by picrotoxinin. In addition, the GABA(A) receptor antagonists (bicuculline, picrotoxinin and SR 95531) protected cells from high [K(+)](o)-triggered excitotoxicity and reduced extracellular glutamate concentration. The Cl(-) channel blockers niflumic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid also exerted a neuroprotective effect and reduced extracellular glutamate concentration, even though they did not reduce high [K(+)](o)-induced (36)Cl(-) influx. Primary cultures of cerebellar granule cells also contain a population of GABAergic neurons that released GABA in response to high [K(+)](o). Chronic treatment of primary cultures with kainic acid abolished GABA release and rendered granule cells insensitive to high [K(+)](o) exposure, even though NMDA receptors were functional. Altogether, these results demonstrate that, under conditions of membrane depolarization, low micromolar concentrations of extracellular glutamate might induce an excitotoxic process through both NMDA and GABA(A) receptors and niflumic acid-sensitive Cl(-) channels.     

Potassium-induced enhancement of persistent inward current in hippocampal neurons in isolation and in tissue slices

Previous work suggested a role for the voltage-dependent persistent sodium current, I(Na,P), in the generation of seizures and spreading depression (SD). Ordinarily, I(Na,P) is small in hippocampal neurons. We investigated the effect of raising external K(+) concentration, [K(+)](o), on whole-cell persistent inward current in freshly isolated hippocampal CA1 pyramidal neurons. I(Na,P) was identified by TTX-sensitivity and dependence on external Na(+) concentration. When none of the ion channels were blocked, I(Na,P) was not usually detectable, probably because competing K(+) current masked it, but after raising [K(+)](o) I(Na,P) appeared, while K(+) currents diminished. With K(+) channels blocked, I(Na,P) could usually be evoked in control solution and raising [K(+)](o) caused its reversible increase in most cells. The increase did not depend on external calcium [Ca(2+)](o). In CA1 pyramidal neurons in hippocampal slices a TTX-sensitive persistent inward current was always recorded and when [K(+)](o) was raised, it was reversibly enhanced. Strong depolarization evoked irregular current fluctuations, which were also augmented in high [K(+)](o). The findings support a role of potassium-mediated positive feedback in the generation of seizures and spreading depression.     

Allopregnanolone modulates spontaneous GABA release via presynaptic Cl- permeability in rat preoptic nerve terminals

The endogenous neurosteroid 3alpha-hydroxy-5alpha-pregnane-20-one (allopregnanolone) affects presynaptic nerve terminals and thereby increases the frequency of spontaneous GABA release. The present study aimed at clarifying the mechanisms underlying this presynaptic neurosteroid action, by recording the frequency of spontaneous GABA-mediated inhibitory postsynaptic currents (sIPSCs) in neurons from the medial preoptic nucleus (MPN) of rat. Acutely dissociated neurons with functional adhering nerve terminals were studied by perforated-patch recording under voltage-clamp conditions. It was shown that the sIPSC frequency increased with the external K(+) concentration ([K(+)](o)). Further, the effect of allopregnanolone on the sIPSC frequency was strongly dependent on [K(+)](o). In a [K(+)](o) of 5 mM, 2.0 microM allopregnanolone caused a clear increase in sIPSC frequency. However, the effect declined rapidly with increased [K(+)](o) and at high [K(+)](o) allopregnanolone reduced the sIPSC frequency. The effect of allopregnanolone was also strongly dependent on the external Cl(-) concentration ([Cl(-)](o)). In a reduced [Cl(-)](o) (40 mM, but with a standard [K(+)](o) of 5 mM), the effect on sIPSC frequency was larger than that in the standard [Cl(-)](o) of 146 mM. The dependence of the effect of allopregnanolone on [K(+)](o) and on estimated presynaptic membrane potential was also altered by the reduction in [Cl(-)](o). As in standard [Cl(-)](o), the effect in low [Cl(-)](o) declined when [K(+)](o) was raised, but reversed at a higher [K(+)](o). The GABA(A) receptor agonist muscimol also potentiated the sIPSC frequency. Altogether, the results suggest that allopregnanolone exerts its presynaptic effect by increasing the presynaptic Cl(-) permeability, most likely via GABA(A) receptors.