Coupling of AMPA receptors with the Na/Ca exchanger in cultured rat astrocytes

Astrocytes exhibit three transmembrane Ca²⁺ influx pathways: voltage-gated Ca²⁺ channels (VGCCs), the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) class of glutamate receptors, and Na⁺/Ca²⁺ exchangers. Each of these pathways is thought to be capable of mediating a significant increase in Ca²⁺ concentration ([Ca²⁺]_i); however, the relative importance of each and their interdependence in the regulation astrocyte [Ca²⁺]_i is not known. We demonstrate here that 100 μM AMPA in the presence of 100 μM cyclothiazide (CTZ) causes an increase in [Ca²⁺]_i in cultured cerebral astrocytes that requires transmembrane Ca²⁺ influx. This increase of [Ca²⁺]_i is blocked by 100 μM benzamil or 0.5 μM U-73122, which inhibit reverse-mode operation of the Na⁺/Ca²⁺ exchanger by independent mechanisms. This response does not require Ca²⁺ influx through VGCCs, nor does it depend upon a significant Ca²⁺ influx through AMPA receptors (AMPARs). Additionally, AMPA in the presence of CTZ causes a depletion of thapsigargin-sensitive intracellular Ca²⁺ stores, although depletion of these Ca²⁺ stores does not decrease the peak [Ca²⁺]_i response to AMPA. We propose that activation of AMPARs in astrocytes can cause [Ca²⁺]_i to increase through the reverse mode operation of the Na⁺/Ca²⁺ exchanger with an associated release of Ca²⁺ from intracellular stores. This proposed mechanism requires neither Ca²⁺-permeant AMPARs nor the activation of VGCCs to be effective.     

Effect of the removal of extracellular Ca on the response of cytosolic concentrations of Ca to ouabain in carotid body glomus cells of adult rabbits

Effect of the removal of extracellular Ca²⁺ on the response of cytosolic concentrations of Ca²⁺ ([Ca²⁺]_i) to ouabain, an Na⁺/K⁺ exchanger antagonist, was examined in clusters of cultured carotid body glomus cells of adult rabbits using fura-2AM and microfluorometry. Application of ouabain (10 mM) induced a sustained increase in [Ca²⁺]_i (mean±S.E.M.; 38±5% increase, n=16) in 55% of tested cells (n=29). The ouabain-induced [Ca²⁺]_i increase was abolished by the removal of extracellular Na⁺. D600 (50 μM), an L-type voltage-gated Ca²⁺ channel antagonist, inhibited the [Ca²⁺]_i increase by 57±7% (n=4). Removal of extracellular Ca²⁺ eliminated the [Ca²⁺]_i increase, but subsequent washing out of ouabain in Ca²⁺-free solution produced a rise in [Ca²⁺]_i (62±8% increase, n=6, P<0.05), referred to as a [Ca²⁺]_i rise after Ca²⁺-free/ouabain. The magnitude of the [Ca²⁺]_i rise was larger than that of ouabain-induced [Ca²⁺]_i increase. D600 (5 μM) inhibited the [Ca²⁺]_i rise after Ca²⁺-free/ouabain by 83±10% (n=4). These results suggest that ouabain-induced [Ca²⁺]_i increase was due to Ca²⁺ entry involving L-type Ca²⁺ channels which could be activated by cytosolic Na⁺ accumulation. Ca²⁺ removal might modify the [Ca²⁺]_i response, resulting in the occurrence of a rise in [Ca²⁺]_i after Ca²⁺-free/ouabain which mostly involved L-type Ca²⁺ channels.     

The ouabain-induced [Ca]i increase in leech Retzius neurones is mediated by voltage-dependent Ca channels

In leech Retzius neurones the inhibition of the Na⁺–K⁺ pump by ouabain causes an increase in the cytosolic free calcium concentration ([Ca²⁺]_i). To elucidate the mechanism of this increase we investigated the changes in [Ca²⁺]_i (measured by Fura-2) and in membrane potential that were induced by inhibiting the Na⁺–K⁺ pump in bathing solutions of different ionic composition. The results show that Na⁺–K⁺ pump inhibition induced a [Ca²⁺]_i increase only if the cells depolarized sufficiently in the presence of extracellular Ca²⁺. Specifically, the relationship between [Ca²⁺]_i and the membrane potential upon Na⁺–K⁺ pump inhibition closely matched the corresponding relationship upon activation of the voltage-dependent Ca²⁺ channels by raising the extracellular K⁺ concentration. It is concluded that the [Ca²⁺]_i increase caused by inhibiting the Na⁺–K⁺ pump in leech Retzius neurones is exclusively due to Ca²⁺ influx through voltage-dependent Ca²⁺ channels.     

Heterogeneity in cytosolic calcium responses to hypoxia in carotid body cells

Previous investigators have reported that intracellular pH responds to hypoxia with a heterogenous pattern in individual glomus cells of the carotid body. The aim of the present study was to examine whether hypoxia had similar effects on cytosolic calcium ([Ca²⁺]_i) in glomus cells, and if so, whether a heterogenous response pattern is also seen in other cell types. Experiments were performed on glomus cells from adult rat carotid bodies, rat pheochromocytoma (PC12) and vascular smooth muscle (A7r5) cells. Changes in [Ca²⁺]_i in individual cells were determined by fluorescence imaging using Fura-2. Glomus cells were identified by catecholamine fluorescence. [Ca²⁺]_i in glomus cells increased in response to hypoxia (pO₂ = 35 ± 8mmHg; 5 min), whereas hypoxia induced decreases in [Ca²⁺]_i were not seen. Increases in [Ca²⁺]_i were observed in 20% of the isolated cells and strings of cells, but clustered glomus cells never responded. The magnitude of the calcium change in responding cells was proportional to the hypoxic stimulus. Under a given hypoxic challenge, there were marked variations in the response pattern between glomus cells. The response pattern characteristic of any given cell was reproducible. At comparable levels of hypoxia, PC12 cells also responded with an increase in [Ca²⁺]_i with a heterogenous response pattern similar to that seen in glomus cells. In contrast, increases in [Ca²⁺]_i in A7r5 cells could be seen only with sustained hypoxia ( ∼ 20 min), and little heterogeneity in the response patterns was evident. These results demonstrate that: (a) hypoxia increases cytosolic calcium in glomus cells; (b) response patterns were heterogeneous in individual cells; and (c) the pattern of the hypoxia-induced changes in [Ca²⁺]_i is cell specific. These results suggest that hypoxia-induced increases in [Ca²⁺]_i are faster in secretory than in non-secretory cells.     

K-current modulated by PO2 in type I cells in rat carotid body is not a chemosensor

According to the membrane channel hypothesis of carotid body O₂ chemoreception, hypoxia suppresses K⁺ currents leading to cell depolarization, [Ca²⁺]_i rise, neurosecretion, increased neural discharge from the carotid body. We show here that tetraethylammonium (TEA) plus 4-aminopyridine (4-AP) which suppressed the Ca²⁺ sensitive and other K⁺ currents in rat carotid body type I cells, with and without low [Ca²⁺]_o plus high [Mg²⁺]_o, did not essentially influence low

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effects on [Ca²⁺]_i and chemosensory discharge. Thus, hypoxia may suppress the K⁺ currents in glomus cells but K⁺ current suppression of itself does not lead to chemosensory excitation. Therefore, the hypothesis that K⁺–O₂ current is linked to events in chemoreception is not substantiated. K⁺–O₂ current is an epiphemenon which is not directly linked with O₂ chemoreception.     

Flow-cytometric estimation on glutamate- and kainate-induced increases in intracellular Ca of brain neurons: a technical aspect

Effects of glutamate and kainate on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in a large population (several thousand) of dissociated cerebellar granule cell neurons were evaluated using a flow-cytometer and a combination of two fluorescent dyes, fluo-3-AM for estimating [Ca²⁺]_i and ethidium bromide for removing neurons that had compromised membranes from the cell population examined. The number of neurons responding to glutamate or kainate in augmenting the fluo-3 fluorescence increased in a dose-dependent manner. The number of neurons responding to kainate was much greater than that to glutamate. CNQX, a blocker of non-NMDA receptors, completely blocked the response elicited by kainate while the complete blockade of this glutamate-induced response was made by a combination of MK-801, a NMDA receptor blocker, and CNQX. Nicardipine, a calcium antagonist, decreased the number of neurons responding to glutamate and kainate, suggesting involvement of voltage-dependent calcium channels. These results indicate that the flow-cytometric measurement of glutamate and kainate responses has the potential to provide answers to such questions as what percentage of the population of neurons respond to these amino acids and what is the resulting distribution of [Ca²⁺]_i.     

Relationship between resting cytosolic Ca and responses induced byN-methyl-d-aspartate in hippocampal neurons

Cytosolic calcium concentrations ([Ca²⁺]_i) in cultured hippocampal neurons from rat embryos were measured using fura-2. Neurons with higher resting [Ca²⁺]_i showed greater [Ca²⁺]_i responses toN-methyl-d-aspartate (NMDA) and K⁺ depolarization. There was a strong relationship between resting [Ca²⁺]_i and the maximal changes in [Ca²⁺]_i (Δ[Ca²⁺]_i), which fit the our proposed equation to describe this relationship.     

Ca2+ influx into leech glial cells and neurones caused by pharmacologically distinct glutamate receptors

The effect of glutamatergic agonists on the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) of neuropile glial cells and Retzius neurones in intact segmental ganglia of the medicinal leech Hirudo medicinalis was investigated by using iontophoretically injected fura-2. In physiological Ringer solution the [Ca²⁺]_i levels of both cell types were almost the ssame (glial cells: 58 ± 30 nM, n = 51; Retzius neurones: 61 ± 27 nM, n = 64). In both cell types glutamate, kainate, and quisqualate induced an increase in [Ca²⁺]_i which was inhibited by 6,7-dinitroquinoxaline-2,3-dione (DNQX). This increase was caused by a Ca²⁺ influx from the extracellular space because the response was greatly diminished upon removal of extracellular Ca²⁺. The glutamate receptors of neuropile glial cells and Retzius neurones differed with respect to the relative effectiveness of the agonists used, as well as with regard to the inhibitory strenght of DNQX. In Retzius neurones the agonist-induced [Ca²⁺]_i increase was abolished after replacing extracellular Na⁺ by organic cations or by mM amounts of Ni²⁺, whereas in glial cells the [Ca²⁺]_i increase was largely preserved under both conditions. It is concluded that in Retzius neurones the Ca²⁺ influx is predominantly mediated by voltage-dependent Ca²⁺ channels, whereas in neuropile glial cells the major influx occurs via the ion channels that are associated with the glutamate receptors.     

Intracellular acidification induced by membrane depolarization in rat hippocampal slices: roles of intracellular Ca and glycolysis

To elucidate the mechanism of pH_i changes induced by membrane depolarization, the variations in pH_i and [Ca²⁺]_i induced by a number of depolarizing agents, including high K⁺, veratridine, N-methyl-

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-aspartate (NMDA) and ouabain, were investigated in rat hippocampal slices by the fluorophotometrical technique using BCECF or fura-2. All of these depolarizing agents elicited a decrease in pH_i and an elevation of intracellular calcium ([Ca²⁺]_i) in the CA1 pyramidal cell layer. The increases in [Ca²⁺]_i caused by the depolarizing agents almost completely disappeared in the absence of Ca²⁺ (0 mM Ca²⁺ with 1 mM EGTA). In Ca²⁺ free media, pH_i acid shifts produced by high K⁺, veratridine or NMDA were attenuated by 10–25%, and those produced by ouabain decreased by 50%. Glucose-substitution with equimolar amounts of pyruvate suppressed by two-thirds the pH_i acid shifts induced by both high K⁺ and NMDA. Furthermore, lactate contents were significantly increased in hippocampal slices by exposure to high K⁺, veratridine or NMDA but not by ouabain. These results suggest that the intracellular acidification produced by these depolarizing agents, with the exception of ouabain, is mainly due to lactate accumulation which may occur as a result of accelerated glycolysis mediated by increased Na⁺–K⁺ ATPase activity. A Ca²⁺-dependent process may also contribute to the intracellular acidification induced by membrane depolarization. Since an increase in H⁺ concentration can attenuate neuronal activity, glycolytic acid production induced by membrane depolarization may contribute to the mechanism that prevents excessive neuronal excitation.     

Activation of ATP receptor increases the cytosolic Ca concentration in nucleus accumbens neurons of rat brain

ATP increased the cytosolic Ca²⁺ concentration ([Ca]_i) in nucleus accumbens neurons acutely dissociated from rat brain. The ATP response was dependent on external Ca²⁺ and Na⁺, and was blocked by voltage-dependent Ca²⁺ channel blockers. The results suggest that the ATP-induced depolarization increases Ca²⁺ influx resulting in the increase in [Ca]_i.     

Measurement of intracellular Ca in the bullfrog sympathetic ganglion cells using fura-2 fluorescence

The intracellular free ([Ca²⁺]_i) of the bullfrog sympathetic ganglion cell was measured with fura-2 fluorescence under various conditions, and compared with changes in membrane potential recorded with an intracellular electrode. The [Ca²⁺]_i was 109 nM on average under the resting condition and increased by raising the extracellular K⁺, stimulating repetitively the pre- or post-ganglionic nerve, or by applying acetylcholine or muscarine. Since all these procedures depolarized the cell membrane, most of the rise in [Ca²⁺]_i could be the result of opening of voltage-dependent Ca²⁺ channels. However, Ca²⁺ entries through nicotinic acetylcholine receptor channels and the channel activated by the muscarinic acetylcholine receptor were also indicated by considering the threshold for the opening of voltage-dependent Ca²⁺ channels (for both entries) or a limited number of the cells showing the latter response.     

Involvement of Na+–Ca2+ Exchanger in Reperfusion-induced Delayed Cell Death of Cultured Rat Astrocytes

In some cells, Ca²⁺ depletion induces an increase in intracellular Ca²⁺ ([Ca²⁺]_i) after reperfusion with Ca²⁺-containing solution, but the mechanism for the reperfusion injury is not fully elucidated. Using an antisense strategy we studied the role of the Na⁺-Ca²⁺ exchanger in reperfusion injury in cultured rat astrocytes. When astrocytes were perfused in Ca²⁺-free medium for 15–60 min, a persistent increase in [Ca²⁺]_i was observed immediately after reperfusion with Ca²⁺-containing medium, and the number of surviving cells decreased 3–5 days latter. The increase in [Ca²⁺]_i was enhanced by low extracellular Na⁺ ([Na⁺]_o) during reperfusion and blocked by the inhibitors of the Na⁺-Ca²⁺ exchanger amiloride and 3,4-dichlorobenzamil, but not by the Ca²⁺ channel antagonists nifedipine, Cd²⁺ and Ni²⁺. Treatment of astrocytes with antisense, but not sense, oligodeoxynucleotide to the Na⁺-Ca²⁺ exchanger decreased Na⁺–Ca²⁺ exchanger protein level and exchange activity. The antisense oligomer attenuated reperfusion-induced increase in [Ca²⁺]ⁱ and cell toxicity. The Na⁺-Ca²⁺ exchange inhibitors 3,4-dichlorobenzamil and ascorbic acid protected astrocytes from reperfusion injury partially, while the stimulators sodium nitroprusside and 8-bromo-cyclic GMP and low [Na⁺]_o exacerbated the injury. Pretreatment of astrocytes with ouabain and monensin caused similar delayed glial cell death. These findings suggest that Ca²⁺ entry via the Na⁺–Ca²⁺ exchanger plays an important role in reperfusion-induced delayed glial cell death.     

In vitro dopamine biosynthesis in the median eminence of rat hypothalamic slices: Involvement of voltage-dependent Ca channels

Equimolar replacement of Na⁺ in medium with choline chloride or sucrose and experimental manipulations known to increase [Na⁺]_i, such as ouabain addition and K⁺ deprivation from medium, caused a marked increase in in vitro DOPA synthesis in the median eminence of rat hypothalamic slices in a Ca²⁺-dependent manner. These results suggest that a Na⁺−Ca²⁺ exchange mechanism is closely involved in the regulation of dopamine biosynthesis in tuberoinfundibular neurons.     

Voltage‐induced Ca2+ release by ryanodine receptors causes neuropeptide secretion from nerve terminals

Depolarisation‐secretion coupling is assumed to be dependent only on extracellular calcium ([Ca²⁺]_o). Ryanodine receptor (RyR)‐sensitive stores in hypothalamic neurohypophysial system (HNS) terminals produce sparks of intracellular calcium ([Ca²⁺]_i) that are voltage‐dependent. We hypothesised that voltage‐elicited increases in intraterminal calcium are crucial for neuropeptide secretion from presynaptic terminals, whether from influx through voltage‐gated calcium channels and/or from such voltage‐sensitive ryanodine‐mediated calcium stores. Increases in [Ca²⁺]_i upon depolarisation in the presence of voltage‐gated calcium channel blockers, or in the absence of [Ca²⁺]_o, still give rise to neuropeptide secretion from HNS terminals. Even in 0 [Ca²⁺]_o, there was nonetheless an increase in capacitance suggesting exocytosis upon depolarisation. This was blocked by antagonist concentrations of ryanodine, as was peptide secretion elicited by high K⁺ in 0 [Ca²⁺]_o. Furthermore, such depolarisations lead to increases in [Ca²⁺]_i. Pre‐incubation with BAPTA‐AM resulted in > 50% inhibition of peptide secretion elicited by high K⁺ in 0 [Ca²⁺]_o. Nifedipine but not nicardipine inhibited both the high K⁺ response for neuropeptide secretion and intraterminal calcium, suggesting the involvement of CaV1.1 type channels as sensors in voltage‐induced calcium release. Importantly, RyR antagonists also modulate neuropeptide release under normal physiological conditions. In conclusion, our results indicate that depolarisation‐induced neuropeptide secretion is present in the absence of external calcium, and calcium release from ryanodine‐sensitive internal stores is a significant physiological contributor to neuropeptide secretion from HNS terminals.     

SHORT COMUNICATION Early sodium elevations induced by combined oxygen and glucose deprivation in pyramidal cortical neurons

We investigated the effects of oxygen (O₂)/glucose deprivation on intracellular sodium concentration ([Na⁺]_i) of cortical pyramidal cells in a slice preparation of rat frontal cortex. Intracellular recordings were combined with microfluorometric measurements of [Na⁺]_i using the Na⁺-sensitive dye sodium-binding benzofuran isophthalate (SBFI). Deprivation of O₂/glucose caused an initial membrane hyperpolarization that was followed by a slowly developing large depolarization. Levels of [Na⁺]_i started to increase significantly during the phase of membrane hyperpolarization. Neither tetrodotoxin, a combination of ionotropic and metabotropic glutamate receptor antagonists (d -amino-phosphonovalerate, 6-cyano-7-nitroquinoxaline-2,3-dione plus S-methyl-4-carboxyphenylglycine) nor bepridil, an inhibitor of the Na⁺/Ca²⁺-exchanger, affected these responses to O₂/glucose. The present results demonstrate that, in cortical neurons, O₂/glucose deprivation induces an early rise in [Na⁺]_i which cannot be ascribed to the activity of voltage gated Na⁺-channels, glutamate receptors or of the Na⁺/Ca²⁺-exchanger.     

NMDA-induced increase in [Ca]i andCa uptake in acutely dissociated brain cells derived from adult rats

A preparation of acutely dissociated brain cells derived from adult (3-month-old) rat has been developed under conditions preserving the metabolic integrity of the cells and the function of N-methyl-d-aspartate (NMDA) receptors. The effects of glutamate and NMDA on [Ca²⁺]_i measured with fluo3 and⁴⁵Ca²⁺ uptake have been studied on preparations derived from hippocampus and cerebral cortex. Glutamate (100 μM) and N-methyl-dl-aspartate (200 μM) increased [Ca²⁺]_i by 26-12 nM and 23-9 nM after 90 s in cerebral cortex and hippocampus, and stimulated⁴⁵Ca²⁺ uptake about 16–10% in the same regions. The increases in [Ca²⁺]_i and⁴⁵Ca²⁺ uptake were inhibited by 40% in the presence of 1 mM MgCl₂ and by 90–50% in the presence of MK-801. The results indicate (a) that a large fraction of the [Ca²⁺]_i response to glutamate in freshly dissociated brain cells from the adult rat involves NMDA receptors, (b) when compared with results in newborn rats, there is a substantial blunting of the [Ca²⁺]_i increase in adult age.     

Glutamate increases the [Ca]i but stimulates Ca-independent release of [H]GABA in cultured chick retina cells

The effect of glutamate of [Ca²⁺]_i and on [³H]γ-aminobutyric acid (GABA) release was studied on cultured chick embryonic retina cells. It was observed that glutamate (100 μM) increases the [Ca²⁺]_i by Ca²⁺ influx through Ca²⁺ channels sensitive to nitrendipine, but not to ω-conotoxin GVIA (ω-Cg Tx) (50%), and by other channels insensitive to either Ca²⁺ channel blocker. Mobilization of Ca²⁺ by glutamate required the presence of external Na⁺, suggesting that Na⁺ mobilization through the ionotropic glutamate receptors is necessary for the Ca²⁺ channels to open. The increase in [Ca²⁺]_i was not related to the release of [³H]GABA induced by glutamate, suggesting that the pathway for the entry of Ca²⁺ triggered by glutamate does not lead to exocytosis. In fact, the glutamate-induced release of [³H]GABA was significantly depressed by Ca_o²⁺, but it was dependent on Na_o⁺, just as was observed for the [³H]GABA release induced by veratridine (50 μM). The veratridine-induced release could be fully inhibited by TTX, but this toxin had no effect on the glutamate-induced [³H]GABA release. Both veratridine- and glutamate-induced [³H]GABA release were inhibited by 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid (NNC-711), a blocker of the GABA carrier. Blockade of the NMDA and non-NMDA glutamate receptors with MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively, almost completely blocked the release of [³H]GABA evoked by glutamate. Continuous depolarization with 50 mM K⁺ induced maximal release of [³H]GABA of about 1.5%, which is much smaller than the release evoked by glutamate under the same conditions (6.0–6.5%). Glycine (3 μM) stimulated [³H]GABA release induced by 50 mM K⁺, and this effect was blocked by MK-801, suggesting that the effect of K⁺ on [³H]GABA release was partially mediated through the NMDA receptor which probably was stimulated by glutamate released by K⁺ depolarization. We conclude that glutamate induces Ca²⁺-independent release of [³H]GABA through reversal of the GABA carrier due to Na⁺ entry through the NMDA and non-NMDA, TTX-insensitive, channels. Furthermore the GABA carrier seems to be inhibited by Ca²⁺ entering by the pathways open by glutamate. This Ca²⁺ does not lead to exocytosis, probably because the Ca²⁺ channels used are located at sites far from the active zones.     

Selective enhancement by basic fibroblast growth factor of NMDA receptor-mediated increase of intracellular Ca concentration in hippocampal neurons

The short-term effect of bFGF on intracellular Ca²⁺ concentration ([Ca²⁺]_i) of hippocampal neurons was investigated using dissociated cell cultures. Changes in [Ca²⁺]_i were measured by microfluorometrically monitoring the fluorescence intesities from indivudual neurons loaded with fura-2. Perfusion of bFGF (20 ng/ml) alone did not affect the basal level of [Ca²⁺]_i in hippocampal neurons, but clearly enhanced the [Ca²⁺]_i increase induced by NMDA. Quisqualate or KCl-induced [Ca²⁺]_i increase was not influenced by bFGF. These results suggest that bFGF selectively enhances the NMDA receptor-mediated response in hippocampal neurons.     

INVOLVEMENT OF Na+-K+-2Cl− COTRANSPORTERS IN HYPERTONICITY-INDUCED RISE IN INTRACELLULAR CALCIUM CONCENTRATION

A hypertonic saline containing propylene glycol facilitates calcium (Ca²⁺) influx through voltage-dependent Ca²⁺ channels. The present study performed experiments to elucidate the mechanism by which Na⁺-K⁺-2Cl^? cotransporters participate in the rise in the intracellular calcium concentration ([Ca²⁺]i) under the hypertonic condition. Both furosemide and ethacryonic acid significantly decreased the [Ca²⁺]i raised by hypertonicity. Similarly, Na⁺-, K⁺-, or Cl^?-free saline also reduced it. Both norepinephrine and dopamine significantly enhanced the rise in [Ca²⁺]i. In conclusion, the findings obtained indicate that the Na⁺-K⁺-2Cl^? cotransporters evoke cell depolarization and that this depolarization raises the [Ca²⁺]i by activating voltage-dependent Ca²⁺ channels.     

The action of valproate on spontaneous epileptiform activity in the absence of synaptic transmission and on evoked changes in [Ca]0 and [K]0 in the hippocampal slice

The effects of valproate (VPA) on neuronal excitability and on changes in extracellular potassium ([K⁺]₀) and calcium ([Ca²⁺]₀) were investigated with ion selective-reference electrode pairs in area CA₁ of rat hippocampal slices. Field potential responses to single ortho- and antidromic stimuli were unaltered by VPA (1–5 mM). The afferent volley evoked in the Schaffer-commissural fibers was also unaffected. In contrast, VPA (1 mM) depressed frequency potentiation and paired pulse facilitation markedly. Decreases in [Ca²⁺]₀ induced either by repetitive stimulation or by application of the excitatory amino acids N-methyl-d-aspartate and quisqualate were reduced, and the latter results suggest that VPA interferes with postsynaptic Ca²⁺ entry. When synaptic transmission was blocked by lowering [Ca²⁺]₀ (0.2 mM) and elevating [Mg²⁺]₀ (7 mM), prolonged afterdischarges elicited by antidromic stimulation were blocked by VPA. VPA also suppressed the spontaneous epileptiform activity seen when [Ca²⁺]₀ was lowered to 0.2 mM, without elevating [Mg²⁺]₀. The amplitudes of the rises in [K⁺]₀ induced by repetitive orthodromic stimulation were only slightly depressed and those elicited by antidromic stimulation were generally unaltered by VPA, as were laminar profiles of stimulus-evoked [K⁺]₀ signals. These results indicate that VPA has membrane actions in addition to known effects on excitatory and inhibitory transmitter pools.