Effect of dantrolene on KCl- or NMDA-induced intracellular Ca2+ changes and spontaneous Ca2+ oscillation in cultured rat frontal cortical neurons

Summary Dantrolene has been known to affect intracellular Ca²⁺ concentration ([Ca²⁺]_i) by inhibiting Ca²⁺ release from intracellular stores in cultured neurons. We were interested in examining this property of dantrolene in influencing the [Ca²⁺]_i affected by the NMDA receptor ligands, KCl, L-type Ca²⁺ channel blocker nifedipine, and two other intracellular Ca²⁺-mobilizing agents caffeine and bradykinin. Effect of dantrolene on the spontaneous oscillation of [Ca²⁺]_i was also examined. Dantrolene in M concentrations dose-dependently inhibited the increase in [Ca²⁺]_i elicited by NMDA and KCl. AP-5, MK-801 (NMDA antagonists), and nifedipine respectively reduced the NMDA and KCl-induced increase in [Ca²⁺]_i. Dantrolene, added to the buffer solution together with the antagonists or nifedipine, caused a further reduction in [Ca²⁺]_i to a degree similar to that seen with dantrolene alone inhibiting the increase in [Ca₂₊]_i caused by NMDA or KCl. At 30 M, dantrolene partially inhibited caffeine-induced increase in [Ca²⁺]_i whereas it has no effect on the bradykinin-induced change in [Ca²⁺]_i. The spontaneous oscillation of [Ca²⁺]_i in frontal cortical neurons was reduced both in amplitude and in base line concentration in the presence of 10 M dantrolene. Our results indicate that dantrolene's mobilizing effects on intracellular Ca²⁺ stores operate independently from the influxed Ca²⁺ and that a component of the apparent increase in [Ca²⁺]_i elicited by NMDA or KCl represents a dantrolene-sensitive Ca₂₊ release from intracellular stores. Results also suggest that dantrolene does not affect the IP₃-gated release of intracellular Ca²⁺ and that the spontaneous Ca²⁺ oscillation is, at least partially, under the control of Ca²⁺ mobilization from internal stores.Abbreviations AP-5 (±)-2-amino-5-phosphonopentanoic acid - AMPA amino-3-hydroxy-5-methyl-isoxazole-4-propionate - BSS balanced salt solution - CNS central nervous system - CICR Ca²⁺-induced Ca²⁺ release - DCKA 5,7-dichlorokynurenate - DNasel deoxyribonuclease I - DMEM Dulbecco's Modified Eagle's Medium - EGTA ethylene glycol-bis(-aminoethyl ether)N,N,N,N,-tetraacetic acid - FCS fetal calf serum - fura-2-AM 1-(2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy-2-ethane-N,N,N,N-te-traacetic acid, pentaacetoxymethyl ester - HEPES N-[2-hydroxyethyl] piperazine-N-[2-ethanesulfonic acid] - [Ca ²⁺] _i intracellular free Ca²⁺ concentration - LTP long-term potantiation - MK-801 (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]-cyclohepten-5,10-imine hydrogen maleate - NMDA N-methyl-D-aspartate     

Mechanism of synchronized Ca oscillations in cortical neurons

Dissociated rat cortical neurons reassociate in vitro to form synaptically connected networks. Removal of Mg²⁺ from the extracellular medium then induces neurons in the network to undergo synchronized oscillations of cytoplasmic calcium. Previous studies have shown that these calcium oscillations involve the activation of NMDA receptors and that the rising phase of each calcium spike is coincident with a brief burst of action potentials (Robinson et al., Jpn. J. Physiol. 43 (Suppl. 1) (1993) S125–130; Robinson et al., J. Neurophysiol. 70 (1993) 1606–1616; Murphy et al., J. Neurosci. 12 (1992) 4834–4845). We have found that these calcium oscillations are dependent on an influx of extracellular calcium but are independent of mobilization of calcium from intracellular stores. The influx of extracellular Ca²⁺ occurs primarily through L-type voltage-gated calcium channels (VGCCs), since diltiazem inhibits calcium oscillations under all conditions. On the other hand, N-, P/Q-, and T-type VGCCs are not required for calcium oscillations, although inhibitors of these channels may act as partial antagonists. In addition to removal of Mg²⁺, oscillations can also be induced by the inhibition of voltage-gated K⁺ channels with 4-aminopyridine (4-AP), a treatment known to increase neurotransmitter release. In the presence of 4-AP, synchronized calcium oscillations become independent of NMDA receptor activation, although they continue to require activation of AMPA/KA receptors. A model for the mechanism of neuronal calcium oscillations and the reason for their synchrony is presented.     

Glutamate selectively increases the high-threshold Ca channel current in sensory and hippocampal neurons

Previous studies resulted in conflicting conclusions that glutamate application either decreases or increases the activity of Ca²⁺ channels in hippocampal neurons. We studied whole-cell Ca²⁺ currents (I_Ca) in chick dorsal root ganglion neurons and rat hippocampal cells. For both cell types glutamate (1–30 μM) increased high-threshold Ca²⁺ current. It was independent of the charge carriers, Ca²⁺ or Ba²⁺. Low-threshold Ca²⁺ channel current and the fast sodium current were not changed with glutamate application. The effect developed within 1–2 min and then further facilitated after washout of the agonist. A second application of glutamate produced no additional increase in _ICa. No changes in the time-course of whole-cell currents were observed, suggesting that glutamate recruits ‘sleepy’ Ca²⁺ channels. Whatever its mechanism, overlasting increase of I_Ca by glutamate may be important in neuronal plasticity.     

Dibutyryl cGMP raises cytosolic concentrations of Ca in cultured nodose ganglion neurons of the rabbit

The effect of dibutyryl cGMP (dbcGMP), a membrane permeant cGMP analogue, on cytosolic concentrations of Ca²⁺ ([Ca²⁺]_i) was studied in cultured nodose ganglion neurons of the rabbit using fura-2AM and microfluorometry. Application of dbcGMP (10–1000 μM) increased [Ca²⁺]_i in 42% of neurons (n=67). The effect was observed in a dose-dependent fashion. The threshold dose was 100 μM and the increase at 500 μM averaged 117±8%. Removal of extracellular Ca²⁺ abolished the dbcGMP effect. Application of Ni²⁺ (1 mM) or neomycin (50 μM), a non-L-type voltage-gated Ca²⁺ channel (VGCC) antagonist, eliminated the dbcGMP effect. ω-conotoxin GVIA (2 μM), the N-type Ca²⁺ channel antagonist, or L-type Ca²⁺ channel antagonists (D600, 50 μM, or nifedipine, 10 μM) did not alter the dbcGMP effect. Ryanodine (10 μM) did not alter the effect of dbcGMP. Therefore, cGMP could play a part of role of an intracellular messenger in primary sensory neurons of the autonomic nervous system.     

Homeostatic compensation maintains Ca2+ signaling functions in Purkinje neurons in the leaner mutant mouse

Several human neurological disorders have been associated with mutations in the gene coding for the alpha1 subunit of the P/Q type voltage-gated calcium channel (alpha1A/Ca(v)2.1). Mutations in this gene also occur in a number of neurologically affected mouse strains, including leaner (tg(la)/tg(la)). Because the P-type calcium current is very prominent in cerebellar Purkinje neurons, these cells from mice with alpha1 subunit mutations make excellent models for the investigation of the functional consequences of native mutations in a voltage-gated calcium channel of mammalian central nervous system. In this review, we describe the impact of altered channel function on cellular calcium homeostasis and signaling. Remarkably, calcium buffering functions of the endoplasmic reticulum and calcium-binding proteins appear to be regulated in order to compensate for altered calcium influx through the mutant channels. Although this compensation may serve to maintain calcium signaling functions, such as calcium-induced calcium release, it remains uncertain whether such compensation alleviates or contributes to the behavioral phenotype.     

Intrinsic membrane potential oscillations in hippocampal neurons in vitro

Membrane potential oscillations (MPOs) of 2-10 Hz and up to 6 mV were found in almost all stable hippocampal CA1 and CA3 neurons in the in vitro slice preparation. MPOs were prominent for pyramidal cells but less pronounced in putative interneurons. MPOs were activated at threshold depolarizations that evoked a spike and the frequency of the MPOs increased with the level of depolarization. MPOs were distinct from and seemed to regulate spiking, with a spike often riding near the top of a depolarizing MPO wave. Analysis of the periodicity of the oscillations indicate that the period of MPOs did not depend on the afterhyperpolarization (AHP) following a single spike. MPOs persisted in low (0-0.1 mM) Ca2+ medium, with or without Cd2+ (0.2 mM), when synaptic transmission was blocked. Choline-substituted low-Na+ (0-26 mM) medium, 3 microM tetrodotoxin (TTX) or intracellular injection of QX-314 reduced or abolished the fast Na(+)-spike and reduced inward anomalous rectification. About 40% of CA1 neurons had no MPOs after Na+ currents were blocked, suggesting that these MPOs were Na(+)-dependent. In about 60% of the cells, a large depolarization activated Ca(2+)-dependent MPOs and slow spikes. MPOs were not critically affected by extracellular Ba2+ or Cs2+, or by 0.2 mM 4-aminopyridine, with or without 2 mM tetraethylammonium (TEA). However, in 5-10 mM TEA medium, MPOs were mostly replaced by 0.2-3 Hz spontaneous bursts of wide-duration spikes followed by large AHPs. Low Ca2+, Cd2+ medium greatly reduced the spike width but not the spike-bursts. In conclusion, each cycle of an MPO in normal medium probably consists of a depolarization phase mediated by Na+ currents, possibly mixed with Ca2+ currents activated at a higher depolarization. The repolarization/hyperpolarization phase may be mediated by Na+/Ca2+ current inactivation and partly by TEA-sensitive, possibly the delayed rectifier, K+ currents. The presence of prominent intrinsic, low-threshold MPOs in all hippocampal pyramidal neurons suggests that MPOs may play an important role in information processing in the hippocampus.     

NMDA-evoked consumption and recovery of mitochondrially targeted aequorin suggests increased Ca2+ uptake by a subset of mitochondria in hippocampal neurons

Activation of NMDA receptors produces large increases in cytosolic Ca(2+) that are taken up into mitochondria. We used recombinant aequorin targeted to mitochondria to report changes in matrix Ca(2+) in rat hippocampal neurons in culture. Upon binding Ca(2+), aequorin emits a photon in a one-shot reaction that consumes the indicator. Here we show that stimulation with NMDA produced a mitochondrial Ca(2+) response that rapidly inactivated. However, following a 30-min recovery period the response was restored, suggesting the presence of a pool of indicator that was not exposed to high Ca(2+) during the initial stimulus. We speculate that aequorin distant from the Ca(2+) source was protected from microdomains of high Ca(2+) near the plasmalemma and that this aequorin moved, either by movement of individual mitochondria or via the mitochondrial tubular network, to replenish consumed indicator during the recovery time. A large Ca(2+) increase in a subset of mitochondria could produce local changes in energy metabolism, regional Ca(2+) buffering, and foci that initiate neurotoxic processes.     

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.     

Effects of GABA on presumed presynaptic Ca entry in hippocampal slices

Evoked field potentials and changes in [Ca²⁺]_o were measured in the ‘in vitro’ hippocampal slice of the rat. When [Ca] in the perfusion medium was lowered to 0.2 mM synaptic transmission from Schaffer collateral/comissural fibers was blocked. Nevertheless, repetitive stimulation of afferent fibers still resulted in detectable decreases of [Ca²⁺]_o. In contrast to findings in normal medium these decreases in [Ca²⁺]_o could be larger in stratum radiatum than in stratum pyramidale, so mimicking the spatial distribution of activated afferent fibers. These findings suggest, that the loss of extracellular Ca²⁺ in low Ca²⁺ media is predominantly due to entry into presynaptic terminals. This permits to study effects of drugs on presynaptic endings. We found that iontophoretic application of GABA is capable to block this presumed presynaptic Ca²⁺ entry without affecting the electrical activity of the afferent fibers. This suggests, that presynaptic GABA receptors occur also in the Schaffer collateral/commissural fiber system.     

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.     

Prostaglandins block a Ca-dependent slow spike afterhyperpolarization independent of effects on Ca influx in visceral afferent neurons

The blockade of a slow Ca2+-activated K+-dependent afterhyperpolarization (AHPs) in rabbit visceral sensory neurons by the prostaglandins, PGE1 and PGD2, was investigated to determine whether the blockade was indirectly due to a reduction in Ca2+ influx. The prostaglandins (PGs) could block the AHPs in the absence of any change in Ca2+-dependent spikes elicited in the presence of tetrodotoxin and tetraethylammonium bromide. A PG-induced decrease in Ca2+-dependent spike width observed in some neurons was temporally dissociated from the PG-induced block of the AHPs. In addition, a slow afterhyperpolarization produced by the application of the Ca2+ ionophore, A23187, was blocked by the PGs. It is concluded that a reduction in Ca2+ influx is not responsible for the PG-induced blockade of the AHPs.     

Histamine modulates reactivity of hippocampal CA3 neurons to afferent stimulation in vitro

Intracellular activity was recorded from hippocampal CA3 pyramidal cells maintained in vitro. Histamine (HA) produced a slow depolarization associated with minimal conductance changes. In addition, there was an increase in action potential discharge rates and the emergence of bursting firing patterns. EPSP size increased by about 50% and spontaneous dendritic spikes were observed. These effects were markedly reduced by retrodotoxin. Extracellular recording of population spikes revealed a marked difference between CA1 and CA3 regions; in the former HA produced an increase in population spike size whereas in the latter this increase was larger and was associated with the appearance of secondary and tertiary population spikes. It is suggested that HA produces its effects by enhancing release of neurotransmitters from excitatory synapses on the recorded neurons.     

Paraoxon suppresses Ca(2+) spike and afterhyperpolarization in snail neurons: Relevance to the hyperexcitability induction

The effects of organophosphate (OP) paraoxon, active metabolite of parathion, were studied on the Ca(2+) and Ba(2+) spikes and on the excitability of the neuronal soma membranes of land snail (Caucasotachea atrolabiata). Paraoxon (0.3 muM) reversibly decreased the duration and amplitude of Ca(2+) and Ba(2+) spikes. It also reduced the duration and the amplitude of the afterhyperpolarization (AHP) that follows spikes, leading to a significant increase in the frequency of Ca(2+) spikes. Pretreatment with atropine and hexamethonium, selective blockers of muscarinic and nicotinic receptors, respectively, did not prevent the effects of paraoxon on Ca(2+) spikes. Intracellular injection of the calcium chelator BAPTA dramatically decreased the duration and amplitude of AHP and increased the duration and frequency of Ca(2+) spikes. In the presence of BAPTA, paraoxon decreased the duration of the Ca(2+) spikes without affecting their frequency. Apamin, a neurotoxin from bee venom, known to selectively block small conductance of calcium-activated potassium channels (SK), significantly decreased the duration and amplitude of the AHP, an effect that was associated with an increase in spike frequency. In the presence of apamin, bath application of paraoxon reduced the duration of Ca(2+) spike and AHP and increased the firing frequency of nerve cells. In summary, these data suggest that exposure to submicromolar concentration of paraoxon may directly affect membrane excitability. Suppression of Ca(2+) entry during the action potential would down regulate Ca(2+)-activated K(+) channels leading to a reduction of the AHP and an increase in cell firing.     

Repetitive application of caffeine sensitizes caffeine-induced Ca release in bullfrog sympathetic ganglion neurons

Cytosolic free calcium concentration ([Ca(2+)](i)) was recorded from cultured bullfrog sympathetic ganglion cells loaded with the Ca(2+)-indicator Fura-2 or Fura-6F. Repetitive application of caffeine at a low concentration, which either failed to produce any [Ca(2+)](i) elevation or induced a small gradual increase in [Ca(2+)](i) at first challenge, produced a drastic increase in the amplitude of Ca(2+) release (caffeine response). The caffeine response eventually reached peak amplitude and then remained constant even if caffeine application were continued. This augmentation was maintained for up to 2 h, and was achieved not only by repetitive application but also by a long exposure of caffeine. However, this augmentation was neither achieved by repetitive administration of high K(+)-solution, nor caused by inhibition of phosphodiesterase by caffeine. The repetitive or sustained application of caffeine is suggested to increase the caffeine sensitivity of the calcium release channel to calcium, thus causing the potentiation of the caffeine response.     

Platelet-activating factor induced Ca signaling in human microglia

Increases in intracellular Ca(2+) concentration in human microglial cells in response to platelet-activating factor (PAF) were studied using Ca(2+)-sensitive fluorescence microscopy. In normal physiological solution (PSS), PAF-induced transient increases in [Ca2+](i) which recovered to baseline values within 200 s. Application of PAF in zero-Ca(2+) solution caused the peak response to be decreased to a value near 20% of that recorded in PSS suggesting a primary contribution of Ca(2+) influx for the [Ca2+](i) increase in PSS. To investigate PAF-induced Ca(2+) influx, the contents of intracellular stores were modulated using the SERCA blocker cyclopiazonic acid (CPA). The Ca(2+) signal induced by CPA (10 microM) in zero-Ca(2+) solution showed a peak response about 20% of the amplitude in the presence of external Ca(2+), suggesting the latter response included significant contributions from store-operated Ca(2+) entry. The influx of divalent cations with PAF or CPA was directly measured using Mn(2+) quenching of the fluorescence signal. Although both PAF and CPA induced a similar degree of Mn(2+) influx over time, the PAF effect was very rapid, whereas the CPA action was delayed and only evident about 200 s after application. Overall, the results show that the primary source of the PAF-induced increase of [Ca2+](i) in human microglia was the influx of Ca(2+) from the extracellular space and intracellular Ca(2+)-release contributed only a small part of the total Ca(2+) signal. Nevertheless, Ca(2+)-release induced by PAF (or CPA) serves as an important factor in controlling Ca(2+) entry presumably mediated by activation of store-operated-Ca(2+) channels.     

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.     

Identification of calcium channels involved in neuronal injury in rat hippocampal slices subjected to oxygen and glucose deprivation

The presynaptic Ca²⁺-influx affecting glutamate release during neuropathological processes is mediated via voltage-sensitive calcium channels (VSCCs). There is controversy, however, over the fractional contribution of the specific channel types involved. We have addressed this by investigating the protective effects of various VSCC blockers on oxygen and glucose-deprived rat hippocampal slices. The viability of treated and non-treated slices was assayed electrophysiologically by measuring the evoked population spike (PS) amplitude in the stratum pyramidale of the CA1 region and by imaging slices loaded with fluorochrome dyes specific for dead (ethidium homodimer) and live (calcein) cells using confocal microscopy. PS amplitudes were significantly (P<0.01) depressed from 4.4±0.2 mV (n=38) to 0.2±0.1 mV (n=40) after the deprivation insult. Responses from deprived slices treated with ω-conotoxin MVIIC (100 nM; 4.2±0.5 mV; n=20) were not significantly different from control, non-deprived slice responses. In contrast, deprived slices treated with either L-type (0.1 or 1 μM nimodipine) or N-type (0.1 or 3 μM ω-conotoxin MVIIA) blockers showed no significant protection. The viability of CA1 neurons as revealed by the fluorescence live/dead confocal viability assay was consistent with the electrophysiological measurements. By comparison with previous studies using P- and Q-type blockers to attempt neuroprotection against the same deprivation insult, the rank order in which specific Ca²⁺-channel types contribute to neuronal death due to oxygen and glucose deprivation was determined to be Q>N>>P>L. © 1997 Elsevier Science B.V. All rights reserved.     

Involvement of the glutamate transporter and the sodium–calcium exchanger in the hypoxia-induced increase in intracellular Ca in rat hippocampal slices

Hippocampal slices prepared from adult rats were loaded with fura-2 and the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in the CA1 pyramidal cell layer was measured. Hypoxia (oxygen–glucose deprivation) elicited a gradual increase in [Ca²⁺]_i in normal Krebs solution. At high extracellular sodium concentrations ([Na⁺]_o), the hypoxia-induced response was attenuated. In contrast, hypoxia in low [Na⁺]_o elicited a significantly enhanced response. This exaggerated response to hypoxia at a low [Na⁺]_o was reversed by pre-incubation of the slice at a low [Na⁺]_o prior to the hypoxic insult. The attenuation of the response to hypoxia by high [Na⁺]_o was no longer observed in the presence of antagonist to glutamate transporter. However, antagonist to Na⁺–Ca²⁺ exchanger only slightly influenced the effects of high [Na⁺]_o. These observations suggest that disturbance of the transmembrane gradient of Na⁺ concentrations is an important factor in hypoxia-induced neuronal damage and corroborates the participation of the glutamate transporter in hypoxia-induced neuronal injury. In addition, the excess release of glutamate during hypoxia is due to a reversal of Na⁺-dependent glutamate transporter rather than an exocytotic process.     

Effect of nilvadipine on the voltage-dependent Ca channels in rat hippocampal CA1 pyramidal neurons

Effects of nilvadipine on the low- and high-voltage activated Ca²⁺ currents (LVA and HVA I_Ca, respectively) were compared with other organic Ca²⁺ antagonists in acutely dissociated rat hippocampal CA1 pyramidal neurons. The inhibitory effects of nilvadipine, amlodipine and flunarizine on LVA I_Ca were concentration- and use-dependent. The apparent half-maximum inhibitory concentrations (IC₅₀s) at every 1- and 30-s stimulation were 6.3×10⁻⁷ M and 1.8×10⁻⁶ M for flunarizine, 1.9×10⁻⁶ M and 7.6×10⁻⁶ M for nilvadipine, and 4.0×10⁻⁶ M and 8.0×10⁻⁶ M for amlodipine, respectively. Thus, the strength of the use-dependence was in the sequence of nilvadipine>flunarizine>amlodipine. Nilvadipine also inhibited the HVA I_Ca in a concentration-dependent manner with an IC₅₀ of 1.5×10⁻⁷ M. The hippocampal CA1 neurons were observed to have five pharmacologically distinct HVA Ca²⁺ channel subtypes consisting of L-, N-, P-, Q- and R-types. Nilvadipine selectively inhibited the L-type Ca²⁺ channel current which comprised 34% of the total HVA I_Ca. On the other hand, amlodipine non-selectively inhibited the HVA Ca²⁺ channel subtypes. These results suggest that the inhibitory effect of nilvadipine on the neuronal Ca²⁺ influx through both LVA and HVA L-type Ca²⁺ channels, in combination with the cerebral vasodilatory action, may prevent neuronal damage during ischemia.