Late expression of Na+/H+ exchanger 1 (NHE1) and neuroprotective effects of NHE inhibitor in the gerbil hippocampal CA1 region induced by transient ischemia

Although acidosis may be involved in neuronal death, the participation of Na⁺/H⁺ exchanger (NHE) in delayed neuronal death in the hippocampal CA1 region induced by transient forebrain ischemia has not been well established. In the present study, we investigated the chronological alterations of NHE1 in the hippocampal CA1 region using a gerbil model after ischemia/reperfusion. In the sham-operated group, NHE1 immunoreactivity was weakly detected in the CA1 region. Two and 3 days after ischemia/reperfusion, NHE1 immunoreactivity was observed in glial components, not in neurons, in the CA1 region. Four days after ischemia/reperfusion, NHE1 immunoreactivity was markedly increased in CA1 pyramidal neurons as well as glial cells. These glial cells were identified as astrocytes based on double immunofluorescence staining. Western blot analysis also showed that NHE protein level in the CA1 region began to increase 2 days after ischemia/reperfusion. The treatment of 10 mg/kg 5-(N-ethyl-N-isopropyl) amiloride, a NHE inhibitor, significantly reduced the ischemia-induced hyperactivity 1day after ischemia/reperfusion. In addition, NHE inhibitor potently protected CA1 pyramidal neurons from ischemic damage, and NHE inhibitor attenuated the activation of astrocytes and microglia in the ischemic CA1 region. In addition, NHE inhibitor treatment blocked Na⁺/Ca²⁺ exchanger 1 immunoreactivity in the CA1 region after transient forebrain ischemia. These results suggest that NHE1 may play a role in the delayed death, and the treatment with NHE inhibitor protects neurons from ischemic damage.     

Histochemical study of Ca2+-ATPase activity in ischemic CA1 pyramidal neurons in the gerbil hippocampus

Although cytosolic Ca²⁺ accumulation plays a pivotal role in delayed neuronal death, there have been no investigations on the role of the cellular Ca²⁺ export system in this novel phenomenon. To clarify the function of the Ca²⁺-pump in delayed neuronal death, the plasma membrane Ca²⁺-ATPase activity of CA1 pyramidal neurons was investigated ultracytochemically in normal and ischemic gerbil hippocampus. To correlate enzyme activity with delayed neuronal death, histochemical detection was performed at various recirculation times after 5 min of ischemia produced by occlusion of the bilateral carotid arteries. At 10 min after ischemia, CA1 pyramidal neurons showed weak Ca²⁺-ATPase activity. Although enzyme activity had almost fully recovered 2 h after ischemia, it was reduced again 6 h after ischemia. Thereafter, Ca²⁺-ATPase activity on the plasma membrance of CA1 pyramidal neurons decreased progressively, losing its localization on day 3. On day 4 following ischemia, reaction products were diffusely scattered throughout the whole cell body. Our results indicate that, after once having recovered from ischemic damage, severe disturbance of the membrane Ca²⁺ export system proceeds from the early stage of delayed neuronal death and disturbs the re-export of accumulated cytosolic Ca²⁺, which might contribute to delayed neuronal death. Occult disruption of Ca²⁺ homeostasis seems to occur from an extremely early stage of delayed neuronal death in CA1 pyramidal cells.     

Veratridine delays apoptotic neuronal death induced by NGF deprivation through a Na+-dependent mechanism in cultured rat sympathetic neurons

Superior-cervical ganglion (SCG) cells dissociated from newborn rats depend on nerve growth factor (NGF) for survival. Membrane depolarization with elevated K+ is known to prevent neuronal death following NGF deprivation and/or to promote survival via a Ca2+-dependent mechanism. Here we have exploited the possibility of whether or not a Na+-dependent pathway for neuronal survival is present in these cells. Veratridine (ec50=40 nM), a voltage-dependent Na+ channel activator, significantly delayed the onset of apoptotic cell death in NGF-deprived SCG neurons that had been cultured for 7 days in the presence of NGF. This effect was blocked completely by Na+ channel blockers including tetrodotoxin (TTX, 1 μM), benzamil (25 μM) and flunarizine (1 μM), but was not attenuated by nimodipine (1 μM), an L-type Ca²⁺ channel blocker. The saving effect of veratridine on cultured neurons was observed even in low Ca²⁺ media (0–1.0 mM), but was completely abolished in a low Na⁺ medium (38 mM). Sodium-binding benzofuran isophthalate was employed as a fluorescent probe for monitoring the level of cytoplasmic free Na⁺, which revealed a sustained increase in its level (12.9 mM, 307% of that of control) in response to veratridine (0.75 μM). The TTX or flunarizine completely blocked veratridine-induced Na⁺ influx in these cultured neurons. Moreover, no appreciable increase in intracellular Ca²⁺ was detected under these conditions. Though Na⁺ channels were effectual in SCG neurons which were freshly isolated from newborn rats, the Na⁺-dependent saving effect of veratridine was not observed in these young neurons. These lines of evidence suggest that the death-suppressing effect of veratridine on cultured SCG neurons depends on the Na⁺ influx via voltage-dependent Na⁺ channels, and suggests the presence of Na⁺-dependent regulatory mechanism(s) in neuronal survival.     

Improvement of ischemic damage in gerbil hippocampal neurons by procaine

Acute cerebral ischemia induces membrane depolarization in the neuron, thereby incurring the simultaneous influx of various ions such as Na⁺ and Ca²⁺. Since procaine possesses the ability to inhibit the release of Ca²⁺ from intracellular Ca²⁺ stores to the cytosol as well as the ability to block Na⁺ channels, the effects of procaine on ischemia were investigated in the present study in gerbils both in vivo and in vitro. The histologic outcome was evaluated 7 days after 3 min of transient forebrain ischemia by assessing delayed neuronal death in hippocampal CA1 pyramidal cells in animals administered procaine (0.2, 0.4, or 2 μmol) intracerebroventricularly 10 min before ischemia and in animals given saline. The changes in the direct-current potential shift in the hippocampal CA1 area were measured using an identical animal model. A hypoxia-induced intracellular Ca²⁺ increase was evaluated by in vitro microfluorometry in gerbil hippocampal slices, and the effects of procaine (10, 50, and 100 μmol/l) on the Ca²⁺ accumulation were examined. Additionally, the effect of procaine (100 μmol/l) in a Ca²⁺-free condition was investigated. The histologic outcome was improved and the onset of the ischemia-induced membrane depolarization was prolonged by the preischemic administration of procaine. The increase in the intracellular concentration of Ca²⁺ induced by the in vitro hypoxia was suppressed by the perfusion of procaine-containing mediums (50 and 100 μmol/l), regarding both the initiation and the extent of the increase. A hypoxia-induced intracellular Ca²⁺ elevation in the Ca²⁺-free condition was observed, and the perfusion with procaine (100 μmol/l) inhibited this elevation. Procaine helps protect neurons from ischemia by suppressing the direct-current potential shift and by inhibiting the release of Ca²⁺ from the intracellular Ca²⁺ stores, as well as by inhibiting the influx of Ca²⁺ from the extracellular space.     

Immunologic localization and kinetic characterization of a Na/Ca exchanger in neuronal and non-neuronal cells

The plasma membrane Na⁺/Ca²⁺ exchanger is believed to play a role in the regulation of Ca²⁺ fluxes in neurons, though the lack of specific inhibitors has limited the delineation of its precise contribution. We recently reported the development of antibodies against a 36-kDa brain synaptic membrane protein which immunoprecipitated exchanger activity from solubilized membranes. In the present study we examined the kinetics of the Na⁺/Ca²⁺ exchanger in primary neurons in culture, in a neuronal hybrid cell line (NCB-20), and in a fibroblast-like cell line (CV-1) to see whether the level of exchanger activity correlated with the degree of immunostaining produced by our antibodies. The V_max was determined for each cell type and found to be highest in primary neurons. Exchanger activity increased in primary neurons between days 1 and 6 in culture, but no such time-dependent change occurred in either of the cell lines. Immunoblot analysis of the three cell types probed with the anti-36-kDa protein antibodies revealed significantly greater immunostaining in the primary neurons compared with the other two cell types. Intensity of staining of neurons also increased significantly between days 1 and 6 in culture. Immunocytochemistry showed significant labelling of the primary neurons on the neuritic processes and points of contact between cells. The NCB-20 and CV-1 cells showed considerably lower levels of immunoreactivity. The antibodies immunoextracted 90% of the exchanger activity in the primary neurons and 70 and 50% of the activity in NCB-20 and CV-1 cells respectively. Thus the expression of the 36-kDa protein appears to be closely associated with the Na⁺/Ca²⁺ exchanger in neuronal cells and, possibly to a lesser extent, in non-neuronal cells.     

Ischemia-related changes in naive and mutant forms of ubiquitin and neuroprotective effects of ubiquitin in the hippocampus following experimental transient ischemic damage

Ubiquitin binds to short-lived proteins and denatured proteins produced by various forms of injury. The loss of ubiquitin leads to an accumulation of abnormal proteins and may affect cellular structure and function. The aim of the present study is to observe the chronological changes in ubiquitin naive form and its mutant form (ubiquitin⁺¹) in the hippocampal CA1 region (CA1) after transient cerebral ischemia in gerbils. Delayed neuronal death in the CA1 was confirmed 4 days after ischemic insult with NeuN immunohistochemistry. Ubiquitin immunoreactivity and protein level in the CA1 were lowest at 12 h after ischemia/reperfusion; thereafter, they were increased with time. Ubiquitin⁺¹ immunoreactivity and protein levels in the CA1 were slightly decreased at 3 h after ischemia/reperfusion, and they were significantly increased 1 day after ischemia/reperfusion. In addition, ubiquitin and ubiquitin⁺¹ immunoreaction was expressed in astrocytes after delayed neuronal death in the ischemic CA1. To elucidate the protective effect of ubiquitin on ischemic damage, the animals were treated with ubiquitin (1.5 mg/kg body weight) intravenously via the femoral vein. Ubiquitin treatment significantly reduced ischemia-induced locomotor hyperactivity, neuronal death and reactive gliosis such as astrocytes and microglia. In addition, 5 days after ubiquitin treatment in the ischemic group, ubiquitin immunoreactivity was similar to that in the ubiquitin-treated sham group, however, ubiquitin⁺¹ immunoreactivity was higher than that in the ubiquitin-treated sham group. These findings indicate that the depletion of ubiquitin and the accumulation of ubiquitin⁺¹ in CA1 pyramidal neurons after transient cerebral ischemia may inhibit ubiquitin proteolytic pathway and this leads to delayed neuronal death of CA1 pyramidal neurons directly or indirectly after transient cerebral ischemia.     

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.     

Effects of blockade of voltage-sensitive Ca/Na channels by a novel phenylpyrimidine derivative, NS-7, on CREB phosphorylation in focal cerebral ischemia in the rat

NS-7 is a novel blocker of voltage-sensitive Ca²⁺ and Na⁺ channels, and it significantly reduces infarct size after occlusion of the middle cerebral artery. Persistent activation of cyclic AMP response element binding protein (CREB), which can be induced by increase in intracellular Ca²⁺ concentrations or other second messengers, has recently been found to be closely associated with neuronal survival in cerebral ischemia. The present study was therefore undertaken to evaluate the neuroprotective effects of NS-7 by analyzing changes in CREB phosphorylation in a focal cerebral ischemia model. CREB phosphorylation in the brain of rats was investigated immunohistochemically at 3.5–48-h recirculation after 1.5-h occlusion of the middle cerebral artery. NS-7 (1 mg/kg; NS-7 group) or saline (saline group) was intravenously injected 5 min after the start of recirculation. The NS-7 group showed significantly milder activation of CREB phosphorylation in various cortical regions after 3.5 h of recirculation than the saline group. The inner border zone of ischemia in the NS-7 group subsequently exhibited a moderate, but persistent, increase in number of phosphorylated CREB-positive neurons with no apparent histological damage. By contrast, the saline group displayed a marked, but only transient, increase in number of immunopositive neurons in this border zone after 3.5 h of recirculation, and this was followed by clear suppression of CREB phosphorylation and subsequent loss of normal neurons. These findings suggest that: (1) the marked enhancement of CREB phosphorylation in the acute post-ischemic phase may be triggered largely by an influx of calcium ions as a result of activation of the voltage-sensitive Ca²⁺ and Na⁺ channels; and that (2) the neuroprotective effects of NS-7 may be accompanied by persistent activation of CREB phosphorylation in the inner border zone of ischemia.     

Neuronal damage,glial response and cerebral metabolism after hypothermic forebrain ischemia in the rat

Summary We investigated the effect of 30°C whole body hypothermia on neuronal injury, astroglial reactivity and intracellular pH in rats subjected to 15 min of forebrain ischemia. Experimental groups included: (1) normothermic ischemia (n=8), ischemia induced under 37°C body temperature, (2) hypothermic ischemia (n=6), ischemia induced under 30°C body temperature. Cerebral intracellular pH was measured using in vivo ³¹P NMR spectroscopy over 7 days. Neuronal injury and astrocytic reactivity were evaluated using hematoxylin and eosin staining, and immunoreactivity to glial fibrillary acidic protein, respectively. Normothermic animals revealed significant alkalosis (P<0.01) at 48 h after ischemia compared to the pre-ischemic value. No significant intracellular pH change was detected after ischemia in the hypothermic group. Ischemic neuronal injury was prevented in the hypothermic animals, compared to the severe neuronal injury found in the normothermic animals (P<0.01). The marked astrocytosis of normothermic animals was significantly inhibited in the hypothermic animals (P<0.01). Our data indicate, that hypothermia significantly inhibits neuronal injury as well as post-ischemic alkalois and astrocytosis, induced by 15 min of forebrain ischemia in the rat.Supported by NINDS grants NS23393 and NS29463     

Enhancement in activities of large conductance calcium-activated potassium channels in CA1 pyramidal neurons of rat hippocampus after transient forebrain ischemia

It has been reported previously that the neuronal excitability persistently suppresses and the amplitude of fast afterhyperpolarization (fAHP) increases in CA1 pyramidal cells of rat hippocampus following transient forebrain ischemia. To understand the conductance mechanisms underlying these post-ischemic electrophysiological alterations, we compared differences in activities of large conductance Ca²⁺-activated potassium (BK_Ca) channels in CA1 pyramidal cells acutely dissociated from hippocampus before and after ischemia by using inside-out configuration of patch clamp techniques. (1) The unitary conductance of BK_Ca channels in post-ischemic neurons (295 pS) was higher than that in control neurons (245 pS) in symmetrical 140/140 mM K⁺ in inside-out patch; (2) the membrane depolarization for an e-fold increase in open probability (P_o) showed no significant differences between two groups while the membrane potential required to produce one-half of the maximum P_o was more negative after ischemia, indicating no obvious changes in channel voltage dependence; (3) the [Ca²⁺]_i required to half activate BK_Ca channels was only 1 μM in post-ischemic whereas 2 μM in control neurons, indicating an increase in [Ca²⁺]_i sensitivity after ischemia; and (4) BK_Ca channels had a longer open time and a shorter closed time after ischemia without significant differences in open frequency as compared to control. The present results indicate that enhanced activity of BK_Ca channels in CA1 pyramidal neurons after ischemia may partially contribute to the post-ischemic decrease in neuronal excitability and increase in fAHP.     

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.     

BDNF potentiates spontaneous Ca oscillations in cultured hippocampal neurons

Brain-derived neurotrophic factor (BDNF) is thought to regulate neuronal plasticity in developing and matured neurons, although the molecular mechanisms are less well characterized. We monitored changes in the intracellular calcium (Ca²⁺) levels induced by BDNF using a fluorescence Ca²⁺ indicator (Fluo-3) by means of confocal laser microscopy in rat cultured hippocampal neurons. BDNF acutely potentiated spontaneous Ca²⁺ oscillations in dendrites and also in the soma of several neurons, although it increased intracellular Ca²⁺ in only selective proportion of resting neurons without Ca²⁺ oscillations. The potentiation was observed both in the frequency and the amplitude of Ca²⁺ oscillations, completely blocked by K-252a, and significantly reduced by 2-aminophosphonovaleric acid. These findings suggest that BDNF increases glutamate release and N-methyl-

Full-size image

-aspartate (NMDA) channel-gated Ca²⁺ influx via TrkB and regulates the frequency and the amplitude of Ca²⁺ oscillations. BDNF may have the potential to modulate spontaneous Ca²⁺ oscillations to regulate neuronal plasticity in developing hippocampal neurons.     

Developmental lead exposure impairs extinction of conditioned fear in young adult rats

Pavlovian fear conditioning is a model of emotional learning in which a neutral stimulus such as a tone is paired with an aversive stimulus such as a foot shock. Presentation of a tone with a foot shock in a context (test box) elicits a freezing response representative of stereotypic fear behavior. After conditioning has occurred, presentation of the context (test box) or tone in the absence of the unconditioned stimulus (shock) causes extinction of the fear response. Rats chronically exposed to environmentally relevant levels of lead (Pb²⁺) and controls were tested in a fear-conditioning (FC) paradigm at 50 days of age (PN50). Littermates to FC rats received an immediate shock (IS) when placed in the test box with no tone. Blood Pb²⁺ levels in control and Pb²⁺-exposed animals were (mean ± S.E.M.): 0.76 ± 0.11 (n = 15) and 25.8 ± 1.28 μg/dL (n = 14). Freezing behavior was recorded during acquisition (day of training) or during 4 consecutive extinction days. Control and Pb²⁺-exposed FC rats exhibited the same level of freezing time on the acquisition day. No freezing behavior occurred in IS rats regardless of treatment. Presentation of context 24 h later produced a freezing response on both control and Pb²⁺-exposed FC rats but not in IS rats. When tested in the extinction phase, Pb²⁺-exposed FC rats exhibited deficits in extinction compared to control FC rats. That is, when presented with context on 4 consecutive days after acquisition of the fear response, Pb²⁺-exposed FC rats exhibited a greater freezing response than control FC rats. These findings indicate that chronic Pb²⁺ exposure produces a deficit in extinction learning and the animals remain more fearful than controls.     

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.     

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.     

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.     

Re-evaluation of acute neurotoxic effects of Cd on mesencephalic trigeminal neurons of the adult rat

The mechanism of Cd²⁺ neurotoxicity, which is considered to be secondary to changes in blood vessels, was re-evaluated in dissociated mesencephalic trigeminal (Me5) neurons of the adult rat. Cd²⁺ induced morphological changes in Me5 neurons at 0.1 and 1 mM but not at 0.01 mM. The changes appeared predominantly in the cytoplasm: destruction of the cytoplasmic organelles, swelling and vacuolization of the cell body, and finally resulted in cell lysis. These observations indicate necrosis rather than apoptosis, and no sign of degraded nuclear DNA, characteristic to apoptosis, was detected by the TUNEL technique. Using a Ca²⁺-sensitive dye Indo-1, Cd²⁺ was found to elevate the intracellular Ca²⁺ concentration [Ca²⁺]_i (both in the cytoplasm and the nucleus). Both the elevation in [Ca²⁺]_i and the morphological alteration were inhibited either by removing Ca²⁺ from the bathing medium or by the application of BAPTA/AM (10 μM), a membrane-permeable intracellular Ca²⁺ chelator. Furthermore, neither morphological changes nor elevation in [Ca²⁺]_i by Cd²⁺ occurred in the presence of Zn²⁺. It is concluded that (1) Cd²⁺ can directly affect nerve cells, (2) toxicity of Cd²⁺ on Me5 neurons is mediated by continuous elevation in [Ca²⁺]_i, (3) Cd²⁺ induces necrotic cell death, and (4) Cd²⁺ neurotoxicity can be antagonized by Zn²⁺.     

Erratum to: Differential alterations in the distribution of voltage-gated calcium channels in aged rat cerebellum: [Brain Research 903 (2001) 247–252]

In the present study, we have investigated the spatial and temporal distribution of voltage-gated calcium channels in the gerbil model of global cerebral ischemia using immunohistochemistry. Distinct localizations of P-type (α_1A), N-type (α_1B), and L-type (α_1C and α_1D) Ca²⁺ channels were observed in the hippocampus at days 1–5 after ischemic injury. However, increased expression of N-type Ca²⁺ channels was detectable in brain regions vulnerable to ischemia only at days 2 and 3 after ischemic injury. The pyramidal cell bodies of CA1-3 areas and the granule cell bodies of the dentate gyrus were intensely stained at days 2 and 3 following ischemic injury. Transient changes in N-type Ca²⁺ channel expression were also observed in the affected cerebral cortex and striatum at days 2 and 3 after ischemic injury. Although the present study has not addressed the multiple mechanisms contributing to the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) increase in the ischemic brain, the first demonstration of the transient increase in N-type Ca²⁺ channels may prove useful for future investigations.     

Differential effect of a dihydropyridine derivative to Ca entry pathways in neuronal preparations

Nicardipine is one of the 1,4-dihydropyridine derivatives known as blockers for the voltage-dependent Ca²⁺ channels in muscle cells. The effects of nicardipine on the neuronal functions were studied in several neuronal preparations including clonal rat pheochromocytoma (PC12) cells, rat brain synaptosomes and slices. Nicardipine failed to block the Ca²⁺-dependent action potentials and the after-spike hyperpolarizations evoked by intracellularly injected current pulses in rat pheochromocytoma cells, while the high K^+- stimulated Ca²⁺ influx and ATP release were dose-dependently inhibited in the same cells. In rat cerebral synaptosomes and cortical slices, nicardipine showed no blockade on the high K⁺-stimulated Ca²⁺ influx and transmitter releases. It was then suggested that the voltage-dependent Ca²⁺ channels are polymorphic among tissues or even in a single cell from the viewpoint of dihydropyridine susceptibility.     

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