Long-lasting Depolarization Shift Research Articles

Involvement of Ca2+ Channels in Abnormal Excitability of Hippocampal CA3 Pyramidal Cells in Noda Epileptic Rats

Noda epileptic rat (NER) is a mutant rat, which spontaneously exhibits a tonic-clonic convulsion from 14 weeks of age. An intracellular recording study was performed to elucidate the abnormal excitability of NER hippocampal CA3 neurons. The recorded neurons were classified into two groups, group A and B neurons, according to the responses to a single stimulation of mossy fibers. In group A neurons, a stimulus elicited a long-lasting depolarization shift accompanying repetitive firings followed by after-hyperpolarization. In group B neurons, the same stimulus elicited a single spike without a long-lasting depolarization shift. Bath application of 1 mM Cd2+, a nonselective Ca2+ channel blocker, completely inhibited the abnormal excitation in group A neurons. We further examined the character of Ca2+ spikes in NER CA3 neurons. Ca2+ spikes were completely blocked by 10 μM Cd2+ in group A neurons, but not in either group B or control neurons, suggesting that Ca2+ channels in NER group A neurons have the hypersensitivity to Cd2+. Analysis using subtype specific blockers of Ca2+ channel raised the possible involvement of T-type Ca2+ channels. These results suggest that Ca2+ channel dysfunction is involved in the abnormal excitability of CA3 pyramidal neurons and pathogenesis of epilepsy in NER.

Modulation of abnormal synaptic transmission in hippocampal CA3 neurons of spontaneously epileptic rats (SERs) by levetiracetam

Levetiracetam (LEV) inhibits partial refractory epilepsy in human, and both convulsive and absence-like seizures in the spontaneously epileptic rat (SER). Two-thirds of hippocampal CA3 neurons in SER show a long-lasting depolarization shift, with accompanying repetitive firing upon mossy fiber stimulation. This abnormal excitability is probably attributable to abnormalities in the L-type Ca(2+) channels. We performed electrophysiological studies to elucidate the mechanism underlying the antiepileptic effects of LEV via intracellular recording from the hippocampal CA3 neurons in slice preparations of SER and non-epileptic Wistar rats. LEV (100 μM) inhibited the depolarization shift with repetitive firing by mossy fiber stimulation (MFS), without affecting the first spike in SER CA3 neurons. At a higher dose (1mM), LEV suppressed the first spike in all SER neurons (including the CA3 neurons which showed only a single action potential by MFS), while the single action potential of Wistar rat CA3 neurons remained unaffected. SER CA3 neurons with MFS-induced abnormal firing exhibited a higher number of repetitive spikes when a depolarization pulse was applied in the SER CA3 neurons. LEV (100 μM, 1mM) reduced the repetitive firing induced by a depolarization pulse applied without affecting Ca(2+) spike in SER neurons. LEV is known not to bind glutamate and gamma-aminobutyric acid (GABA) receptors. These findings suggest that the therapeutic concentration of LEV inhibits abnormal firing of the CA3 neurons by modulating abnormal synaptic transmission and abnormal Na(+) channels in SER.

Voltage‐dependent Calcium Channel Abnormalities in Hippocampal CA3 Neurons of Spontaneously Epileptic Rats

Hippocampal CA3 neurons of spontaneously epileptic rats (SER; zi/zi, tm/tm), which show both absence-like seizures and tonic convulsions, exhibit a long-lasting depolarization shift with repetitive firing with a single stimulation of mossy fibers. Therefore a whole-cell patch-clamp study using temporarily dissociated hippocampal CA3 neurons from SER was performed to elucidate whether such abnormal excitability was due to abnormalities in voltage-dependent Ca(2+) channels (VDCCs). Hippocampal CA3 neurons were temporarily dissociated with enzymatic and mechanical treatments. In a voltage-clamp mode with whole-cell recording, depolarizing step pulses were applied to induce Ca(2+) currents in the presence of tetrodotoxin and tetraethylammonium. The threshold level of the Ca(2+) current induced by depolarizing pulses was found to be lower in hippocampal CA3 neurons of SER compared with those of control Wistar rats. In addition, the Ca(2+) current peak amplitude was greater, and decay of the current was weaker in CA3 neurons of SER than in those of normal Wistar rats. These findings suggest that enhancements of Ca(2+) influx into hippocampal CA3 neurons due to the easier activation properties of VDCCs, as well as a decrease in decay, are involved in SER epileptic seizures.

Involvement of Ca(2+) channels in abnormal excitability of hippocampal CA3 pyramidal cells in noda epileptic rats.

Noda epileptic rat (NER) is a mutant rat, which spontaneously exhibits a tonic-clonic convulsion from 14 weeks of age. An intracellular recording study was performed to elucidate the abnormal excitability of NER hippocampal CA3 neurons. The recorded neurons were classified into two groups, group A and B neurons, according to the responses to a single stimulation of mossy fibers. In group A neurons, a stimulus elicited a long-lasting depolarization shift accompanying repetitive firings followed by after-hyperpolarization. In group B neurons, the same stimulus elicited a single spike without a long-lasting depolarization shift. Bath application of 1 mM Cd(2+), a nonselective Ca(2+) channel blocker, completely inhibited the abnormal excitation in group A neurons. We further examined the character of Ca(2+) spikes in NER CA3 neurons. Ca(2+) spikes were completely blocked by 10 microM Cd(2+) in group A neurons, but not in either group B or control neurons, suggesting that Ca(2+) channels in NER group A neurons have the hypersensitivity to Cd(2+). Analysis using subtype specific blockers of Ca(2+) channel raised the possible involvement of T-type Ca(2+) channels. These results suggest that Ca(2+) channel dysfunction is involved in the abnormal excitability of CA3 pyramidal neurons and pathogenesis of epilepsy in NER.

Epileptiform burst discharges in hippocampal CA3 neurons of young but not mature Noda epileptic rats (NER)

Noda epileptic rat (NER), originally found in a colony of Crj; Wistar rats, shows spontaneous tonic–clonic convulsion characterized by the appearance of high voltage polyspikes in cortical and hippocampal EEG once every 2–3 days after 2–4 months of age. Electrophysiological studies using hippocampal slice preparations of NER were performed to determine whether hippocampal neurons have abnormal excitability. When a single stimulus (1–25 V) was delivered to the mossy fibers of NER at 4–6 weeks old before they showed any seizures, a long-lasting depolarization shift (DS) accompanied by repetitive firings and after-hyperpolarization following the abnormal firing was observed in seven of 14 hippocampal CA3 neurons. A lower stimulation intensity evoked DS and abnormal firing in three of nine CA3 neurons of NER at 10–15 weeks old which had already showed seizures at 10–15 weeks of age. However, the abnormal firing was not observed in any 10 neurons of the animals at more than 20 weeks old nor in Wistar rats. The input impedances of CA3 neurons in NER with abnormal firing were lower than those without abnormal firing and those in Wistar rats. The abnormal excitability obtained in NER at an age when it did not display any seizures suggests that the hippocampus may play a role in epileptogenicity in NER.

Effects of vigabatrin on epileptiform abnormal discharges in hippocampal CA3 neurons of spontaneously epileptic rats (SER)

Vigabatrin, a γ-amino butyric acid (GABA) transaminase inhibitor, is known to inhibit partial epilepsy in humans. The spontaneously epileptic rat (SER), a double mutant ( zi/ zi, tm/ tm), exhibits both tonic convulsion and absence-like seizures from the age of 8 weeks. Hippocampal CA3 pyramidal neurons in SER show a long-lasting depolarization shift with accompanying repetitive firing when a single stimulus is delivered to the mossy fibers in slice preparations. The effects of vigabatrin on the abnormal excitability of hippocampal CA3 pyramidal neurons in SER were examined to elucidate the mechanism underlying the antiepileptic action of the drug. Intracellular recordings were performed in 24 hippocampal slice preparations of 20 SER aged 8–17 weeks old. Bath application of vigabatrin (1 mM) inhibited the depolarizing shifts with repetitive firing induced by mossy fiber stimulation in 15 min without affecting the first spike and resting membrane potentials in hippocampal CA3 neurons of SER. A higher dose of vigabatrin (10 mM) sometimes inhibited the first spike. However, vigabatrin at doses up to 10 mM did not significantly affect the single action potential elicited by stimulation of the mossy fibers in the hippocampal CA3 neurons of age-matched Wistar rats. In addition, application of vigabatrin (10 mM) did not significantly affect the firing induced by depolarizing pulse applied in the CA3 neurons of the SER, nor the miniature excitatory postsynaptic potential (mEPSP) recorded in the CA3 neurons of SER. The inhibitory effect of vigabatrin (1 mM) on the mossy fiber stimulation-induced depolarization shift with repetitive firing was blocked by concomitant application of bicuculline (10 μM), a GABA A receptor antagonist. These findings strongly suggested that GABA increased by inhibition of GABA transaminase with vigabatrin inhibits abnormal excitation of hippocampal CA3 neurons of SER via GABA A receptors, although the possibility that the drug acted directly on the GABA A receptors of CA3 neurons could not be completely excluded.

Enhanced calcium influx in hippocampal CA3 neurons of spontaneously epileptic rats.

The spontaneously epileptic rat (SER: tm/tm, zi/zi) shows both absence-like seizures and tonic convulsions. Our previous electrophysiologic studies have demonstrated that SER has abnormal excitability of hippocampal CA3 neurons, which shows a long-lasting depolarization shift by a single stimulation of mossy fibers, probably resulting from the Ca2+ channel abnorrmalities. The present study was performed to determine whether Ca2+ influx is actually enhanced in the CA3 area of SER. Hippocampal slices were prepared from normal Wistar rats and SER aged 11-16 weeks old, when the epileptic seizures had been observed, and loaded with fura-2AM. Intracellular Ca2+ concentration ([Ca2+]i) was monitored as the ratio of fluorescence intensities excited at wavelengths of 340 and 380 nm (RF340/F380) with photometric devices. High K+ (10-60 mM) applied to the bath for 2 min increased [Ca2+]i in hippocampal CA1, CA3, and dentate gyrus (DG) areas of both the normal rats and SER in a concentration-dependent manner. However, the high K+-induced increase in [Ca2+]i was significantly more pronounced in the CA3 area of the SER than in that of the normal animals, whereas there were no significant differences in high K+-induced increases of [Ca2+]i in CA1 or DG between the SER and controls. The high K+-induced increases in [Ca2+]i of CA1, CA3, and DG were inhibited by nifedipine (1 to approximately 10 nM), a Ca2+ channel antagonist in both SER and controls. However, the inhibition of the high K+-induced increase in [Ca2+]i by nifedipine (1 nM) was significantly greater in the CA3 area of SER than that of controls. These findings suggest that Ca2+ influx through the L-type Ca2+ channels is much greater in the CA3 area of SER than in that of normal animals and is involved in the epileptic seizures of the SER.

Suppression by topiramate of epileptiform burst discharges in hippocampal CA3 neurons of spontaneously epileptic rat in vitro

Topiramate, a novel antiepileptic drug, inhibits the seizures of spontaneously epileptic rat (SER), a double mutant ( zi/ zi, tm/ tm) which exhibits both tonic convulsion and absence-like seizures from the age of 8-weeks. Hippocampal CA3 pyramidal neurons in SER show a long-lasting depolarization shift with accompanying repetitive firing when a single electrostimulation is delivered to the mossy fibers in vitro. The effects of topiramate on the excitability of CA3 pyramidal neurons in SER were examined to elucidate the mechanism underlying the antiepileptic action. Intracellular recordings were performed in 23 hippocampal slice preparations of 16 SER aged 8–17 weeks. Topiramate (10–100 μM) dose-dependently inhibited the depolarizing shifts with repetitive firing induced by mossy fiber stimulation without affecting the first spike and resting membrane potentials in hippocampal CA3 neurons of SER. Higher dose of topiramate (100 μM) sometimes inhibited the first spike, and decreased excitatory postsynaptic potentials in the SER CA3 neurons. However, topiramate up to 100 μM did not affect the single action potential elicited by the stimulation in the hippocampal CA3 neurons of age-matched Wistar rat devoid of the seizure. Application of topiramate (100 μM) did not significantly affect the firing induced by depolarizing pulse applied in the CA3 neurons of the SER. In addition, topiramate (100 μM) had no effects on the Ca 2+ spike induced by intracellularly applied depolarizing pulse in the presence of tetrodotoxin and tetraethylammonium. In contrast, a dose-dependent inhibition of depolarization and repetitive firing induced by bath application of glutamate in CA3 pyramidal neurons was obtained with topiramate (10–100 μM). Furthermore, topiramate (100 μM) decreased the number of miniature postsynaptic potential of CA3 pyramidal neurons of SER. In patch clamp whole cell recording using acutely dissociated hippocampal CA3 neurons from SER aged 8-weeks and age-matched normal Wistar rats, there were no remarkable effects on voltage dependent Ca 2+ current with topiramate up to 300 μM in either animal; the current was completely blocked by Cd 2+ at a concentration of 1 mM. These findings suggest that topiramate inhibits release of glutamate from the nerve terminals and/or abnormal firing of the CA3 pyramidal neurons of SER by mainly blocking glutamate receptors in the neurons.

てんかん動物(SERとNER)を用いた抗てんかん薬の評価

Epileptic model animals, Spontaneously epileptic rat (SER) and Noda epileptic rats (NER), were examined to determine if they were useful for the evaluation of acute and chronic antiepileptic drugs. SER is a double mutant (zi/zi, tm/tm) exhibits tonic convulsion and absence-like seizures. The antiepileptic profile of a single application of conventional drugs in SER was similar to those found in human epilepsy. Novel antiepileptics such as topiramate, clobazam, and piracetam were successfully evaluated with SER for their anticonvulsant effects. The antiepileptic effects of repeated administrations of novel drugs could also be evaluated with this animal. The long-lasting effects of the antiepileptics were similar to those in human epilepsy. In SER hippocampal slices, long-lasting depolarization shift accompanying repetitive firing obtained in CA3 pyramidal cells with a single stimulation of the mossy fibers was inhibited by topiramate, but the firing induced by applying a depolarizing pulse in the cell was not affected, suggesting that the drug interferred with synaptic transmission in the mossy fiber-CA3 system of SER. Thus SER can also be empolyed for elucidating the mechanisms underlying the antiepileptic effects of the drug in question. Recently, a novel epileptic model animal NER, was developed. The clinical features resemble the tonicclonic seizures in human epilepsy. Multiple high voltage spikes appear in cotical EEG during seizures. The audiogenic seizures were induced by acoustic priming of a bell sound which had been given weekly to the NER since 3 weeks of age. This audiogenic seizure was inhibited by the conventional antiepileptics. Thus, both SER and NER are useful models for evaluating the acute and chronic antiepileptic effects of novel drugs.

Pharmacological Application of Spontaneously Epileptic Rat(SER).

The spontaneously epileptic rat (SER) is a double mutant (zi/zi, tm/tm) obtained by mating zitter homozygote (zi/zi) with tremor heterozygote (tm/ + ) . This animal exhibits both tonic convulsion and absence-like seizures characterized by a sudden appearance of 5-7 Hz spikes and wave complex in cortical and hippocampal EEG. This study was performed to elucidate the usefulness of SER in evaluating of acute and long-term effects of antiepileptic drugs. Moreover, activities of the central inhibitory system and abnormal excitability of neurons related to the epileptic seizures were investigated. (1) From recordings of implanted electrode in cerebral cortex and hippocampus, phenytoin and carbamazepine (agents for tonic-clonic grand mal seizure) inhibited tonic convulsion without affecting absence-like seizures, whereas trimethadione and ethosuximide (agents effective for absence seizure, petit mal epilepsy) suppressed absence-like seizures without any effects on tonic convulsion. Both seizures were inhibited by phenobarbital, valproate and diazepam, agents which are effective for grand and petit mal epilepsy. This antiepileptic profile suggests that tonic and absence-like seizures in SER correspond to human grand and petit mal seizures. Therefore, SER is a useful model for evaluating the acute effects of novel antiepileptics. (2) The group of SER, which were orally administered with 1 % phenobarbital-containing pellet displayed a marked increase in body weight and longer a life span than the control group with a plasma concentration of 35-50 μg/ml. Similar to the futile results of chronic administration of phenobarbital in human absence epilepsy, absence-like seizures in SER were not affected by this drug, although tonic convulsion was inhibited. Long-term effects of valproate (1 and 5 %) were similar to those of phenobarbital except for its efficacy on absence-like seizures. Therefore, SER is also useful for the evaluation of long-term effects of antiepileptics. (3) Both tonic and absence-like seizures in SER were inhibited by TRH and its analog, CNK-602A, and dopamine agonists, methamphetamine and apomorphine. These results suggest that dopaminergic pathways act as an inhibitory system on the seizure-producing mechanism. Further, SER can be used for investigations of the central inhibitory system involved in the regulation of seizures. (4) In hippocampal slice preparations, a single stimulus given to the mossy fiber induced a longlasting depolarization shift with repetitive firings in CA3 field pyramidal neurons of the SER but not zitter or wistar rats. This abnormal excitation of the neurons were completely blocked by verapamil, a calcium channel blocker. These findings suggest that abnormal calcium channel activities in the CA3 pyramidal neurons are involved in the seizures of SER. Thus, SER is a useful animal model for investigating abnormalities related to epileptic seizures.

Nonsynaptic epileptogenesis in the mammalian hippocampus in vitro. I. Development of seizurelike activity in low extracellular calcium.

Epileptiform activity induced in rat hippocampal slices by lowering extracellular Ca2+ concentration ([Ca2+]o) was studied with extracellular and intracellular recordings. Perfusing the slices with low Ca2+ (less than or equal to 0.2 mM) or EGTA-containing solutions blocked the synaptic responses of hippocampal pyramidal cells (HPCs). Despite the block, spontaneous paroxysms, termed seizurelike events (SLEs), appeared in the CA1 area and then recurred regularly at a stable frequency. Transient hypoxia accelerated their development and increased their frequency. When [Ca2+]o was raised in a stepwise manner, the SLEs disappeared at 0.3 mM. With extracellular recording from the CA1 stratum pyramidale, a SLE was characterized by a large negative shift in the field potential, which lasted for several seconds. During this period a large population of CA1 neurons discharged intensely and often in synchrony, as concluded from the frequent appearance of population spikes. Synchronization, however, was not a necessary precursor for the development of paroxysmal activity, but seemed to be the end result of massive neuronal excitation. The cellular counterpart of a SLE, as revealed by intracellular recording from HPCs in the discharge zone of the paroxysms, was a long-lasting depolarization shift (LDS) of up to 20 mV. This was accompanied by accelerated firing of the neuron. A prolonged after-hyperpolarization succeeded each LDS and arrested cell firing. Brief (approximately 50 ms) bursts were commonly observed before LDS onset. Single electrical stimuli applied focally to the stratum pyramidale or alveus evoked paroxysms identical to the spontaneous SLEs, provided they surpassed a critical threshold intensity. Subthreshold stimuli elicited only small local responses, whereas stimuli of varied suprathreshold intensities evoked the same maximal SLEs. Thus the buildup of a SLE is an all or nothing or a regenerative process, which mobilizes the majority, if not all, of the local neuronal population. Each SLE was followed by absolute and relative refractory periods during which focal stimulation was, respectively, ineffective and less effective in evoking a maximal SLE. In most slices the spontaneous SLEs commenced at a "focus" located in the CA1a subarea (near the subiculum). SLEs evoked by focal stimulation arose near the stimulating electrode. From their site of origin the paroxysmal discharges spread transversely through the entire CA1 area at a mean velocity of 1.74 mm/s. Consequently, the discharge zone of a SLE could encompass for several seconds the entire CA1 area.(ABSTRACT TRUNCATED AT 400 WORDS)