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 Ca(2+) influx with mossy fiber stimulation in hippocampal CA3 neurons of spontaneously epileptic rats

This study was performed to determine whether the intracellular Ca²⁺ concentration ([Ca²⁺]_i) is increased in hippocampal CA3 neurons of spontaneously epileptic rats (SER) which show both absence-like and convulsive seizures using hippocampal slices loaded with Calcium Green-1 when a weak single stimulation is given to the mossy fiber. [Ca²⁺]_i in the CA3 area was significantly increased after a single stimulus to mossy fibers in SER, while no changes were detected in normal Wistar rats. These findings suggest the existence of an abnormality in the Ca²⁺ channel in the SER CA3 region and that this is probably responsible for epileptic seizures.     

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.     

Voltage-dependent calcium channel abnormalities in hippocampal CA3 neurons of spontaneously epileptic rats

PURPOSE: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.     

Enhanced Calcium Influx in Hippocampal CA3 Neurons of Spontaneously Epileptic Rats

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.     

Effects of hemoglobin and its breakdown products on synaptic transmission in rat hippocampal CA1 neurons

During head injuries and hemorrhagic stroke, blood is released into the extravascular space. The pooled erythrocytes get lysed and hemoglobin is released into the intracranial cavities. Therefore, neurons may be exposed to hemoglobin and/or its breakdown products, hemin and iron, for long periods of time. In this study, the electrophysiological actions of these agents on synaptic transmission in rat hippocampal CA1 pyramidal neurons were studied using extracellular field- and whole cell patch-recordings. Previously our laboratory reported that commercially available hemoglobin produced a dose dependent suppression of synaptic transmission in hippocampal CA1 neurons. In the present study, however, we found that this depression was caused by impurities present in the hemoglobin samples. Commercially available hemoglobin and methemoglobin did not have a significant effect on synaptic transmission. Although, reduced-hemoglobin prepared using a method described by Martin et al. [J. Pharm. Exp. Ther. 232 (1985) 708], produced a significant depression of synaptic transients, these effects were due to contamination with bisulfite that was present due to the reducing procedure. Therefore, the technique of Martin et al. was inadequate in removing the reducing agents or their breakdown products. A number of studies in literature used commercial samples of hemoglobin or reduced hemoglobin prepared using the method of Martin et al. Our observations indicate that it would be important to determine if contaminants, rather than hemoglobin, are responsible for the observed effects in these studies. Unlike hemoglobin, its breakdown products, ferrous chloride and hemin, produced an irreversible and significant depression of field excitatory postsynaptic potentials. The relevance of these effects in neurological complications that follow head injuries and hemorrhagic stroke awaits further investigation.     

Relation between responsiveness to neurotransmitters and complexity of epileptiform activity in rat hippocampal CA1 neurons

PURPOSE: Our previous works suggested that sensitivity of neurons with chaotic firing patterns to stimuli is significantly greater than that in neurons with periodic firing patterns, which shows that responsiveness of neurons may depend on the complexity of the firing series. This study was performed to determine the relation between responsiveness of the hippocampal CA1 neurons with epileptiform activity (EA) to neurotransmitters and their complexity of firing series. METHODS: Firing series of CA1 neurons were recorded extracellularly in rat hippocampal slice. Approximate entropy was used to describe the complexity of the interspike interval (ISI) series. EA was induced by local application of penicillin (1,000 IU/ml). The change of firing rate induced by neurotransmitters (glutamate and gamma-aminobutyric acid) was compared with that of the degree of complexity of ISI series in the process of EA. RESULTS: The excitatory responses to glutamate and the inhibitory responses to gamma-aminobutyric acid in CA1 neurons appeared to be decreased during the process of penicillin-induced EA. However, during this same process, the approximate entropy of the ISI series also was decreased significantly. CONCLUSIONS: The results suggest that the reduced responses to neurotransmitters of the CA1 neurons appear to be closely related to the onset of EA. Furthermore, these neurons show that the changes in responsiveness are closely parallel to the decrease of degree of complexity of firing series during penicillin epileptogenesis.     

Heterogeneous distribution of tetrodotoxin-sensitive calcium-conducting channels in rat hippocampal CA1 neurons

Voltage-dependent Ca2+ currents (ICas) in neurons can be classified into T-, N- and L-types. In the CA1 pyramidal neurons freshly isolated from rat hippocampus we found an additional tetrodotoxin (TTX)-sensitive Ca2+ current (termed 'TTX-ICa') which passed through the Na+ channel. The TTX-ICa showed a heterogeneous distribution in the dorsal site of Ca1 region.     

Effects of tissue preincubation and hypoxia on CA3 hippocampal neurons in the in vitro slice preparation

Using the in vitro hippocampal slice preparation, we studied the electrophysiological properties of pyramidal cells in tissue that was 'preincubated' (2-6 h in a large, static volume of oxygenated bathing medium) before being placed in an interface chamber for study. Striking differences were found in 'preincubated' vs 'non-preincubated' CA3 cells. The preincubated cells had more negative resting potentials, higher input resistance, lower threshold for stimulus-evoked burst discharge and larger hyperpolarizing afterpotentials. Cells in the preincubated CA3 region were also more likely to show spontaneous synchronized burst discharge, but were relatively resistant to hypoxia-induced spreading depression. CA1 cells were less dramatically affected by preincubation, showing little difference from their non-preincubated counterparts. Possible mechanisms involved in the CA3 preincubation effect, including glial buffering alterations and changes in Na+, K+-ATPase activity, are discussed.     

N-acetyl-L-aspartate activates hippocampal CA3 neurons in rodent slice preparations

High N-acetyl-L-aspartate (NAA) levels prevail as a free amino acid in vertebrate brains. NAA is synthesized from aspartate and acetyl Co-A, or is liberated by the hydrolyzation of N-acetyl-L-aspartyl-glutamate in mitochondria before being metabolized by aspartoacylase to aspartate and acetate in the cytosol of glial cells. The tremor rat (tm/tm), derived from a Kyoto-Wistar colony, shows absence-like seizures with 5- to 7-Hz spike-wave-like complexes in cortical and hippocampal electroencephalograms (EEG). Genomic microdeletion was found within the aspartoacylase-encoding tm critical region, where an increase in the NAA level was noted. Intracerebroventricular NAA induced absence-like seizures, convulsive seizures or both in epileptic EEG of Wistar rats. NAA activated the hippocampal CA3 neurons of Wistar rats via the metabotropic glutamate receptor (mGluR) in acutely dissociated hippocampal CA3 neurons. The mechanism of NAA action on CA3 neurons was examined with intracellular recording of Wistar and tremor rat hippocampal slices to evaluate the role of NAA in neuronal networks. Bath application of NAA (10 microM-1mM) dose-dependently induced depolarization in CA3 neurons of Wistar and tremor rats. Cadmium (a Ca(2+) channel antagonist) and GDEE (an ionotropic glutamate receptor antagonist) did not affect NAA-induced depolarization. Although ACPD (a nonspecific mGluR agonist) induced similar depolarizations in CA3 neurons, MCPG (a mGluR antagonist) inhibited NAA-induced depolarization. These results suggest that NAA probably activates hippocampal CA3 neurons via the mGluR in a neuronal network.     

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.     

Blockade by magnesium of sodium currents in acutely isolated hippocampal CA1 neurons of rat

The effects of magnesium (MgSO(4)) on sodium currents (Na(+) currents) in freshly dissociated rat hippocampal neurons were studied using the whole-cell patch clamp techniques. MgSO(4) caused a concentration-dependent and voltage-dependent reversible decrease of Na(+) currents. The half-blocking concentration (IC(50)) of MgSO(4) on Na(+) currents was 4.05 mM. But the action was frequency-independent. In addition, 4 mM MgSO(4) shifted the steady state activation curve of Na(+) currents toward positive potential (control V(h)=-55.83+/-6.79 mV, MgSO(4)V(h)=-34.15+/-6.18 mV, n=8, P相似文献   

The influence of electric fields on the epileptiform bursts induced by high potassium in CA3 region of rat hippocampal slice

Abstract

The effects of pulsed direct current (de) electric fields on the frequency of spontaneous bursting in a model epileptic focus were studied. The high potassium hippocampal slice model was used to generate spontaneous burst firing activity similar to interictal spikes in the pyramidal cell layer of CA3. Electric fields were generated from platinum subdural electrodes placed in the perfusion bath. Three hundred and seventy-eight experimental trials were performed on 10 hippocampal slices from 10 rats and the effects of field polarity, field strength and duration of stimuli on firing frequency was examined. Hippocampal slices were oriented horizontally with the CA3 layer towards the positive electrodel the average interburst interval did not correlate significantly with polarity of the delivering pulses (one-way ANOVA, p = 0.96). Average interburst interval showed a significant correlation with pulse duration of200 and 400msec (p = 0.030 and p = 0.004, respectively). As a function of field strength, there were significant average interval changes for fields of 33, 46, and 73 mV/mm (p = 0.024, P = 0.001 and p = 0.001, respectively). In conclusion, CA3 burst firing activity in high potassium concentration can therefore be altered by electric fields. [Neural Res 1998; 20: 542-548]     

Evidence for neuronal interactions by electrical field effects in the CA3 and dentate regions of rat hippocampal slices

During population spikes in slices of rat hippocampus, transmembrane differential recordings (intracellular minus extracellular) revealed electrical field-effect depolarizations in CA3 pyramidal and dentate granule neurons. The field-effect depolarizations were not seen in single-ended recordings, and were consistently shown to increase neuronal excitability. Therefore, as previously shown for the CA1 area, large population spikes from CA3 pyramidal and dentate granule cells are associated with transmembrane depolarizations that increase neuronal excitability.     

Blockade of U50488H on sodium currents in acutely isolated mice hippocampal CA3 pyramidal neurons

The effect of opioid agonist U50488H on Na(+) currents was examined in freshly dissociated hippocampal neurons of mice using the whole-cell patch clamp technique. U50488H (1-100 microM) caused a concentration dependent reversible inhibition of the voltage-activated sodium currents. IC50 of 15.5 microM and Hill constant of 1.4 were calculated respectively. The inhibitory actions of U50488H on I(Na) were still observed in the presence of 30 microM naloxone. Moreover, under the action of U50488H, repetitive stimulation induced further inhibition which was frequency-dependent. The activation curve did not change before and after application of 10 microM U50488H. However, after exposure to 10 microM U50488H and repetitive depolarizing at 10 Hz, frequency-dependent inhibition occurred, and a mean shift of half-activation membrane potential by +20 mV could be induced. The inactivation curve was significantly changed toward negative membrane potential with 10 microM U50488H, and further negative shift was observed after repetitive depolarizing at 10 Hz. Our results indicate that U50488H could directly inhibit neuronal Na(+) currents without involvement in the activation of kappa-opioid receptors.     

Preferential inhibition of L- and N-type calcium channels in the rat hippocampal neurons by cilnidipine

The effect of a dihydropyridine Ca²⁺ antagonist, cilnidipine, on voltage-dependent Ca²⁺ channels was studied in acutely dissociated rat CA1 pyramidal neurons using the nystatin-perforated patch recording configuration under voltage-clamp conditions. Cilnidipine had no effect on low-voltage-activated (LVA) Ca²⁺ channels at the low concentrations under 10⁻⁶ M. On the other hand, cilnidipine inhibited the high-voltage-activated (HVA) Ca²⁺ current (I_Ca) in a concentration-dependent manner and the inhibition curve showed a step-wise pattern; cilnidipine selectively reduced only L-type HVA I_Ca at the low concentrations under 10⁻⁷ and 10⁻⁶ M cilnidipine blocked not only L- but also N-type HVA I_Ca. At the high concentration over 10⁻⁶ M cilnidipine non-selectively blocked the T-type LVA and P/Q- and R-type HVA Ca²⁺ channels. This is the first report that cilnidipine at lower concentration of 10⁻⁶ M blocks both L- and N-type HVA I_Ca in the hippocampal neurons.     

Activation by N-acetyl-L-aspartate of acutely dissociated hippocampal neurons in rats via metabotropic glutamate receptors

PURPOSE: We previously reported that an increase in the N-acetyl-L-aspartate (NAA) level due to the lack of aspartoacylase gene was found in the brain of the tremor rat (tm/tm), which is a mutant with a causative gene named tm that shows epileptic seizures. Therefore, NAA is suggested to be one of the factors involved in the induction of epileptic seizures. Patch-clamp studies were performed to determine whether NAA produces an excitatory effect on acutely dissociated rat hippocampal neurons. METHODS: Acutely dissociated hippocampal neurons were prepared from normal Wistar rats aged 3-4 weeks. NAA-induced currents were investigated by using the whole-cell voltage-clamp recording technique. RESULTS: Application of NAA at concentrations of 100 nM to 1 mM through a U-tube for 2 s produced an inward current in a concentration-dependent manner at a holding potential of -60 mV. When the current-voltage relation was examined, the reversal potential of the NAA-induced current was found to be approximately 0 mV. The NAA-induced current was inhibited by bath application of the metabotropic glutamate receptor (mGluR) antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG) and by intracellular application of guanosine 5'-O-(2-thiodiphosphate) (GDP-betaS), a nonhydrolyzable GDP analogue. However, the NAA-induced current remained unaffected by glutamic acid diethyl ester, a non-N-methyl-D-aspartate (NMDA)-subtype ionotropic glutamate receptor antagonist, or the voltage-dependent ion channel blockers tetrodotoxin, CdCl2, and tetraethylammonium-chloride. Conversely, the mGluR agonist, trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD) also induced an inward current, with a reversal potential of 0 mV. The ACPD-induced current also was inhibited by MCPG. CONCLUSIONS: These results suggest that NAA acts on the G protein-coupled mGluRs to induce an inward current that results in excitation of the neurons, thereby contributing to the occurrence of epileptic seizures.     

Chaotic responses of the hippocampal CA3 region to a mossy fiber stimulation in vitro

Undoubted evidence of chaotic activity of a biological neural network are given. Spontaneous epileptiform bursts of a neuron population in the CA3 region of rat hippocampal slices were caused in a perfusing medium with 2 mM penicillin and 8 mM K⁺ ions, and responses of field potential in the CA3 region to a periodic mossy fiber stimulation were investigated. Phase-locked and chaotic responses occur depending on stimulus parameters; for example, when the frequency of the stimulation increases, 1:1 phase-locking bifurcates to chaos through 1:2 phase-locking. The chaotic responses show a broad-band spectrum, and their trajectories in the three-dimensional phase space (V(t), V(t + τ), V(t + 2τ)) reconstruct a strange attractor. Lyapunov exponents of the strange attractors estimated by the Wolf's algorithm are positive. Moreover, one-dimensional strobomaps obtained from the chaotic responses show a non-invertible function. Since the slope of each strobomap at their fixed point is more negative than −1, the fixed points are unstable. These are undoubted evidences for chaotic responses of the CA3 region in hippocampal slices maintained in vitro. Cross-correlation functions between field potential responses which were simultaneously observed at different sites show that the responses are spatially coherent throughout the CA3 region even when the responses are chaotic.     

Early dendritic changes in hippocampal pyramidal neurones (field CA1) of rats subjected to acute soman intoxication: a light microscopic study

In rats poisoned with soman (s.c. 100 μg/kg), a potent inhibitor of cholinesterase (ChE), the numbers of dendritic spines of Golgi impregnated hippocampal pyramidal cells (CA1 sector) were evaluated within the first hour of the intoxication. Animals that experienced convulsions showed a rapid and striking decrease in the density of dendritic spines which could be reduced by nearly 80% of the controls in the basal dendrites 60 min post-soman exposure. Although the exact mechanisms cannot be determined from the present study, it is suggested that the spine loss may represent: (1) the first sign of the seizure-related neuronal changes which are known to occur later during soman intoxication; and (2) the expression of the ‘dendrotoxic’ effects produced by certain non-cholingergic excitatory transmitters such as glutamate.