Norihito Yamada

Antiepileptogenic and anticonvulsant effects of NBQX, a selective AMPA receptor antagonist, in the rat kindling model of epilepsy

To investigate the role of non-NMDA receptors in epileptic seizures, we examined the antiepileptogenic and anticonvulsant effects of NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)-quinoxaline), a potent and selective AMPA receptor antagonist, in the rat kindling model. Systemic administration of 10-40 mg/kg NBQX significantly and dose dependently suppressed previously kindled seizures from the amygdala (AM), assessed in terms of the motor seizure stage and afterdischarge (AD) duration. The maximal effects were observed at 0.5-1 h after drug injection. When the intensity of electrical stimulation was increased to twice the generalized seizure-triggering threshold (GST), the anticonvulsant effects of NBQX on AM-kindled seizures were not reversed, suggesting that the effects were not due to non-specific elevation of the GST. In contrast to AM-kindled seizures, 20-40 mg/kg NBQX significantly suppressed only the motor seizure stage without reducing the AD duration of previously hippocampal-kindled seizures. Daily administration of 15 or 30 mg/kg NBQX prior to each electrical stimulation of the AM markedly and significantly suppressed the development of kindling. During drug sessions, the growth of the AD duration was blocked almost completely, while the waveform of ADs became more complex. These results indicate that NBQX has potent antiepileptogenic and anticonvulsant actions on kindling, at least from the AM and that non-NMDA receptors have an important role in seizure propagation.

Time-Dependent Changes in Neurotrophic Factor mRNA Expression after Kindling and Long-Term Potentiation in Rats

We compared the time-dependent changes in messenger ribonucleic acid (mRNA) levels for two neurotrophic factors after amygdala-kindled seizures and hippocampal long-term potentiation (LTP) in rats in vivo. The brain-derived neurotrophic factor (BDNF) mRNA levels in the bilateral granule cell layer of the dentate gyrus, increased significantly 1-4 h after stage 5 kindled seizures. Nerve growth factor (NGF) mRNA levels increased throughout the bilateral limbic regions more gradually than those of BDNF mRNA. The maximum levels in the dentate gyrus ipsilateral to stimulation (BDNF mRNA: 493%, NGF mRNA: 199% of control levels) occurred 2 h after seizures. As observed with kindling, BDNF and NGF mRNA expression increased in the dentate gyrus ipsilateral to stimulation also increased following LTP induced by the perforant path stimulation, with maximum levels occurring 2 h and 4 h, respectively, after stimulation, when they reached 284% and 189% of the control levels, respectively. These results suggest that BDNF and NGF are involved in enhancement of synaptic efficacy in the granule cells of the dentate gyrus in the hippocampus in kindling, not related to the neuronal excitability associated with seizure activity.

Effects of lamotrigine and conventional antiepileptic drugs on amygdala- and hippocampal-kindled seizures in rats.

We investigated the anticonvulsant and adverse behavioral effects of lamotrigine (LTG), a novel antiepileptic drug (AED), as well as other conventional AEDs on kindled seizures in rats. We also applied an anticonvulsive dose of LTG in vivo to rats in which the hippocampus had been subjected to long-term potentiation (LTP). LTG potently attenuated limbic-kindled seizures in a dose-dependent fashion, at doses at which animals showed no adverse behavioral effects. LTG was effective in preventing kindled seizures for up to 24 h after a single i.p. administration. The anticonvulsant effects of LTG were reversed when the stimulus current was raised to two or three times the generalized seizure-triggering threshold. Among the AEDs examined, valproate and LTG were the only drugs that engendered a potent anticonvulsant effect without concomitant adverse behavioral effects. Although all of the other AEDs exhibited anticonvulsant effects with various potencies, they produced adverse effects such as sedation or motor ataxia. Furthermore, an anticonvulsant dose of LTG did not affect either the induction or maintenance of tetanus-induced LTP in the hippocampus. These results indicate that LTG potently suppresses limbic-kindled seizures by raising the seizure triggering-threshold in the kindling focus at doses that do not affect LTP in the hippocampus.

Long-lasting enhancement of metabotropic excitatory amino acid receptor-mediated polyphosphoinositide hydrolysis in the amygdala/ pyriform cortex of deep prepiriform cortical kindled rats

We have previously demonstrated that ibotenate (IBO)-stimulated polyphosphoinositide (PPI) hydrolysis is increased for a long period in the amygdala/pyriform cortex (AM/PC) of amygdala (AM)- and hippocampal (HIPP)-kindled rats. This finding indicates that enhanced function of the PPI-coupled excitatory amino acid (EAA) receptor may be associated with the long-lasting seizure susceptibility of kindling. The present study further examined PPI hydrolysis induced by trans-ACPD, a selective agonist of the metabotropic EAA receptor, as well as by IBO in brain slices of rats kindled from the deep prepiriform cortex (DPC). IBO-stimulated accumulation of [3H]inositol monophosphate ([3H]InsP) was significantly increased in the AM/PC by 162 (P less than 0.0001), 130 (P less than 0.005) and 81% (P less than 0.03) at 24 h, 7 days and 28 days, respectively, after the last kindled seizure, whereas it was increased significantly only at 24 h after the last seizure in the HIPP and did not change at any time in the limbic forebrain (LFB). The IBO-stimulated accumulation of [3H]InsP was significantly increased by 55% (P less than 0.01) in the AM/PC of partially kindled rats reaching an average stage of 3.7, but not in the AM/PC of those remaining at stage 1, 7 days after the last kindled seizure. Trans-ACPD-stimulated PPI hydrolysis was significantly increased in the AM/PC of DPC-kindled rats by 65 (P less than 0.05) and 45% (P less than 0.005) at 7 and 28 days, respectively, after the last kindled seizure. Cis-ACPD-stimulated PPI hydrolysis was also significantly increased in the AM/PC of DPC-kindled rats by 45 (P less than 0.03) and 30% (P less than 0.04) at 7 and 28 days, respectively, after the last seizure. There was no increase in trans-ACPD- or cis-ACPD-stimulated PPI hydrolysis in the HIPP or LFB. These results further confirm our previous studies showing that the metabotropic EAA receptor-stimulated PPI hydrolysis exhibited a long-lasting increase in the AM/PC irrespective of the primary stimulation site for kindling.

Increased synapsin I immunoreactivity during long-term potentiation in rat hippocampus

The level of synapsin I, a synaptic vesicle protein and marker for synaptic activation, was studied 8 h after long-term potentiation (LTP) induction in rat hippocampus in vivo. Quantitative immunohistochemical analysis revealed that synapsin I increased significantly in the molecular layer of the dentate gyrus and the stratum lucidum of CA3, suggesting activation of the granule cells by synaptic input to the dentate gyrus, and persistent enhancement of glutamate release from the axon terminals of the dentate granule cells.

Lasting increase in excitatory amino acid receptor-mediated polyphosphoinositide hydrolysis in the amygdala/pyriform cortex of amygdala-kindled rats

We previously demonstrated that ibotenate-stimulated polyphosphoinositide hydrolysis, determined as the accumulation of [3H]inositol 1-phosphate, significantly increased in the amygdala/pyriform cortex (AM/PC) 24 h and 7 days after the last seizure in AM-kindled rats. The present study examined whether the increase in ibotenate-stimulated polyphosphoinositide hydrolysis in the AM/PC is longer lasting. AM-kindled rats with a tripolar electrode implanted into the left AM and sham-operated controls were decapitated either 1, 2 or 4 weeks after the last seizure. Ibotenate (10(-3) M)-stimulated accumulation of [3H]inositol 1-phosphate significantly increased by 90% (P less than 0.01), 110% (P less than 0.001) and 73% (P less than 0.05) in the AM/PC 1, 2 and 4 weeks, respectively, after the last seizure. Four weeks after the last seizure, there were significant increases of a similar magnitude in the contralateral (right) AM/PC (by 83%, P less than 0.001) and the ipsilateral (left) AM/PC (by 63%, P less than 0.01). There was no change in the hippocampus or limbic forebrain at any of these times. Also 4 weeks after the last seizure, accumulation of [3H]inositol 1-phosphate significantly (P less than 0.05) increased at ibotenate concentrations of 2 x 10(-4) M, 5 x 10(-4) M and 10(-3) M in the kindled AM/PC. In light of the view that the AM/PC are the crucial brain structures for sustaining seizure susceptibility, the marked and lasting increase in the ibotenate-stimulated polyphosphoinositide hydrolysis coupled to excitatory amino acid receptors in the kindled AM/PC may be associated with the development of kindling and long-term maintenance of kindled events.

Time-dependent and regional expression of GABA transporter mRNAs following amygdala-kindled seizures in rats

To investigate the role played by GABA transporters in epileptic seizures, we examined time-dependent and regional changes in expression of GAT-1 and GAT-3 GABA transporter mRNA in amygdala-kindled rat brain using an in situ hybridization method. GAT-1 mRNA was significantly increased bilaterally in the hippocampal dentate gyrus (111-116%) at 1 h after kindled generalized seizures. GAT-1 mRNA was also significantly increased bilaterally in the hippocampal subfields (CA1-4 and dentate gyrus [110-117%]) at 4 h after kindled seizures. There were no significant changes in GAT-1 mRNA level in the amygdalar nuclei, pyriform cortex or cerebral cortex either ipsilaterally or contralaterally at any time after kindled seizures. In contrast, GAT-3 mRNA was significantly increased bilaterally in the amygdalar nuclei and in the contralateral pyriform cortex and cerebral cortex 1 h after seizures. Since all these changes returned to control levels by 8 or 24 h after kindled seizures, the increases in GABA transporter mRNA appeared to be transient responses to seizure activity. These findings indicate that GAT-1 subtype transporter is specifically involved in seizure activity in the hippocampus, while GAT-3 subtype transporter is mainly involved in seizure activity in the amygdalar nuclei and pyriform cortex following amygdala-kindled generalized seizures.

Effects of YM90K, a selective AMPA receptor antagonist, on amygdala-kindling and long-term hippocampal potentiation in the rat

To investigate the role of alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) type glutamate receptors in epileptic seizures, we examined the antiepileptogenic and anticonvulsant effects of YM90K [6-(1H-imidazol-1-yl)-7-nitro-2,3-(1H,4H)-quinoxalinedione hydrochloride], a potent and selective new AMPA receptor antagonist, in the rat amygdala-kindling model of epilepsy. Pretreatment with YM90K (7.5-30 mg/kg i.p.) markedly retarded the evolution of kindling. Once kindling was established, administration of YM90K (7.5-30 mg/kg i.p.) significantly and dose-dependently suppressed fully kindled seizures. The maximal effects were observed 15-30 min after injection. When the intensity of electrical stimulation was increased to twice the generalized seizure-triggering threshold, the anticonvulsant effects of YM90K were reversed, suggesting that they were due to elevation of the generalized seizure-triggering threshold. Furthermore, an anticonvulsant dose (15 mg/kg) of YM90K affected neither field potentials nor long-term potentiation in the hippocampus in vivo. These results indicate that AMPA receptors play an important role in the seizure expression mechanism and the development of kindling-induced epileptogenesis, and suggest the possible clinical usefulness of AMPA receptor antagonists as antiepileptic drugs.

Increase in ibotenate-stimulated phosphatidylinositol hydrolysis in slices of the amygdala/pyriform cortex and hippocampus of rat by amygdala kindling

Hydrolysis of membrane phospholipid phosphoinositides following ibotenate stimulation of an excitatory amino acid receptor subtype has recently been demonstrated to be a receptor-mediated biochemical response. The present study examined ibotenate-stimulated phosphoinositides hydrolysis, determined as accumulation of [3H]inositol 1-phosphate, in amygdala/pyriform cortical and hippocampal slices of amygdala-kindled rats which exhibited fully developed kindled seizures on 20 consecutive days. Animals which underwent a sham operation were used as controls. Ibotenate (10(-3) M)-stimulated accumulation of [3H]inositol 1-phosphate increased significantly by 191% in the amygdala/pyriform cortex (P less than 0.01) and by 59% in the hippocampus (P less than 0.05) of the amygdala-kindled rats killed 24 h after the last seizure. One week after the last seizure, a similar magnitude of significant increase (by 171%, P less than 0.05) was maintained in the amygdala/pyriform cortex of the amygdala-kindled rats. In contrast, the increase in the hippocampus had attenuated by this time, although accumulation of [3H]inositol 1-phosphate increased significantly (P less than 0.05) when stimulated by 10(-4) M ibotenate. These results suggest that enhancement of ibotenate-stimulated phosphoinositides hydrolysis in the amygdala/pyriform cortex may be associated with the long-lasting seizure susceptibility of amygdala-kindled rats.

Hippocampal kindling enhances excitatory amino acid receptor-mediated polyphosphoinositide hydrolysis in the hippocampus and amygdala/pyriform cortex

We recently demonstrated that a long-lasting increase in ibotenate-stimulated polyphosphoinositide (PPI) hydrolysis in the amygdala/pyriform cortex (AM/PC) is associated with seizure susceptibility of amygdala (AM)-kindled rats. The present study examined (1) whether ibotenate-stimulated PPI hydrolysis would be lastingly enhanced in the hippocampus (HIPP) and AM/PC of the HIPP-kindled rats and (2) whether similar changes would be found in the early stage of HIPP kindling. Although ibotenate-stimulated accumulation of [3H]inositol 1-phosphate ([ 3H]IP1) increased significantly in the HIPP 24 h, 5 days, and 15 days after the last seizure of fully developed HIPP-kindled rats, no statistically significant increase was found in the HIPP 30 days after the last seizure. In the AM/PC, 10(-3) M ibotenate-stimulated [3H]IP1 accumulation significantly increased by 91%, 91%, 86% and 90%, 24 h, 5 days, 15 days and 30 days after the last seizure, respectively. There was no significant increase in ibotenate-stimulated [3H]IP1 accumulation 7 days after the last stimulation in the HIPP and AM/PC of rats which had undergone electrical stimulation only 5 times in the HIPP. These results indicate that (1) PPI hydrolysis coupled to excitatory amino acid receptors increases long-lastingly in the AM/PC regardless of the primary kindled site, and (2) these changes do not occur in the early stage of HIPP-kindling.