Kumatoshi Ishihara

Mu opioid receptor-dependent and independent components in effects of tramadol.

Tramadol is thought to induce analgesia via both opioid and non-opioid pathways, although the precise mechanisms remain to be elucidated. In this study, we investigated the roles of the mu-opioid receptor (MOP) in analgesic and rewarding effects of tramadol by using MOP knockout (KO) mice. Tramadol-induced antinociception, assessed by hot-plate and tail-flick tests, was significantly reduced in heterozygous and homozygous MOP-KO mice when compared with that in wild-type mice. Interestingly, however, tramadol retained its ability to induce significant antinociception in homozygous MOP-KO mice. The tramadol-induced antinociception remaining in homozygous MOP-KO mice was not significantly affected by methysergide, a serotonin receptor antagonist, but was partially blocked by yohimbine, an adrenaline alpha2 receptor antagonist, and both naloxone, a non-selective opioid receptor antagonist, and yohimbine. In addition, antinociceptive effects of an active tramadol metabolite M1 were abolished or remarkably reduced in MOP-KO mice. On the other hand, neither wild-type nor homozygous MOP-KO mice showed significant place preference for tramadol in a conditioned place preference test, although there were slight tendencies toward preference in wild-type mice and avoidance in homozygous MOP-KO mice. These results strongly support the idea suggested in the previous pharmacological studies that MOP and the adrenaline alpha2 receptor mediate most of the analgesic properties of tramadol.

Aniracetam augments, and midazolam inhibits, the long-term potentiation in guinea-pig hippocampal slices

The effects of aniracetam, a nootropic drug, and midazolam, which produces amnesia, on the long-term potentiation (LTP) of population spikes was investigated using hippocampal slices (CA3 area) from the guinea pig. Aniracetam at concentrations of 10(-7) and 10(-8) M, but not at 10(-6) M, significantly augmented LTP. On the other hand, midazolam (10(-9) M) significantly suppressed LTP. The suppressive effect was antagonized by Ro 15-1788 (10(-8) M). Both drugs did not affect the population spikes in the absence of tetanic stimulation at those concentrations. It was suggested that in vitro application of LTP is a feasible model system for evaluating the nootropic activity of drugs.

Inhibition by topiramate of seizures in spontaneously epileptic rats and DBA/2 mice

The effects of topiramate, a novel antiepileptic drug, on tonic and absence-like seizures in spontaneously epileptic rats (SER; zi/zi, tm/tm) and on sound-induced seizures in DBA/2 mice were investigated. Topiramate (20 and 40 mg/kg i.p.) inhibited both tonic and absence-like seizures in a dose-dependent manner, whereas phenytoin (20 mg/kg i.p.) and zonisamide (40 mg/kg i.p.) inhibited only the tonic seizures. The inhibitory effects of topiramate on absence-like seizures were antagonized by pretreatment with haloperidol (0.5 mg/kg i.p.), but those on the tonic seizures remained unaffected. Topiramate inhibited sound-induced seizures in DBA/2 mice (ED50 = 8.6 mg/kg p.o.). These findings suggest that topiramate may be effective for treatment of both convulsive and absence seizures of human epilepsy. The inhibitory effect of topiramate on absence-like seizures in SER may be mediated through the central dopaminergic system.

Attenuation by PACAP of glutamate-induced neurotoxicity in cultured retinal neurons

The effects of pituitary adenylate cyclase activating polypeptides (PACAPs: PACAP27, PACAP38) on glutamate-induced neurotoxicity were examined using cultured retinal neurons obtained from 3- to 5-day old Wistar rats. Cell viability was evaluated by double staining with fluorescein diacetate and propidium iodide. Effects of PACAPs on the increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in retinal neurons was investigated using the Ca(2+) image analyzing system with fura-2. The cAMP contents and the mitogen-activated protein (MAP) kinase activity in retinal cultures were measured by radioimmunoassay. Concomitant application of PACAPs (10 nM-1 microM) with glutamate (1 mM) for 10 min inhibited the delayed death of retinal neurons, which was observed 24 h after glutamate (1 mM) treatment in a dose-dependent manner. Protection by PACAPs (100 nM) against glutamate-induced neurotoxicity was antagonized by PACAP6-38 (1 microM), a PACAP antagonist, and H-89 (1 microM), a protein kinase A (PKA) inhibitor. However, PACAPs did not affect the glutamate-induced increase in [Ca(2+)](i), but PACAPs (1-100 nM) increased the cAMP levels in a dose-dependent manner. In addition, activation of MAP kinase by PACAP38 (1 microM) was inhibited by simultaneous application with H-89 (1 microM). These findings suggest that PACAPs attenuate glutamate-induced delayed neurotoxicity in cultured retinal neurons by activating MAP kinase through the activation of cAMP-stimulated PKA.

Separation of antiepileptogenic and antiseizure effects of levetiracetam in the spontaneously epileptic rat (SER)

Summary:  Purpose: The long‐lasting antiseizure effects of levetiracetam (LEV) have been observed in the spontaneously epileptic rat (SER) that expresses both tonic and absence‐like seizures. Furthermore, the antiepileptogenic effects of LEV in addition to antiseizure effects have been reported in the amygdala‐kindling model in rats. This suggests that the long‐lasting seizure protection of LEV may be at least partly due to its antiepileptogenic effects. Therefore this study aimed to differentiate the antiseizure and potential antiepileptogenic effects of LEV by administering LEV continuously to SERs before the appearance of any seizure expression.

Topiramate reduces abnormally high extracellular levels of glutamate and aspartate in the hippocampus of spontaneously epileptic rats (SER)

The spontaneously epileptic rat (SER), a double mutant, manifests both tonic and absence-like seizures. The effect of topiramate, a novel antiepileptic drug, on the extracellular levels of excitatory amino acids (EAA) in the hippocampus of SER was investigated using in vivo microdialysis. The basal levels of glutamate and aspartate in dialysates of hippocampus in SER were 2- to 3-fold higher than those in normal Wistar rats. Both the dose-response relationship and the time course of the suppression of tonic seizures by topiramate were similar to the attenuation of glutamate level in SER. Topiramate (40 mg/kg i.p.) significantly (P < 0.05) reduced both glutamate and aspartate levels in SER while showing no effect on normal Wistar rats. These findings suggest that topiramate reduces abnormally high extracellular levels of glutamate and aspartate in the hippocampus of SER. This effect may, at least in part, be related to the anticonvulsant activity of topiramate.

Activation by N-Acetyl-l-Aspartate of Acutely Dissociated Hippocampal Neurons in Rats via Metabotropic Glutamate Receptors

Summary:  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.

Epileptic seizures induced by N-acetyl-l-aspartate in rats: in vivo and in vitro studies

Tremor rat (tm/tm), the parent strain of spontaneously epileptic rat (SER: zi/zi, tm/tm), exhibits absence-like seizures characterized by 5-7 Hz spike-wave-like complexes on cortical and hippocampal electroencephalograms (EEG) after 10 weeks of age, prior to development of convulsive seizures. Recently, this animal model has been demonstrated to display a genomic microdeletion within the critical region of tm, where aspartoacylase hydrolyzing N-acetyl-L aspartate (NAA) is located, besides showing the ability to accumulate NAA in the brain. Therefore, the present study was performed to determine the involvement of NAA in the induction of epileptic seizures. When NAA (4 micromol) was applied intracerebroventricularly (i.c.v.) to normal Wistar rats, 4-10 Hz polyspikes and/or spike-wave-like complexes followed by absence-like seizure before persistent 1-5 Hz waxing high-voltage after-discharges were observed on cortical and hippocampal EEG. At a higher dose (8 micromol), NAA induced convulsive seizures. The absence-like seizures with polyspikes and/or spike-wave-like complexes on the EEG were also observed with i.c.v. NAA in premature tremor rats without seizures. The NAA-induced seizures in normal rats were antagonized by i.c.v. glutamic acid diethyl ester, a non-selective glutamate receptor antagonist. In addition, NAA applied to the bath rapidly induced a long-lasting depolarization concomitantly with repetitive firings in hippocampal CA3 neurons of normal rat brain slice preparations. These findings suggest that NAA is involved in the induction of absence-like seizures and/or convulsion, probably via glutamate receptors.

Different susceptibilities of long-term potentiations in CA3 and CA1 regions of guinea pig hippocampal slices to nootropic drugs

Effects of the nootropic drugs, piracetam and bifemelane, on long-term potentiation (LTP) of population spikes in the CA3 and CA1 regions of guinea pig hippocampal slices were investigated. Piracetam (10(-6) to 10(-4) M) and bifemelane (10(-8) to 10(-6) M) significantly augmented the LTP in the CA3 region. The effects of these drugs were inhibited by scopolamine (10(-6) M). However, the LTP in the CA1 region was not affected by piracetam and bifemelane even at highly effective concentrations (10(-5) and 10(-6) M, respectively). Thus, the LTP in the CA3 is more susceptible to nootropic drugs than in the CA1.

Long-lasting antiepileptic effects of levetiracetam against epileptic seizures in the spontaneously epileptic rat (SER): differentiation of levetiracetam from conventional antiepileptic drugs.

Summary:  Purpose: Some evidence suggests that levetiracetam (LEV) possesses antiepileptogenic characteristics. The purpose of this study was to investigate the time course of seizure protection by LEV compared with that of phenytoin (PHT), phenobarbital (PB), valproate (VPA), and carbamazepine (CBZ) in the spontaneously epileptic rat (SER). The SER is a double mutant (tm/tm, zi/zi) showing both tonic convulsions and absence‐like seizures.