Nobuya Ishida

Trimethyltin syndrome as a hippocampal degeneration model: temporal changes and neurochemical features of seizure susceptibility and learning impairment

The effects of trimethyltin on the hippocampus were investigated in terms of changes in histology, depth electroencephalography, learning acquisition and memory retention, choline acetyltransferase and neuropeptides, and seizure-induced c-fos messenger RNA expression. The results were as follows. (1) Morphologically, trimethyltin produced a progressive loss of hippocampal CA3 and CA4 pyramidal cells, starting from four days after peroral treatment with trimethyltin hydroxide (9 mg/kg), as described previously. (2) Neurophysiologically, the increased seizure susceptibility to pentylenetetrazol treatment reached a maximum at four days post-trimethyltin and then declined after five days post-trimethyltin. The maximal seizure susceptibility at four days post-trimethyltin was confirmed by the immediate and long-lasting appearance of spike discharge in the hippocampus. However, this was not verified by the expression of c-fos messenger RNA in the hippocampus, which was comparable between trimethyltin-treated and control rats. (3) Behaviorally, the time-courses of aggression and learning impairment were similar to that of the seizure susceptibility. (4) Neurochemically, trimethyltin treatment caused changes of neurochemical markers, which were manifested by the elevation of neuropeptide Y content in the entorhinal cortex, and of choline acetyltransferase in the hippocampal CA3 subfield. Trimethyltin may offer potential as a tool for investigations on the relationship between neuronal death in the hippocampus and the development of seizure susceptibility and learning impairment. Alterations in glucocorticoids, glutamate and neuropeptides may all contribute to the manifestation of the trimethyltin syndrome.

Epileptic seizure of El mouse initiates at the parietal cortex: depth EEG observation in freely moving condition using buffer amplifier

The initiation site of seizure discharges and the relationship between behavioral manifestations and electroencephalography were investigated in the El mouse, a hereditary epilepsy model. The chronic depth electrodes were implanted stereotaxically into the frontal cortex, parietal cortex, temporal cortex, hippocampus, striatum, amygdaloid complex, non-specific nuclei of thalamus and substantia nigra. Electrical activities were recorded in freely moving condition with use of the buffer amplifier devised in the laboratory and behaviors were monitored simultaneously. Seizure spike discharges started in the parietal cortex and spread out into other brain areas. When the hippocampus was involved, the tonic convulsion occurred behaviorally. The paper describes the first direct evidence of the initiation and propagation of seizure discharges in the brain of El mouse.

Potent and long-lasting anticonvulsant effects of 1-naphthylacetyl spermine, an analogue of Joro spider toxin, against amygdaloid kindled seizures in rats.

The anticonvulsant effect of 1-naphthylacetyl spermine (1-NA-Spm), an analogue of Joro spider toxin, against amygdaloid kindled seizures was studied in rats. 1-NA-Spm (10, 20 and 40 micrograms/rat) dose-dependently improved kindled seizures and shortened the afterdischarge duration 30 min after the administration. The anticonvulsant effect was observed even one day after the drug, and then gradually disappeared within 4 days. The present findings demonstrate that 1-NA-Spm acts as a potent and long-acting anticonvulsant against amygdaloid kindled seizures, and also suggest, together with the previous findings, that the calcium-permeable AMPA receptors, which are selectively antagonized by 1-NA-Spm, play a critical role in the seizure generation mechanism of amygdaloid kindling.

An analogue of Joro spider toxin selectively suppresses hippocampal epileptic discharges induced by quisqualate.

The anticonvulsant effect of 1-naphthylacetyl spermine, an analogue of Joro spider toxin (JSTX), was studied against seizures induced by quisqualate (QUIS), a non-NMDA agonist, as assessed electrophysiologically and behaviorally in freely moving rats. Electrodes were implanted into right dorsal hippocampus and an injection cannula for drugs into right ventricle. The pretreatment with JSTX analogue significantly inhibited both of QUIS-induced hippocampal discharges (80-11%) and generalized tonic clonic seizures (100-33%) in a dose-dependent manner, whereas JSTX had no effect on seizures induced by quinolinate, a NMDA agonist. The paper provides the first direct evidence that the JSTX analogue exerts a potent and selective suppression of hippocampal epileptic discharges mediated by non-N-methyl-D-aspartate (non-NMDA) receptors.

Characterization of Audiogenic-Like Seizures in Naive Rats Evoked by Activation of AMPA and NMDA Receptors in the Inferior Colliculus

The role of glutamate receptors in the inferior colliculus (IC) in audiogenic and audiogenic-like seizures was investigated in adult rats with transient neonatal hypothyroidism by 0.02% propylthiouracil (PTU) treatment through mothers milk (PTU rats) and in naive rats treated intracisternally with N-methyl-d-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole-proprionic acid (AMPA), or cyclothiazide, an inhibitor of rapid AMPA receptor desensitization. All rats showed audiogenic or audiogenic-like seizures characterized by running fit (RF) and generalized tonic-clonic seizures (GTCS). While systemically administered MK-801 inhibited GTCS, intracisternally administered NBQX inhibited RF and GTCS in both audiogenic and audiogenic-like seizures. Auditory stimulation shortened the latency to GTCS induced by AMPA, but not NMDA, at a subclinical dose and further elongated the shortened duration of RF, but not GTCS, induced by MK-801 pretreatment. Furthermore, Northern blot analysis was used to evaluate the expression of the immediate-early gene c-fos in the IC following induction of audiogenic or audiogenic-like seizures. The significant induction of c-fos mRNA by audiogenic seizures in PTU rats or by AMPA- or cyclothiazide-induced seizures in naive rats was prominent in the IC. MK-801 suppressed c-fos mRNA expression in the IC induced by audiogenic seizures in PTU rats or by AMPA-induced seizures in naive rats. NBQX suppressed the expression of c-fos mRNA in the IC induced by AMPA-induced seizures but did not suppress c-fos mRNA in PTU rats or rats with cyclothiazide-induced seizures. Auditory stimuli failed to affect c-fos mRNA induction by AMPA. The present study suggests that audiogenic-like seizures can be reproduced by glutamate receptor agonists in which AMPA receptors are primarily linked to the initiation of audiogenic seizures (RF) while NMDA receptors presumably located within the IC are involved in the propagation of GTCS in audiogenic seizures.

Expression of c-fos mRNA after audiogenic seizure in adult rats with neonatal hypothyroidism

In situ hybridization histochemistry was used to evaluate the expression of the immediate-early gene c-fos following the induction of audiogenic seizures in adult rats with transient neonatal hypothyroidism. The rats treated with 0.02% propylthiouracil (PTU) through mothers milk during days 0-19 after delivery showed a high incidence of seizures to auditory stimulation at the age of 4 months. The significant induction of c-fos mRNA by audiogenic seizures is prominent in several brain areas including central gray, peripeduncular nucleus, inferior colliculus, septal nucleus, bed nucleus of stria terminalis, and dorsomedial hypothalamus. However, the expression of c-fos mRNA was comparable in neocortex, dorsal hippocampus and medial geniculate body between control rats and PTU-treated, seizure-induced rats. These results confirm the previous report on the c-fos expression following audiogenic seizure sensitized during development by a loud noise [20]. The present results indicate that the neonatal PTU treatment may provide a useful tool for studying the mechanism underlying the seizure susceptibility and development after maturation.

Pharmacological Characterization of EEG Spikes and Seizures Induced by a Specific Calcium‐Permeable AMPA Receptor Antagonist, 1‐Naphthylacetyl Spermine (1‐NA‐Spm)

Purpose,: It is known that AMPA receptor has a crucial role in seimre generation and epileptogcnesis. Joro spider toxin (JSTX) sclectively blocks the calciurn‐permeable AMPA receptor a s a channel blocker. We have reported that a synthetic JSTX analoguc, 1 ‐NA‐Spin, suppresses amygdaloid and hippocampal kindled seizures in a dosedependent manner. This analogue induces EEG spikes both on the kindled animals and on the niiivc animals. The spike‐inducing effect of I ‐NA‐Spm is not directly related to the anticonvulsant effects of this drug, because time‐course of the inhibitory effects on the kindled seiurcs is different from that of the spike‐inducing effect. The drug soinctiines produces myoclonus and generalized seizures coinciding with the spike induction. To elucidate the mechanism underlying these proconvulsant cffects, participants of the AMPA, NMDA, and GABA transmitter systems to the I ‐NA‐Spin induced effects were tested.

NMDA receptors in the inferior colliculus are critically involved in audiogenic seizures in the adult rats with neonatal hypothyroidism

The effects of N-methyl-d-aspartate (NMDA) and non-NMDA receptor antagonists were compared on audiogenic seizures in the rats neonatally exposed to propylthiouracil (PTU). The rats treated with 0.02% PTU through mothers milk during days 0-19 after delivery showed a high incidence of audiogenic seizures consisting of running fit (RF) followed by generalized tonic-clonic seizure (GTCS) after matured. The systemic administration with MK-801, a NMDA receptor antagonist dose-dependently inhibited both RF and GTCS. NBQX (6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione), a non-NMDA receptor antagonist, when systemically administered, failed to block audiogenic seizures. Audiogenic seizures caused a marked induction of c-fos messenger RNA (mRNA) in septal nucleus, bed nucleus of stria terminalis, amygdaloid nuclei, peripeduncular nucleus, and inferior colliculus, which was almost completely blocked by the pretreatment with MK-801. Bilateral microinjection of MK-801 into the inferior colliculus showed a tendency for inhibiting GTCS, but not RF, whereas CPP (3-(R)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid), a competitive NMDA receptor antagonist produced a significant inhibition against both RF and GTCS. These NMDA receptor antagonists administered into cisterna ambience, the floor of which is composed of inferior colliculus and neighboring structures, have shown potent blocking effects on both RF and GTCS. The present results suggest that NMDA receptors in the inferior colliculus, presumably in the subnucleus of external cortex may play the critical role in the initiation of audiogenic seizures in PTU-treated rats.

Anticonvulsant Actions of Glutamate Receptor Antagonists Against Audiogenic Seizures in Adult Rats with Neonatal Hypothyroidism.

It has been reported that rats exposed neonatally to propylthiouracil (PTU), an antithyroid agent, develop marked susceptibility to audiogenic seizures after maturation. The immediate early gene, c-fos, is rapidly and transiently induced by a variety of stimuli, and seizures represent one of the stimuli that induce drastic expression of c-fos in the brain. We have reported the specific expression of c-fos messenger RNA (mRNA) after generalized tonicclonic seizures in PTU rats. In this study, we examined the effect of an N-methyl-D-aspartate (NMDA)receptor antagonist on c-fos mRNA expression after audiogenic seizures by means of in situ hybridization. Six pregnant Sprague-Dawley rats were housed individually and allowed to litter normally. On the day of delivery (day 0), all pups were collected, randomized, and then redelivered to each dam (9-10 pups per dam). Four dams received 0.02% PTU in the drinking water from days 0 through 19 of postparturition (PTU rats), and two dams received water only (control). At age 7-8 weeks, the seizure susceptibility was confirmed by auditory stimulation for 80 s. Whereas control rats showed no behavioral change, all the PTU rats exhibited running fits followed by tonic-clonic seizures. At age 4 months, six PTU-treated and six control rats were killed 30 rnin and 60 min (n = 3 each) after the initiation of auditory stimulation. Furthermore, six PTU-treated rats were pretreated intraperitoneally with MK-801 (1 mg/kg) or the vehicle (n = 3 each) 30 rnin before auditory stimulation and killed 30 rnin after the auditory stimulation. The brains were quickly removed and stored at -70°C until in situ hybridization. Sixteen-millimeter sections were hybridized with a [35S]UTP-labeled antisense riboprobe, which was synthesized with SP6 RNA polymerase from a 405 base-pair fragment (nucleotides 171 1-21 16) of rat clfos cDNA. The specificity of the probes was confirmed by the absence of signals in sections hybridized with sense probes and Northern blot hybridization. After hybridization, the sections were rinsed, air dried, and then exposed to Kodak X-OMAT film for 1 week at room temperature. Relative optical density values for the autoradiograms were determined by computer-assisted densitometry . The robust seizure-induced expression of c-fos mRNA both 30 rnin and 60 rnin after auditory stimulation was confirmed in the central gray, peripeduncular nucleus, inferior colliculus, septal nucleus, red nucleus of stria terminalis, and dorsomedial hypothalamus. In the neocortex, dorsal hippocampus, and medial geniculate body, the c-fos mRNA induction was comparable in control and PTU-treated rats after auditory stimulation. Pretreatment with MK801 abolished both the behavioral manifestation of generalized seizures and the expression of c-fos mRNA in specific areas, such as the inferior colliculus, septal nucleus, bed nucleus of stria terminalis, and amygdaloid nuclei. Although preliminary, our results suggest that NMDA receptors in the inferior colliculus and related structures are involved in the mechanisms underlying the development of audiogenic seizures.

Glutamate Receptor Activation in the Brainstein of Audiogenic Seizure‐Susceptible Rats

Purpose: This study was conducted to assess the effect of NMDA receptor antagonists (MK‐80 I and CPP) and a non‐NMDA receptor antagonist (NBQX), administered into inferior colliculus (IC) or into the ambient cistern on audiogenic seizures i n rats neonatally exposed to propylthiourecil (PTU). Furthermore, the role of glutamatc rcceptor subtypes i n audiogenic seizures was cxanincd by analysis of behavioral manifcstations induced hy inttacisternal injection of NMDA or AMPA in naive rats.