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Abstract

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*Shin-ichi Yoshioka *Division of Neuropsychiatry, Faculty of Medicine, Tottori University, Yonago, Japan .

Purpose: In humans, prenatal or postnatal brain insults such as hyperthermia, x-ray irradiation, malnutrition, or exposure to toxic substances may produce brain defects. These brain defects result in an increased postnatal seizure susceptibility. Methotrexate (MTX), a folic acid antagonist, interferes with DNA synthesis and is teratogenic or embryotoxic in humans as well as in experimental animals. In addition, MTX exposure during pregnancy is known to result in brain malformations. The aim of this study was to determine whether in utero exposure to MTX in rats modifies the development of amygdaloid kindling at immature ages and the persistence of seizure susceptibility during development.

Methods: Pregnant Wistar rats received a single intraperitoneal injection of MTX (5 mg/kg) dissolved in saline or 1 ml/kg of vehicle at gestational day 15 (day 15 of embryonic development). On day 12 after birth (postnatal day 12; PN12), pup rats were stereotaxically implanted with a twisted bipolar electrode into the right amygdala under pentobarbital (PTB) anesthesia. A reference electrode was placed in the frontal sinus. On PN 14, after the determination of the afterdischarge threshold (ADT), the rats received electrical stimulation at an ADT level of 60 Hz sine wave for 1 s once every 30 min until the appearance of five stage 5 seizures. Subsequently, the electrodes were removed, and the rats were allowed to grow. On PN 63, bipolar electrodes were again implanted into the ipsilateral amygdala under PTB anesthesia. A reference electrode was placed in the frontal bone. According to whether 70-day-old rats were given MTX treatment and/or initial kindling on PN 14, four groups were designated as vehicle-treatment plus kindling group (CC), MTX-treatment plus kindling group (MC), vehicle-treatment plus rekindling group (CK), and MTX-treatment plus rekindling group (MK). After the determination of the ADT on PN 70, the rats received the same electrical stimulation at the ADT level for 1 s once daily until the appearance of five stage 5 seizures. The behavioral seizure stages of amygdaloid kindling were assessed by using the classification proposed by Moshé for immature rats and by Racine for adult rats. Electrographic recording in the right amygdala and cortex also was performed before and during kindling stimulation and the duration of the afterdischarge (AD) elicited at each stimulation was noted.

Results: In the 14-day-old pup rats, prenatal MTX treatment decreased significantly the number of stimulations required to reach the first stage 5 seizure compared with vehicle-treated rats. In addition, the cumulative AD durations elicited up to the tenth kindling stimulation after the initial stimulation (cumulative × 10 ADDs) were significantly shorter in MTX-treated rats than in control rats. When the rats were kindled on PN 70, no significant difference was found in the number of stimulations needed to induce the first stage 5 seizure between the CC and the CK groups. However, the number of stimulations required to induce the first stage 5 seizure was significantly lower in the MK group than in the CC and CK groups. Additionally, the cumulative ×10 ADDs was significantly longer in the MK group than in the CC group.

Conclusions: These results suggest that the susceptibility to kindling-induced seizure in immature rats is enhanced by in utero exposure to MTX, and that the enhanced seizure susceptibility acquired during the immature age persists into maturity.

Abstract

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*Shin-ichi Imamura, ‡Shigeya Tanaka, †Koichi Akaike, †Hideshi Tojo, †Akira Sano, and *Jun-ichi Kuratsu Departments of *Neurosurgery and †Neuropsychiatry, University of Kagoshima, Faculty of Medicine, Kagoshima ; and ‡Tanaka Neurosurgical Clinic, Kagoshima, Japan .

Purpose: This study was designed to unveil the effect of hippocampal transection (hTx) on spontaneous amygdalar seizures (sAS).

Methods: Intrahippocampal monoamines were measured by using high-performance liquid chromatography on 18 rats with acute kainic acid (KA)-induced amygdalar seizures: a group that did not develop sAS (controls, n = 6); a group that developed sAS and did not undergo hTx (SP, n = 6); and a group that developed sAS and underwent hTx (SPT, n = 6).

Results: Dopamine (DA)/dihydroxyphenyl acetic acid (DOPAC) was lower, and 5-hydroxytryptamine (5-HT)/5-hydroxy-indole acetic acid (5-HIAA) was higher in SP than in controls. Conversely, DA/DOPAC became higher again in SPT compared with SP, whereas 5-HT/HIAA remained unchanged.

Conclusions: Hippocampal transection is probably associated with the changes of DA/DOPAC.

Abstract

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*Hiroshi Gohma, †Mituru Kuwamura, *Takashi Kuramoto, ‡Takahiro Seki, §Reuben Matalon, ∥Kazuhiro Kitada, ¶Masashi Sasa, and *Tadao Serikawa *Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto ; †Department of Veterinary Pathology, Graduate School of Agriculture and Biological Sciences ; ‡Department of Molecular and Pharmacological Neuroscience, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima ; §Department of Pediatrics and Human Biological Chemistry and Genetics, Children's Hospital, Galveston , ∥Center for Advanced Science and Technology, Hokkaido University, Sapporo , and ¶Nagisa Hospital, Maikata, Japan .

The Spontaneously Epileptic Rat (SER), a double-mutant for tremor (tm) and zitter (zi) mutations, is one of the most useful tools in epilepsy research, because of its spontaneous occurrences of absence-like and tonic seizures, as well as its genetically defined characteristics. Recently, the tm mutation was identified as a >200-kb genomic deletion including the aspartoacylase (Aspa) gene, and the zi mutation was identified as an 8-bp deletion within the attractin (Atrn) gene. Several lines of evidence suggest that Aspa is the strongest candidate for the epileptic phenotype of the SER. Although the SER has provided a good model for epilepsy research, sterility in this strain has prevented efficient mass production of the SER. Therefore we developed double-mutant mice by crossing Atrn mutant mice with the Aspa knockout mice. The Aspa/Atrn double-mutant mice exhibited absence-like and tonic seizures that were characterized by 5-7/s spike-wave–like complexes and low-voltage fast waves on the EEGs, respectively, and showed more exaggerated vacuolation in the CNS than did its parental strains. The double-mutant mice were fertile, in contrast to the sterile SER. The data on double-mutant mice supported the previous conclusion that defects in the CNS and the abnormal EEG observed in the SER were caused by functional losses of Aspa and Atrn genes. The Aspa/Atrn double-mutant mice developed in this study can be produced more efficiently than the SER, because of smaller size and greater fertility. Accordingly, the double-mutant mice can be used more extensively in epilepsy research for activities such as evaluation of antiepileptic drugs.