Koji Mui

Distribution of Fos- and Jun-related proteins and activator protein-1 composite factors in mouse brain induced by neuroleptics

The mechanisms by which the direct actions of neuroleptics are translated into therapeutic effects are unknown. We immunocytochemically investigated the expression of Fos- and Jun-related proteins and examined activator protein-1 DNA-binding activity in ddY mouse brain 120 min after the administration of haloperidol (1 mg/kg), (-)-sulpiride (20 mg/kg) and a selective dopamine D1 receptor antagonist, SCH23390 (1 mg/kg). The densities of Fos-, FosB-, Fra-1-, Jun- and JunD-immunoreactive nuclei induced by haloperidol and sulpiride in the hippocampus, piriform cortex and accumbens nucleus were higher than those in the control groups. The same regions showed higher densities of FosB-, Fra-1- and JunD-immunoreactive nuclei induced by SCH23390 compared with the control groups. We investigated further the activator protein-1 composite factors using super gel shift assays. These results suggested that induced Fos, FosB, Fra-1, Jun and JunD proteins constitute the activator protein-1 complex after the administration of haloperidol and sulpiride. In contrast, FosB, Fra-1 and JunD appear to constitute the activator protein-1 complex after the administration of SCH23390. Therefore, the diversity of activator protein-1 composite factors suggests that various kinds of gene are induced to act by some neuroleptics.

Comparison of the effects of dopamine D1 and D2 receptor antagonists on nerve growth factor mRNA expression

Regulation of the expression of the nerve growth factor (NGF) gene has been reported previously to be mediated by the interaction of c-fos with an activator protein-1 (AP-1) binding site present in the first intron on the NGF gene. Using an RNase protection assay and in situ hybridization, we examined the effects of dopamine D1 and D2 receptor antagonists on NGF mRNA. Haloperidol (0.1-8 mg/kg) and (-)-sulpiride (10-100 mg/kg), induced NGF mRNA in a dose-dependent fashion in the hippocampus, piriform cortex, striatum and nucleus accumbens. The haloperidol (1 mg/kg)- and (-)-sulpiride (20 mg/kg)-induced NGF mRNA expression attained a maximum level 120 min after injection and returned to control levels 24 h later. Prior administration of the protein synthesis inhibitor cycloheximide blocked the haloperidol- and (-)-sulpiride-mediated induction of NGF mRNA. In contrast, R-(-)-8-chloro-2,3,4,5-tetrahydro-3,1-methyl-5-phenyl-11-3-benzyoepin e-7-ol (SCH23390) did not induce NGF mRNA expression in either a dose-dependent or time-dependent manner. Our previous studies have shown that haloperidol and (-)-sulpiride induce the expression of c-fos and c-jun mRNAs and increase their AP-1 DNA binding activities. Thus, the data suggest that neuroleptics induce NGF gene expression by increasing AP-1 DNA binding activity.

Alterations of Glutamate Transporter Gene Expression in Epileptic EL Mice Brains

Intmduc%ion: Glutamate is considered to be an important excitatory amino acid and has been implicated in the pathophysiology of cpilcpsy. Glutamate transporters help to maintain the extracellular glutamate concentration and have been identified i n inousc brains, incl tiding GLT‐I and GLAST in astroglia and EAAC‐I in neurons. In an amygdala‐kindling study, alteration of glutamate transporter proteins showed that GLAST protein was down‐regulated in the piriform cortcx/amygclala rcgion of the amygdala‐kindled Iiits. In contrast, kindling induced an increase in EAAC‐ I levels in the pirilorrn cortex/amygdala and hippocampus. No changes i n GLT‐I wcrc obscrvcd in any region. The EL mouse, an inbred mutant strain, is considered to be a model for human complex partial seidures. This study was carried out to reveal the relationship bctwccn cpilcptogcncsis and glutamate transporters in EL mice.

Sequence complexity of polyadenylated RNA from seizure-susceptible El mouse brain polysomes.

The hybridization kinetics in the complementary DNAs (cDNA) to polyadenylated messenger RNA (mRNA) in the brain of seizure-susceptible El mice were examined to elucidate the correlation between seizure susceptibility and genetic expression. Homologous and heterologous saturation hybridizations indicated that cDNA of seizure-nonsusceptible ddY mice hybridized with less mRNA in seizure-experienced El(+) or seizure-nonexperienced El(o) mice than that in ddY mice, cDNA of El(+) mice hybridized to a lesser extent with mRNA of El(o) mice than that of ddY or El(+) mice, and no difference was observed with cDNA hybridization results of El(o) mice to individual mRNAs of ddY, El(+) or El(o) mice. The results suggest that some sequences found in mRNA of ddY are lacking in both El strains and are responsible for the inbred predisposition, while those present in El(+) mRNA but missing in El(o) may associate with seizure susceptibility, since common sequences are present in the mRNA of those groups of animal. The sequence diversity among the 3 groups of mice was mainly observed with the rare class of mRNA. Molecular cloning of the cDNA corresponding to this class of mRNA found in the El(+) mice would clarify the gene expression in the brains of epileptics.

Nuclear polyadenylate polymerase activity in the brain of seizure-prone EL mice.

Abstract Nuclear polyadenylate polymerase form I activity in the brains of seizure‐prone EL mice was significantly higher than in seizure‐non‐susceptible progenitor ddY mice. This finding may be essential in acquiring susceptibility to seizures, since there was no significant difference between EL(S) mice and those that did not receive stimulation, EL(NS) mice. Lower form II enzymatic activity was observed in both groups of EL mice but not in ddY mice. Moreover, significantly lower activities of form II 7 days after seizures were found in EL(S) mice compared with EL(NS) mice, suggesting that this is a consequence of repeated seizures. The activity of form I enzyme decreased immediately and at 30 and 60 min after seizures, then returned to control levels at 100 min. Form II enzymatic activity was significantly decreased only at 30 min after seizures, implying that seizures exerted a later effect on form II enzyme. These changes may cause a decrease in the rate of polyadenylation in the brain; thus, alteration of post‐transcriptional events, including messenger RNA processing and transport, may occur during epileptic seizures.

Effects of a Pentylenetetrazol‐Induced Convulsion on Mouse Cortical Protein Kinase C and Ca2+/Calmodulin‐Dependenl Protein Kinase I1 Activity.

and CBZ, may possibly enhance the transformations of AEDs into epoxide, which may result in increased AED teratogenesis. On the other hand, the effects of AEDs on the detoxification or elimination process of epoxide have not yet been fully clarified. Our study clearly showed that therapeutic concentrations of VPA show inhibitory effects on the activities of EH and most of the GST isozymes, and that these effects are concentration dependent. Therapeutic concentrations of CBZ also inhibited the activity of GST 7-7, found primarly in the placenta. These results suggest that detoxification of the epoxide of AEDs could be inhibited under AED polytherapy by using VPA or CBZ or both. This may partly explain the increased incidence of malformations in offspring exposed to VPA-CBZ, administered with or without other AEDs. PHT, PB, and ZNS had no inhibitory effects on the activities of EH and GST. However, the possibility that PHT and PB have inducing effects on epoxide formation should be taken into account. In this regard, combinations of AEDs should be carefully selected in the treatment of women of childbearing age with epilepsy. (This work was supported by Grant-in-aid No. 05454309 for Scientific Research from the Ministry of Education and Culture and grant from the Hirosaki Research Institute for Neurosciences.)

Translational activity of endogenous and exogenous mRNA in a cell-free translation system from seizure-prone EL mouse brains

7. Watanabe Y, Stone E, McEwen BS. Induction and habituation of crfos and zif/268 by acute and repeated stressors. NeurOReport 1994; 5: 1321-1324. Morgan JI, &hen DR, Hempstead JL, Curran T. Mapping patterns of ~

Genome analysis of El mouse brain using a technique of cDNA-RNA hybridization.

03 expression in the central nervous system after seizure. Science 1987; 237: 192-197. 8. Le Gal La Salle G, Naquet R. Audiogenic seizures evoked in DAB/2 mice induce crfoos oncogene expression into subcortical auditory nuclei. Brain Res. 1990; 5 1 8 308-312. Faingold CL, Naritoku DK, Copley CA et al. Glutamate in the inferior colliculus plays a critical role in audiogenic seizure initiation. Epilepsy Res. 1992; 13: 95-105. 9.

Change in Synaptic Proteins after Seizures of El Mice

The El mouse is a mutant strain of mouse possessing seizure-susceptibility as a model of human epilepsy. Using a technique of unique DNA to mRNA hybridization, we3 have shown that the sequence complexity of mRNAs in the El mouse brain differed from that in ddY, but the difference was relatively less. The present study was taken up to further elucidate the relationship between the genetic expression of mRNAs and seizuresusceptibility using a complementary DNA (cDNA) prepared from poly(A) -mRNAs, and the sequence complexity was estimated by cDNA-mRNA hybridization. Polysomes were isolated from the brains of mice, and RNA was then prepared using the Proteinase-K treatment and the method of phenol-chloroform. Poly(A)-mRNAs were isolated from polysomal RNA by the oligo (dT) -cellulose chr~matography.~ The cDNA was transcribed from poly (A) -mRNAs by the Avean myeloblastosis virus reverse transcriptase.4 The cDNA probe thus synthesized was purified by the Sephadex G-100 chromatography. The cDNA and mRNAs were dissolved in a 0.6 M-NaCl buffer (pH 7.2) containing 0.5% SDS and 10 mM-EDTA.I The reaction mixtures were incubated in capillary tubes at 63OC to the desired RNA concentration time (Rot = multiplied initial concentration in mole per liter by time in sec), rapidly frozen and stored at -2OOC until assayed. The cDNA-RNA hybrids were assayed by the S, nuclease and DEAE-cellulose filter method.5 Fig. 1 shows the homologous hybridization curves of cDNA to mRNAs from the El( + ), El(o) and ddY mouse brains. There are some differences between these curves, indicating that the hybrid values of the El( + ) and El(o) mice were usually higher than those of the ddY mice at a Rot value larger than 100. The hybridization of these cDNAs reached a plateau at a Rot value of 1 O+. The saturation hybridization values were 75, 80 and 67% in the El( + 1, El(o) and ddY mice, respectively (Table 1). The results of the heterologous hybridization values of different cDNA to individual mRNAs from the other two strains of mice in addition to the above homologous saturation values are summarized in Table 1. The heterologous saturation value in cDNA of the El( + ) mice to mRNAs prepared from the El(o) mice was lower than these values to

The Effect of Seizures on Protein Synthesis in El Mouse Brain

We previously showed that protein synthesis in vitro of isolated synaptosomal fractions was increased by seizures of seizuresusceptible El mice.3 The present study was undertaken to investigate the effects of seizures in the El mice on synaptosomal proteins. Seizures of the El mice were induced by a tossed-up stimulation. The seizure-nonsusceptible ddY mice were used as controls. Bemegride (12 mg/kg body weight) was intraperitoneally injected into both the mice and seizures were induced. The synaptosomal fractions were prepared by the method of Booth and Clark: and the proteins were analyzed using a SDS-PAGE electrophoresis. The molecular weight of proteins was determined by a densitometric scan. Figure shows the pattern of SDS-PAGE in the synaptosomal and TCA insoluble proteins isolated immediately after the seizures induced by the tossed-up stimulation and injection of Bemegride. The effects of seizures on percent distribution of 5 major