Correlation between electroconvulsive seizure and HSC70 mRNA induction in mice brain

Neuroscience Letters, 157 (1993) 195-198 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93/$ 06.00

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NSL 09682

Correlation between electroconvulsive seizure and HSC70 mRNA induction in mice brain Masahisa K a n e k o a, Koji Abe b, K y u y a Kogure b, Hiroshi Saito a and Norio Matsuki a "Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, The University of Tokyo, Tokyo (Japan) and bDepartment of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, Sendai (Japan) (Received 22 January 1993; Revised version received 22 April 1993; Accepted 22 April 1993)

Key words: Heat shock protein; Heat shock cognate protein; Mouse; Diazepam; Phenytoin; Electroconvulsive seizure; Time course; Northern blot hybridization The effects of electroconvulsive seizure and anti-convulsant drugs on induction of mRNA of heat shock protein were studied in mouse brain. Electrical shock induced mRNA of heat shock cognate protein (HSC70), but not heat shock protein (HSP70) mRNA. The induction was maximum 1 h after the ECS and continued for several hours, followed by long-lasting depression. Diazepam slightly prevented the ECS, but strongly attenuated the induction of HSC70 mRNA. Whereas phenytoin, which blocked the seizure, did not decrease but delayed the induction of HSC70 mRNA. The present results suggest that HSC70 mRNA level is increased with the ECS and that the induction level did not necessarily correlate the severity of the seizure.

HSP70 is one of the major heat shock proteins induced under various stressful conditions [9]. For example, heat and ischemia are well known stimuli that induce HSP70 and its mRNA in the central nervous system (CNS) [4, 11, 15]. Recently, it has been revealed that the level of the constitutively expressed form of HSP70, called heat shock cognate protein (HSC) 70 mRNA, is also increased by ischemia in vivo [4] or under a rapid cell growing state compared to a cell arresting state obtained by serum starvation in vitro [16]. The physiological role of the induced HSP70 and HSC70 is yet unknown, but the induction pattern of these proteins suggests a significant role of the proteins. Among pathophysiological stressors, epileptic stimuli are known as inducers of HSP70 protein. After kainic acid-induced status epilepticus, HSP70 immunoreactivity was detected in the neurons of the global region in the CNS, where neuronal degeneration was not observed [19]. Inhalation of flurothyl also causes seizure and induces HSP70 [7]. There is little difference in the induction pattern of HSP70 between the two models. In the present study, we employed another type of seizure model, electroconvulsive seizure (ECS). Induction Correspondence: N. Matsuki, Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. Fax: (81) 3-3815-4603.

of mRNA of HSP70 and HSC70 in mouse brain and effects of two anti-convulsant drugs, diazepam and phenytoin, were investigated using Northern blot hybridization. Male ddY mice (Nihon SLC, Japan), aged 7 weeks and weighing 32-39 g, received an intraperitoneal injection of 1 mg/kg diazepam, 10 mg/kg sodium phenytoin, or saline. One hour after the administration, an electroshock (50 mA, 0.4 ms, 100 Hz square wave pulses for 0.1 s) was applied to the mice through a pair of ear clips. The stimulation was nearly fatal and strong enough to cause tonic flexor (TF), tonic extensor (TE), and then clonic convulsion (CL). Each state of convulsion lasted for ca. 1-2, 10-20 and 15-20 s, respectively. Diazepam (1 mg/kg) slightly mitigated the ECS, and the duration of TF, TE, and CL was reduced to less than 1, 5-8 and 3-4 s, respectively. When the dose of diazepam was increased to 3 mg/kg, the drug caused severe sedation and muscle relaxation. Therefore, we did not investigate higher doses of diazepam. Phenytoin (10 mg/kg) strongly attenuated the ECS, and only the state of CL that lasted about 5 s was observed. Rectal temperatures of the mice were monitored before the injection and at the time of sacrifice. The change of rectal temperatures was within 1°C in all animals studied. The mice were killed at 0, 1, 3, 8, 24 and 48 h after the electroshock by decapitation. Then the whole brains

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kind of treatment recovery period(h)

electroshocked C

1 mg/kg diazepam

saline 0

1 3

8 24 48

0

1 3

8 24 48

10 mg/kg sodium phenytoin 0

1 3 8 24 48

Fig. 1. A typical Northern blot analysis of HSC70 m R N A in mouse brain after various period from the electroshock. C, untreated control. Arrowheads represent the ribosomal RNAs.

were quickly dissected within 1.5 min and frozen in liquid nitrogen and stored at - 8 0 °C. Total RNA was prepared by CHAOS R N A extraction method essentially as described by T. Sargent [13]. The amount of RNA was measured spectrophotometrically. For Northern blot analysis, 20/lg of total R N A from a mouse per lane was fractionated on 1.2% agarose gel by the method of Lehrach et al. [5]. Then the R N A was blotted by capillary transfer onto nylon membrane (Gene Screen, DuPont, USA). To make sure that RNA was not resolved and that the same amount of the RNA was loaded in each lane, another gel that was loaded 1/~g of total RNA in each lane was stained with ethidium bromide after electrophoresis. For detection of HSP70 mRNA, the insert fragment of plasmid pGSH3 [K. Abe et al., unpublished data] and 30mer oligonucleotide [11] that are complementary to human and mouse hsp70 coding region, respectively, were used against the Northern blot. For HSC70 m R N A detection, the insert fragment of plasmid pGD3 [14] was also used. This probe is complementary to coding and 3'non-coding region of gerbil hsc70 gene. Oligoprobe was 3'-endlabelled with ~-[32p]-dATR and c D N A probes were radiolabelled with c~-[32P]-dCTP by using of the random primer D N A labeling kit (Takara Shuzo, Japan) and hybridized against Northern blot (total counts = 106 c.p.m./ml) at 65 °C for 16 h in hybridizing solution ( 5 x SSC, 1% sodium dodecylsulfate (SDS), l x D e n h a r d t ' s solution, 50 mM NaPO4, pH = 6.5, 200 pg/ml single strand salmon sperm DNA; 1 x SSC = 150 mM NaC1 + 15 mM sodium citrate) after prehybridization for 6 h with the same solution. Then the filter was washed sequentially with 2 x SSC containing 0.1% SDS for 15 rain at various temperature, room temperature, 42 °C, and then 65 °C. Finally the filter was washed with 0.1 x SSC containing 0.1% SDS at 65 °C for 15 min twice, and exposed to X-ray film (Kodak) for 15 h at -80°C. The autoradiogram was scanned for measuring densitometory. Specificity of the probes were tested using gerbil m R N A derived from 8 h after transient ischemia (data

not shown). This demonstrated that the c D N A probe pGD3 recognized a band that has the previously reported size for HSC70 mRNA, and both the cDNA probe pGSH3 and 30 mer oligonucleotide probe also recognized only one band which is for HSP70 m R N A judging from the size. Analysis of Northern blots revealed that HSC70 m R N A was induced by electroshock (Fig. 1), while HSP70 m R N A was not induced throughout the recovery period (data not shown). The amount of HSC70 m R N A induced by ECS was maximum at 1 h after the ECS, then gradually decreased its expression level and return to nearly the basal level after 8 h (Fig. 2). This indicates that HSC70 m R N A induction is strictly transient: very fast expression and elimination, which may reflect that HSP70s have short half lives [8]. Moreover, HSC70 mRNA expression was below the normal level after 24 h. The decrease may reflect the sedation of animals after the ECS. Lowenstein et al. [7] suggested that HSP70 protein is induced only after status epilepticus, the state that causes irreversible neuronal injury, and that HSP70 protein is induced only when the high-frequency discharge is continued for longer than 2 min. The ECS model in the present study caused a short (< 30 s) seizure that is strictly different from the status epilepticus, and no significant damage of the CNS in this condition is known. Although the ECS used in the present study was nearly fatal, it did not induce HSP70 mRNA. Therefore, it is reasonable to conclude that the ECS model did not induce HSP70 mRNA. On the other hand, HSC70 m R N A was induced by the electroshock. This suggests that HSC70 m R N A can be induced even if the stressor is not sufficient to cause neuronal injury. The present results suggest that the expression of HSC70 m R N A is regulated in the physiological condition. The protective role of HSP70 has been reported previously [12, 20]. HSC70 m R N A may be induced at first to protect against neuronal injury before the induction of HSP70 mRNA. Recently, Wong et al. [21] reported that electrical shock induced HSC70 m R N A in rat hippocampus using

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2.0 1.8 <7 1.6

0 ECS ~ 3 .

•

ECS+diazepam

v ECS+phenytoin

1.4 1.2

< 1.0 ~

0.8 0.6

//

0.4

CO 1 3

8 24 Period from ECS ( hours )

48

Fig. 2. Densitometric analysis of autoradiograms obtained at different periods after electroshock by Northern blot hybridization of HSC70 mRNA as shown in Fig. 1. The level of HSC70 mRNA is expressed as a ratio of the amount of the untreated control preparation. Symbols (©, saline; e, diazepam; v, phenytoin treated 1 h before electroshock) indicate means from three animals. For clarity, only mean values are shown.

in situ hybridization. Our data supported their results and further showed that the induction is transient followed by the long-lasting decrease of expression. Our results also eliminate the possibility that HSP70 mRNA is induced in the CNS except the hippocampus. Why HSC70 mRNA is induced is unknown. Changes in the ATPase level, especially Na+,K+-ATPase, were observed in brain tissues during seizure [10]. Since HSC70 has a function of clathrin uncoating ATPase [6], its expression may be changed by seizure. It is known that c-fos is induced after ECS [1]. Since there is no AP-1 site in the hse70 [16] and hsp70 [2] promoter regions, the induction of these mRNAs is not likely to be related with c-fos. There may be a universal transcription factor(s) that associates with HSC70 mRNA induction. Administration of 1 mg/kg of diazepam reduced the expression of HSC70 mRNA at 1 h (Fig. 1). The HSC70 mRNA level more quickly reduced below the basal level by 3 h and remained low for more than 48 h. On the contrary, phenytoin did not attenuate the ECS-induced increase of HSC70 mRNA, but delayed the onset of the increase (Figs. 1 and 2). The maximum level of HSC70 mRNA was observed 3 h after the ECS. Furthermore, depression of the HSC70 mRNA level after the initial increase was not significant in the mice treated with phenytoin. As mentioned above, the ECS was more ef-

fectively prevented by phenytoin. Therefore, there was apparent dissociation between the severity of the seizure and induction of HSC70 mRNA in the presence of anticonvulsant drugs. The two drugs themselves did not significantly affect the basal level of HSC70 mRNA. Activation o f the 7-aminobutyric acid (GABA) system through the stimulation of benzodiazepine receptor may more effectively control the expression of HSC70 mRNA. It is interesting to investigate the localization of induced HSC70 mRNA in mouse brain treated with diazepam and phenytoin, which may reveal the physiological role of HSC70. The reduction of the HSC70 mRNA level after the initial increase may be related to the sedation of animals. Non-drug-treated and diazepam-treated mice became sedate after the ECS whereas sedation was not observed in mice treated with phenytoin. In conclusion, ECS increased the expression of HSCT0 mRNA but not HSPT0 mRNA, and the induction was transient followed by a long-lasting decrease of the expression. Diazepam and phenytoin differentially affect the induction of HSC70 mRNA.

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