myo-inositol cotransporter mRNA in rat brain

myo-inositol cotransporter mRNA in rat brain

Molecular Brain Research 70 Ž1999. 179–186 www.elsevier.comrlocaterbres Research report Kainic acid-induced seizure upregulates Naqrmyo-inositol cot...

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Molecular Brain Research 70 Ž1999. 179–186 www.elsevier.comrlocaterbres

Research report

Kainic acid-induced seizure upregulates Naqrmyo-inositol cotransporter mRNA in rat brain Masahiro Nonaka a,) , Eiji Kohmura a , Toshihide Yamashita a,b, Atsushi Yamauchi c , Toshiyuki Fujinaka a , Toshiki Yoshimine a , Masaya Tohyama b, Toru Hayakawa a a

b

Department of Neurosurgery, Osaka UniÕersity School of Medicine, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan Departments of Anatomy and Neuroscience, Osaka UniÕersity School of Medicine, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan c First Department of Medicine, Osaka UniÕersity School of Medicine, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan Accepted 30 March 1999

Abstract A major organic osmolyte, myo-inositol protects cells from perturbing effects of high intracellular concentrations of electrolytes. Myo-inositol is accumulated into cells through Naqrmyo-inositol cotransporter ŽSMIT.. In order to investigate the regulation of SMIT in generalized seizure, we employed Northern blot analysis and in situ hybridization to study the changes in SMIT mRNA expression in kainic acid-injected rats. Northern blot analysis demonstrated that SMIT mRNA began to increase in the brain 2 h after onset of seizure, and peaked at 12 h. In situ hybridization revealed rapid increase of SMIT mRNA Ž2 h of seizure. in the CA3 hippocampal pyramidal cells and in the dentate granular cells. Then, at 4–6 h SMIT mRNA expression was observed in the other limbic structure such as amygdala and piriform cortex. Finally, in neocortex and in CA1 pyramidal cells, SMIT mRNA was slowly increased and peaked at 12 h. Microautoradiogram demonstrated that cells expressed SMIT mRNA were mainly neurons. These results suggest that SMIT mRNA is upregulated by kainic acid-induced seizure primarily in structures involved in seizure activity. q 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: Excitotoxicity; Gene expression; Kainic acid ŽKA.; Naqrmyo-inositol cotransporter; Organic osmolyte

1. Introduction Systemic administration of kainic acid ŽKA. causes convulsions resembling human temporal lobe epilepsy w2x. It can cause recurrent limbic-type seizures and also irreversible neuronal damage in certain brain regions w1,15,27–29x. Electrographical analysis from cortical and subcortical structures has confirmed that there are three phases in epileptiform activity patterns. First, localized paroxysmal discharges in the hippocampus occur, then other limbic structures such as amygdala and piriform cortex are involved, and finally generalized to a number of other w2x. Various reports have demonstrated that pyramidal cell layer of CA3 and granular cell layer of dentate gyrus show high affinity to 3 H KA w6,18,34x, which appears to be important for initiation of seizure activity. Both CA3 pyramidal cell layer and dentate granular cell layer )

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show strong epileptiform activity in vitro and in vivo w24,32,36x. In case of intense neuronal excitation, massive influx of Naq, Ca2q, and Cly leads water to flow inward and thus causes cellular swelling w4,16,20,25x. The sustained elevation of intracellular electrolytes is known to disturb the normal function of various enzymes w45x. In such cases, cells, including neuronal cells, accumulate high concentrations of small organic solutes Žorganic osmolytes. which do not perturb the behavior of the enzymes w30,35,41x. The organic osmolytes are consisted of amino acids Žglutamine, glutamate, and taurine., methylamines Žglycerophosphoryl choline and creatine. and polyols Ž myo-inositol.. They have been proven to play an important role in osmoregulation of mammalian brain w9x. Myo-inositol has been identified as one of major osmolytes in the brain w11x. Myo-inositol is accumulated in many organs in the body, especially in the brain, retina and renal medulla, and serves as a non-perturbing osmolyte. Intracellular accumulation of myo-inositol occurs by a

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Naq-dependent, phlorizin-sensitive mechanism w10x. The cDNA for myo-inositol transporter ŽSMIT. has been cloned w14x. SMIT is upregulated by extracellular hypertonicity at the level of transcription w41x. It has been shown that SMIT is also upregulated in various pathological state in the brain, such as acute serum hypernatremia w17x, focal cerebral ischemia w39x, and cryogenic brain injury w40x. The evidence that administration of myo-inositol attenuates seizure activity in the rats w22,37x raise a possibility that myo-inositol plays a role in seizure. However, it is not clear how its transporter is regulated in case of generalized seizure. To examine this issue, we investigated on alteration of regional and cellular distribution of Naqrmyo-inositol cotransporter ŽSMIT. on well-characterized, KA-induced seizure model. 2. Materials and methods 2.1. Animals The experimental model has been described in detail in previous publications w1,15,27–29x. Adult male Sprague– Dawley rats Ž200–250 g, 60–80 d of age. were used. KA ŽSigma. dissolved in normal saline ŽpH adjusted to 7.4. was injected intraperitoneally in a dose of 10 mgrkg. Control animals received the corresponding amount of saline. Animals injected with KA were monitored for 4–6 h to determine the severity of seizures, and only rats exhibiting generalized limbic seizures Žsalivation, rearing, and falling over. were used in this study. The number of the animals tested were as follows: 2 h Ž n s 6., 4 h Ž n s 6., 6 h Ž n s 7., 12 h Ž n s 6., 1 day Ž n s 6., 2 days Ž n s 6., and 7 days Ž n s 5. after seizure began. Those rats were deeply anesthetized with intraperitoneal injection of sodium pentobarbital Ž60 mgrkg., decapitated, and their brains were removed rapidly. Control Žvehicle-injected. rats at each time point Ž n s 3 per time point. and naive control animals Ž n s 6. were also used. At decapitation, blood samples were taken for the measurement of serum Naq, Kq, glucose levels and osmolality measurement. The brains were frozen fresh at y808C. Serial sections Ž10 mm in thickness. were obtained from the frozen brains with a cryostat and stored at y808C until use. All animal experiments described here have been conducted according to the Osaka University Medical School Guideline for the Care and Use of Laboratory Animals. 2.2. Northern blot analysis The entire forebrain of the surviving rats exhibiting seizure was used at 2 h, 4 h, 12 h and 1 day Ž n s 3 for each time interval. after the onset of seizure, as well as saline-injected controls Ž n s 3.. Total RNA was isolated by the acid guanidium isothiocyanate–phenol–chloroform method as described previously w5x. Aliquots Ž40 mg. of RNA were separated on a 1% agarose formaldehyde gel

and was transferred onto a nylon membrane ŽHybond-N w Amersham, Arlington Heights, IL.. A 490-bp rat SMIT cDNA w42x and a nearly full-length rat glyceraldehyde-3phosphate dehydrogenase ŽGAPDH. cDNA were labeled by random priming ŽAmersham. with w a-32 Px dCTP Ž3000 Cirmmol; Amersham.. Hybridization was carried out at 428C overnight in 50% formamide, 5 = SSC Ž1 = SSC is 0.15 M NaCl and 0.015 M sodium citrate; pH 7.0., 0.1% sodium dodecyl sulfate ŽSDS., 50 mM sodium phosphate, 5 = Denhardt’s solution and 100 mgrml salmon sperm DNA. The blots were washed three times at 508C for 30 min in 1 = SSC and 0.8% SDS. The membrane was placed in contact with X-ray film at y808C for five days using an intensifying screen. After quantification of the hybridized probe, it was removed from the membrane to be hybridized with the GAPDH probe. The hybridized probe was removed with a boiling solution of 0.1% SDS poured on the membrane and allowed to cool to room temperature. After the removal of the SMIT probe, hybridization and washing for the GAPDH probe was carried out in the same manner. Finally, X-ray film was placed on the membrane at y808C for 16 h. 2.3. In situ hybridization The antisense riboprobe for SMIT was synthesized from a 490-bps rat SMIT cDNA Žbases 808–1297. w43x insert cloned in the Novagen T-vector. The sense probe for SMIT was synthesized from a 490-bps rat SMIT cDNA insert cloned in the vector pSPORT 1. To synthesize hybridization riboprobes by in vitro transcription, this sequence was first linearized by digestion with restriction endonucleases of EcoR1 for both antisense and sense RNA synthesis. The linearized cDNA was then incubated at 378C for 60 min with a mixture of reagents. This mixture consisted of 2 ml of transcription buffer Ž=5., 0.5 ml of 100 mM dithiothreitol, 0.5 ml of RNase inhibitor, 0.5 ml of 10 mM ATP, CTP and GTP, 5 ml of 35 S UTP ŽNEG-039H, New England Nuclear., 0.5 ml of DNA template Ž1 mgrml. with 1 ml of appropriate RNA polymerase ŽT7 RNA polymerase for antisense; SP6 RNA polymerase for sense probes.. DNA digestion was attained with the addition of 2 ml of DNase and incubation at 378C for 10 min. Efficacy of labeling was estimated by counting radioactivity of the synthesized probes. The sections were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer ŽPB. for 20 min. After washing with PBS, the sections were treated with 10 mgrml of proteinase K in 50 mM Tris–HCl and 5 mM EDTA ŽpH 8.0. for 5 min at room temperature. They were fixed again in the same fixative, then acetylated with acetic anhydride in 0.1 M triethanolamine, rinsed with PBS, dehydrated and air-dried. The 35 S-labeled RNA probes Žsense or antisense. were diluted in hybridization buffer, placed over the sections and covered with siliconized coverslips. Hybridization was performed overnight in a humid chamber at 558C.

M. Nonaka et al.r Molecular Brain Research 70 (1999) 179–186

Fig. 1. Northern blot analysis of SMIT mRNA induction in the forebrain of KA injected rats which demonstrated limbic seizure Ž40 mgrlane.. Transcription level of SMIT mRNA Ž10 kb. started to increase at 2 h, and peaked at 12 h after onset of seizure. Lane 1: control; lane 2: 2 h; lane 3: 4 h; lane 4: 12 h; lane 5: 24 h.

The hybridization buffer consisted of 50% deionized formamide, 0.3 M NaCl, 20 mM Tris–HCl, 5 mM EDTA, 10 mM PB, 10% Dextran sulfate, 1 = Denhardt’s solution, 0.2% sarcosyl, 500 mgrml yeast tRNA, and 200 mgrml herring sperm DNA ŽpH 8.0.. The probe concentration was 5 = 10 5 c.p.m.r150 ml per slide. After hybridization, the

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slides were immersed in 5 = SSC at 558C, and the coverslips were allowed to fall off. The sections were then incubated at 658C in 50% deionized formamide with 2 = SSC for 30 min. After rinsing with RNase buffer w0.5 M NaCl, 10 mM Tris–HCl, 5 mM EDTA ŽpH 8.0.x four times for 10 min each at 378C, the sections were treated with 1 mgrml of RNase A in RNase buffer for 30 min at 378C. After an additional washing in RNase buffer, the slides were incubated in 50% formamide with 2 = SSC for 30 min at 658C, rinsed with 2 = SSC and 0.1 = SSC for 10 min each at room temperature, dehydrated in an ascending alcohol series and air dried. X-ray film was placed on the uncoated sections for 6 days for macroautoradiagraphy. Next, these sections were coated with Ilford K-5 emulsion diluted in distilled water containing 2% glycerin Ž1:1.. The slides were exposed for three weeks in a tightly sealed dark box at 48C, developed in Kodak D-19, fixed with photographic fixer, stained with thionine and coverslipped. After the X-ray macroautoradiogram had been studied, the tissue sections were examined under a regular light microscope Žbright field microautoradiogram. and dark field microscope. For semiquantitative analysis of SMIT mRNA expression, measurement of the optical density of the region of interest of the both sides on the macroautoradiograph were

Fig. 2. Macroautoradiogram of in situ hybridization for SMIT mRNA in coronal section. ŽA. Control; ŽB. 2 h; ŽC. 4 h; ŽD. 12 h. At first Ž2 h., increased transcript of SMIT was found in hippocampal CA3 hippocampal pyramidal cell region and in dentate granular cell layer. After Ž4–6 h., SMIT was expressed in superficial layer of piriform cortex Žarrowhead. and in amygdala. Finally Ž6–12 h., SMIT overexpressed in neocortex.

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performed with Macintosh-based NIH image 1.59 software. In situ hybridization films from control and experimental animals Žtwo sections per animal. were captured using Nikon coolscan, and entire anatomical areas of interest were sampled manually based on rat brain atlas w21x. The optical density of the region of interest was then measured, and the mean values of the counts were used for further analysis. Background signals was determined by sampling three regions outside the section. Average background signals were subtracted from the mean values of the counts of the region of interest. Optical density ratio ŽODR. was calculated by dividing the mean optical density of the control animals. For statistical analysis, analysis of variance ŽANOVA., with subsequent BonferronirDunn multiple comparison tests, was used to compare the data between time points.

We also used results from naive animals as a control for statistical analysis.

3. Results Administration of KA Ž10 mgrkg, i.p.. induced generalized limbic seizure in 66.3% of the animals within 30–90 min after injection, but 15.3% of the animals died within 2 h after the seizure began and were excluded from the study. Rest of the animals Ž18.4%. did not show any seizure behavior, except for ‘wet-dog shakes’. Seizure manifestations included continuous head nodding, recurrent ‘wet-dog shakes’ and rearing, followed by bilateral forelimb clonus, foam at the mouth, and severe secondarily generalized limbic seizures and loss of postural controls.

Fig. 3. Temporal profiles of changes in SMIT mRNA level in various regions. Data are presented as % mRNA change from control. ODR was calculated by dividing the mean optical density of the control animals. Relative values diverting significantly from control are marked by asterisks. Data are shown as means " S.D.

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Fig. 4. Dark-field microautoradiogram of in situ hybridization for SMIT mRNA in hippocampal region. ŽA. Control; ŽB. 2 h; ŽC. 12 h.

Seizure continued at least 2 h. There were no significant differences in the serum Naq, Kq, glucose levels, and osmolality between seized animals and control animals.

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To see whether SMIT mRNA is induced by limbic seizure, total RNA was isolated from the forebrain and hybridized with the rat SMIT cDNA probe. SMIT mRNA dramatically increased after onset of seizure compared with control, and peaked at 12 h ŽFig. 1.. To determine the localization of SMIT expression in the brain after injection of KA, we performed in situ hybridization using an 35 S-labeled cRNA probe. Serial sections were hybridized with both sense and antisense probes to confirm the specificity of the hybridization signals of SMIT mRNA. Consistent with our previous reports w39x, signals were observed only in the sections hybridized with the antisense probes. No significant changes in SMIT mRNA levels were observed in animals which did not show seizure, or in animals only demonstrated ‘wet-dog shake’ Ždata not shown.. In animals that presented seizures, the expression of SMIT mRNA started to increase in CA3 field of hippocampal pyramidal cells, and granular cell layer of dentate, at 2 h of seizure ŽFig. 2B.. Next, SMIT mRNA expressed in limbic structures such as piriform cortex and amygdala ŽFig. 2C.. SMIT signals in neocortex Žparietal cortex. peaked at 12 h ŽFig. 2D.. Semiquantitative evaluation of macroautoradiogram confirmed alteration of SMIT mRNA level in various regions. Fig. 3 shows the time course of the mRNA

Fig. 5. Bright-field microautoradiogram of in situ hybridization for SMIT mRNA. ŽŽA, C. Control; ŽB, D. 2 h after onset of seizure; ŽA, B. dentate granular cells; ŽC, D. CA3 pyramidal cells.. These findings confirm that granular cells ŽB. and pyramidal cells ŽD. mainly express SMIT mRNA. Scale bar s 150 mm.

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Fig. 6. Bright-field microautoradiogram of in situ hybridization for SMIT mRNA. ŽŽA, C. Control; ŽB. 6 h after onset of seizure; ŽD. 12 h after onset of seizure; ŽA, B. superficial layer of piriform cortex; ŽC, D. parietal cortex.. In superficial layer of piriform cortex, cells with large nucleus, presumably neurons, expressed SMIT mRNA ŽB.. Also in parietal cortex, SMIT mRNA tended expressed in cells with large nucleus Žarrowhead.. Scale bar s 150 mm.

expression in the hippocampal region. In dentate gyrus and in CA3, SMIT mRNA level raised approximately 3 to 4-fold from control at 2 h of seizure, and decreased thereafter. Superficial layer of piriform cortex show approximately 5-fold increase from control, peaking at 6 h. In parietal cortex, widespread increase of SMIT signal peaks at 12 h showing 5-fold increase from control. In CA1 pyramidal cell layer, SMIT signal showed 2.5-fold increase from control. Bright field and dark field microautoradiography demonstrated in dentate, granular cells overexpressed SMIT mRNA at 2 h compared to control ŽFigs. 4B and 5B.. In CA3 pyramidal cell layer, cells with large nucleus, presumably neurons mainly expressed SMIT mRNA at 2 h ŽFigs. 4B and 5D.. Also in superficial layer of piriform cortex, cells with large nucleus, presumably neurons mainly expressed SMIT mRNA ŽFig. 6B.. In neocortex Žparietal cortex., also SMIT mRNA tended to express in cells with large nucleus ŽFig. 6D..

4. Discussion Our study demonstrates that KA-induced limbic seizure markedly induced SMIT mRNA in a various brain regions.

These up-regulations were seen in hippocampal pyramidal cell layer in CA3 and dentate granular cell layer in the first phase Ž2 h.. Next, at 4–6 h of seizure SMIT mRNA expression were observed in the other limbic structure such as amygdala and piriform cortex Žsecond phase.. In the third phase Ž6–12 h., expressions were seen in CA1 pyramidal cells and in somatosensory cortex. There are various genes and their products known to express early after KA administration, such as c-fos, c-jun, KROX-20, KROX-24, HSP70 and HSP72 w7,8,23,26,38x. The pattern of the induction of mRNA was different form genes studied before. Furthermore, since dentate granular cells do not show excessive vulnerability against excitotoxicity and seizure, it is unlikely that SMIT expression has strong relationship with cell death. These results suggested that seizure stimulate transcription for SMIT mRNA by different mechanismŽs. from genes studied before. The factor, which is hypothesized to trigger expression of osmoregulatory genes including SMIT, is intracellular ionic strength w33,44x. Extracellular osmolality and intensive neuronal discharge increase intracellular electrolytes. Although the local extracellular osmolality during and after seizure is currently unclear w31x, there is a possibility that SMIT mRNA is upregulated by osmotic changes. Previous reports suggested that extracellular osmolality

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regulate SMIT mRNA. Neural cells, kidney cells, and epithelial cells increase transcription of SMIT when incubated in hypertonic media w35,41x. Also our past observations suggested that SMIT expression reflect regional or local osmolality in the kidney w42,43x, eye w19x, and in the brain w17,40x. Intense neuronal discharge is another important factor that increases intracellular electrolytes after seizure w12,13,25x. Thus, in theory, intense neuronal discharge may trigger SMIT mRNA expression. In the present study, we have demonstrated CA3 pyramidal cells, dentate granular cells and cells in superficial layer of piriform cortex expressed SMIT mRNA. Specific location and large nucleus suggested these cells were mainly neurons. Supporting this theory, these cells have been reported to demonstrate strong epileptiform activity. KA-induced seizures have been shown by electrophysiological methods to originate in the hippocampus and then spread to amygdala and piriform w2x. There is then involvement of neocortex with bilateral synchronous discharges during motor convulsions. Various reports demonstrate that pyramidal cell layer of CA3 and granular cell layer of dentate gyrus show high affinity to 3 H KA w6,18,34x. It has been shown that both in vitro and in vivo, strong epileptiform activity is recorded in these high-binding sites, in CA3 pyramidal cells and dentate granular cells w24,32,36x. We cannot deny the possibility that SMIT may be regulated by other unknown factors. Various transcription factors have been shown to be activated after seizure. These transcription factors may be involved in SMIT mRNA regulation. Previously, it has been shown that administration of myo-inositol attenuates seizure activity w22,37x. Together with our results of SMIT mRNA expression in various regions, myo-inositol seems to play an important role in seizure and neuronal excitation. Although it is not clear at present, SMIT may play a role in protection of excited cells from perturbing effects of increased intracellular electrolytes by increasing myo-inositol. Also, SMIT may supply myo-inositol, which is required as a precursor for phosphoinositide pathway. Inositol triphosphate level was markedly increased after KA seizure w3x, suggesting that seizure increase turnover of myo-inositol for phosphoinositide pathway. Further studies for myo-inositol and seizure may help in better understanding the mechanism of neuronal excitation, and may help develop new therapeutic target for seizure and excitotoxic injury. In summary, we examined localization of SMIT mRNA following KA-induced generalized seizure. In seized rats, SMIT mRNA predominantly increased and peaked rapidly Ž2 h of seizure. in the CA3 hippocampal pyramidal cells and in the dentate granular cells. Then, at 4–6 h of seizure SMIT mRNA expression were observed in the other limbic structure such as amygdala and piriform cortex. Finally, in CA1 pyramidal cells and in the neocortex, SMIT mRNA was slowly increased and peaked at 12 h. These results

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suggest KA-induced seizure upregulates SMIT mRNA primarily in structures involved in seizure activity.

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