Dizocilpine (MK-801) elicits a tetrodotoxinsensitive increase in extracellular release of dopamine in rat medial frontal cortex

Neurochem. Int. Vol. 26, No. 3, pp. 269 279, 1995

~

Pergamon

0197-0186(94)00125-1

Copyright © 1995ElsevierScienceLtd Printedin Great Britain.All rights reserved 01974)186/95$9.50+ 0.00

DIZOCILPINE (MK-801) ELICITS A TETRODOTOXINSENSITIVE INCREASE IN EXTRACELLULAR RELEASE OF DOPAMINE IN RAT MEDIAL FRONTAL CORTEX ATSUSHI KASHIWA j'2, TORU NISHIKAWA l*, KOICHI NISHIJIMA 1'3, ASAMI UMINO ~and KIYOHISA TAKAHASHP LDepartment of Mental Disorder Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira-shi, Tokyo 187, Japan zDepartment of Neuropsychiatry, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan 3Department of Psychiatry, Jichi Medical School, Tochigi-ken 329-04, Japan (Received 13 May 1994 ; accepted 23 August 1994)

Abstract

-We have examined in the rat the effects of a selective non-competitive antagonist for the Nmethyl-D-aspartate (NMDA) type excitatory amino acid receptor, dizocilpine (MK-801), on cortical dopamine (DA) metabolism using an in vivo dialysis technique. An acute intraperitoneal injection of MK801 (0.4-1.25 mg/kg) dramatically increased the concentrations of dopamine, 3,4-dihydroxy-phenylacetic acid and homovanillic acid in the dialysates from the medial frontal cortex in a dose-dependent fashion. Moreover, MK-801 caused a delayed and small augmentation of the cortical extracellular release of 5hydroxyindoleacetic acid. Continuous infusion of tetrodotoxin into the prefrontal region via the microdialysis tube completely blocked the ability of MK-801 (1.25 mg/kg, intraperitoneally) to augment the extracellular release of DA, its metabolites and the serotonin metabolite in the frontal cortex. The present results suggest that MK-801 facilitates DA release in the medial frontal cortex by increasing impulse flow in the DA neurons projecting to the cortical area, adding further support to the view that the NMDA receptor may be involved in the tonic inhibition of frontal cortical DA neurons. It is also proposed that frontal serotonin neurons might be under regulation by excitatory amino acidergic transmission via the NMDA receptor.

Dizocilpine (MK-801 : (+)-5-methyl-10,11-dihydro5H-dibenzo-[a,d]-cyclohepten-5,10-imine maleate) has been shown to be a potent and selective antagonist of the N-methyl-D-aspartate (NMDA) type excitatory amino acid (EAA) receptor(Kemp et al., 1987 ; Wong et al., 1986). Electrophysiological and biochemical studies have revealed that this dibenzocycloheptenimine derivative interrupts N M D A receptormediated neurotransmission in a non-competitive and use-dependent fashion by binding to the phencyclidine (PCP) receptor site within the cation channel of the NMDA receptor complex(Wong et al., 1986, 1988). Since MK-801 and other non-competitive N M D A antagonists acting on the PCP site cause locomotor stimulation and sterotypy like amphetamines which are indirect dopamine (DA) agonists, much attention has been focused on the possible involvement of cer-

ebral hyperdopaminergic activity in these behavioral changes. In fact, mixed DI/D2 DA receptor antagonists or specific antagonists at each receptor subtype have been reported to attenuate some of these behavioral effects of MK-801 and other PCP-like drugs (Clineschmidt et al., 1982; Dall'Olio et al., 1992; Greenberg and Segal, 1985; Hoffman, 1992; Quagazzel et al., 1993) while hyperactivity induced by a very high dose of MK-801 was not reversed by DA antagonists (Carlsson and Carlsson, 1989; Raffa et al., 1989). In support of the dopaminergic components of MK-801- and PCP-induction of abnormal behavior, these drugs produce a marked increase in DA metabolism in cortical and some limbic regions with minimal changes in the striatum (Bowers and Hoffman, 1984; Deutch et al., 1987; Hiramatsu et al., 1989; Rao et al., 1990a,b; L6scher et al., 1991). Although the effects of MK-801 on striatal and limbic * Author to whom all correspondence should be addressed. : .D A neurotransmission have been extensively studied 269

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using in vivo dialysis (Kashihara et al., 1990: Weihmuller el al., 1991; Whitton et al., 1992a; h n p e r a t o et al., 1990), little is known about the exact nature o f MK-801-induced facilitation o f extracellular DA release in the cerebral cortex (Kashiwa el al., 1991 : Wedzony et al., 1993) which might, in part, be related to abnormal behavior elicited by N M D A antagonists (O'Neill and Liebman, 1987). To further characterize the MK-801-induction o f cortical DA release in t'it,o, we have presently investigated the effects o f the systemic application o f MK-801 on the extracellular release of DA and its major metabolites, 3,4-dihydroxy-phenylacetic acid ( D O P A C ) and homovanillic acid (HVA), in the prefrontal cortex o f the freely moving rat using a microdialysis technique. Because MK-801 has been found to affect cerebral serotonin metabolism (L6scher et al., 1991 : Whitton et al., 1992a), we also examined the changes in the in d r o liberation o f the serotonin metabolite 5-hydroxyindoleacetic acid (5HIAA) alter the drug as a comparison. EXPERIMENTAL PROCEDURES

Animals and dru,qs Male Wistar rats (ST strain from Shizuoka Laboratory Animals, Japan) weighing 200-250 g were used. Animals were housed at 22.0_+0.5 C in a humidity controlled room under a 12 h light dark cycle and allowed food and water ad libitum. The present animal experiments were performed in strict accordance with the guidelines of the National Institute of Neuroscience. National Center of Neurology and Psychiatry, and were approved by the Animal Investigation Committee of the Institute. All chemicals and drugs used were of ullrapure quality and commercially available. For systemic administration, MK-801 maleate and ~-methyl-p-tyrosine methyl ester HC1 (~-MT) were dissolved in physiological saline and doubledistilled water, respectively, and were injected i.p. Control animals received saline instead. In some experiments, a sodium channel blocker, tetrodotoxin (TTX) (Westerink et al., 1987), was dissolved in Ringer solution and infused into the medial frontal cortex via a dialysis tube to stop the nerve impulse flow. Doses of these drugs always refer to the free bases. Surgery According to the method previously described (Tanii et al., 1990), rats were anesthetized with pentobarbital (40 mg/kg, i.p.) and mounted on a stereotaxic frame. A straight type cellulose dialysis tubing (3.0 mm in length, 0.16 mm internal diameter, molecular weight cutoff 50,000, EICOM Co. Ltd., Kyoto, Japan) was then implanted into the medial frontal cortex [A+3.2 mm, V+5.2 mm, L+0.6 ram, atlas of Paxinos and Watson (1986)], which is a DA rich portion of the frontal cortex (Bj0orklund and Lindvall, 1984). Dialysis procedures and biochemical analysis Two days after surgery (day 3), the dialysis tube was perfused with a Ringer solution (NaC1, 147 mM; KCI, 4

mM : CaCI2, 2.9 raM) at a rate of 2 pl/min in the freely moving rat. The in Htro recovery of probes for monoamines and their metabolites at this flow rate ranged from 23 27%. Perfusates were collected in the injection valve of the highperformance liquid chromatography (HPLC) equipment which was directly connected to the outlet of the dialysis probe with teflon tubing (400 mm length, 0.1 mm internal diameter). The dialysate samples were automatically injected into the HPLC apparatus every 20 min with the help of an automatic injector (EICOM AS-100, EICOM, Japan) and immediately analyzed for DA. DOPAC, HVA and 5HIAA, which were quantitated by reverse-phase HPLC with electrochemical detection (ECD) as previously described (Tanii et al.. 1990) with minor modifications. Briefly, these substances were separated on a stainless-steel column filled with Nucleosil 5C18 using 0.1 M acetate citrate buffer, pH 4.2, containing 17% methanol, 1.01 mM octanesulphonic acid and 0.03 mM EDTA at a flow rate of 1.0 ml/min. ECD was achieved using a carbon graphite working electrode set at +0.6 V. tti.~tolo.qical controls After completion of each experiment, rats were stunned and decapitated. The brains were removed, frozen quickly on dry ice and stored at - 8 0 C until histological examination. The position of the dialysis probe was verified macroscopically in every case on 150/~m thick serial coronal slices after termination of the experiment. Dala analysis The average of the concentrations of each substance during the period preceding drug treatment (three measurements performed every 20 rain) was used as a control value ( = 100). Individual data are expressed as percentages of this baseline period. The means with SEM of the results obtained on 3 7 animals were calculated using corresponding periods. Areas under the curves (AUC) of the concentration vs time plots for dialysate DA, DOPAC, HVA and 5HIAA at 0 180 rain post-injection were calculated and used as overall measures of the treatment effects (Hjorth and Sharp, 1991 ; Matthews et al., 1990). Statistical comparisons were performed among the groups on the AUC data, using the twotailed Student's t-test, Mann-Whitney's U-test, one-way ANOVA or Kruskal Wallis test followed by a multiple comparison test. RESULTS Characterization o f basal release Basal concentrations o f dialysate DA, D O P A C , HVA and 5 H I A A o f the medial frontal cortex were quite stable from 40 min following the beginning o f perfusion and were (pg/40 /~1 o f perfusate; uncorrected for probe recovery): D A 1.87_+0.23 (12), D O P A C 138+22 (12), H V A 442-+ 10.1 (12), 5 H I A A 3650+622 (12); m e a n - + S E M , ( ) = n). Neither noradrenaline nor serotonin in the dialysates from the brain region were simultaneously detected under our conditions. As shown in Fig. 1, continuous infusion o f TTX ( 10 ~ M) into the medial frontal cortex via the dialysis

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MK-801 elicits a tetrodotoxin-sensitive increase in extracellular release of DA

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Fig. 1. Effects of intra-frontal cortex infusion of TTX on the levels of DA (0), DOPAC ((3), HVA (IS]) and 5HIAA (A) in the dialysates from the medial frontal cortex of the rat. Each value is the mean with SEM of data obtained from 5-7 animals which is expressed as a percentage of the respective average (basal level) of three samples collected before the application of TTX.

Effects o f svstemie administration of MK-801 on extracellular release o f DA, DOPAC, HVA and 5HIAA in the J?ontal cortex

fiber diminished extracellular release of DA, D O P A C , H V A and 5 H I A A to 10, 40, 65 and 80% of the baseline levels, respectively, in the cortical area. D A synthesis inhibition by means of intraperitoneal injection of ~-MT (250 mg/kg, which is a competitive inhibitor of tyrosine hydroxylase ; the rate limiting enzyme for D A biosynthesis) dramatically reduced the release of D A and its metabolites to 15 and 2 0 - 3 0 % of the control values, respectively, without effects on 5 H I A A at 160 min post-injection (Fig. 2).

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Figures 3, 4, 5 and 6 show the time course of changes in the extracellular release of DA, D O P A C , H V A and 5HIAA, respectively, in the frontal cortex of the rat treated with acute MK-801. Table 1 summarizes the data of areas under curves (AUC) for each substance in the dialysates from 0 to 180 min following MK-801 injection.

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TIME(rains) Fig. 3. EtTects of systemic administration of MK-801 on extracellular DA release of rat medial flonlal cortex in the absence (A) or the presence (B) of TTX in the perfusates. Each value is the mean with SEM of data obtained from 3 7 animals (() = n) which is expressed as a percentage of the average (basal level) of three samples collected before the administration of MK-801 (A) or TTX (B). Statistical comparisons were performed among the groups on the overall effects data (AUC ()-180 rain) as described in the Experimental Procedures section. *P < 0.05. **P < 0.01 as compared to saline-injected controls.

Systemic a d m i n i s t r a t i o n of MK-801 (0.4 1.25 mg/kg, i.p.) caused a m a r k e d increase in tile contents of D A [Fig. 3(A)], D O P A C [Fig. 4(A)] and HVA [Fig. 5(A)] m the frontal cortical dialysates in a doserelated manner. The magnitude of the maximal increase in D A content [900% o f the basal levels, Fig. 3(A)] was larger than those of D O P A C [500°A,. Fig. 4(A)] a n d of H V A [350%, Fig. 5(A)]. F u r t h e r m o r e , MK-801-induced a u g m e n t a t i o n of the DA release

reached a peak much more rapidly than those of its metabolites [Figs 3(A)~ 4(A) a n d 5(A)]. The intrafrontal cortex infusion of T T X (10 SM) completely blocked the ability of MK-801 (1.2:5 mg/kg~ i.p.) to increase extracellular D A [Fig. 3(B)], D O P A C {Fig. 4(B)] and H V A [Fig. 5(B)] in the prefrontal region. As indicated in Fig. 6(A), MK-801 produced a slight but significant e n h a n c e m e n t of the extracellular release of 5H1AA in the medial frontal cortex. This

MK-801 elicits a tetrodotoxin-sensitive increase in extracellular release of DA = saline (7) - - o - - MK-801 1.25mg/kg (4) ---D--- MK-801 0.8mg/kg (3) MK-801 0.4mg/kg (3)

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Fig. 4. Effects of systemic administration of M K-801 on extracellular DOPAC release of rat medial frontal cortex in the absence (A) or the presence (B) of TTX in the perfusates. Each value is the mean with SEM of data obtained from 3-7 animals ( ( ) = n) which is expressed as a percentage of the average (basal level) of three samples collected before the administration of MK-801 (A) or TTX (B). Statistical comparisons were performed among the groups on the overall effects data (AUC 0-180 rain) as described in the Experimental Procedures section. *P < 0.05, **P < 0.01 as compared to saline-injected controls. e n h a n c e m e n t was also f o u n d to be TTX-sensitive [Fig. 6(B)], but was delayed as c o m p a r e d to those of D A a n d its metabolites [Fig. 6(A)]. DISCUSSION

The basal levels o f D A , D O P A C , H V A a n d 5 H I A A in the dialysates from the medial frontal cortex are in good agreement with those from previous studies

(Bean a n d R o t h , 1991; C h e n a n d Paredes, 1990; M o g h a d d a m et al., 1990; N a k a h a r a et al., 1992; S e m b a et al., 1992; Tanii et al., 1990). T h e p r e s u m e d D A peak from the cortical dialysate sample co-eluted with the authentic D A in the cortical c h r o m a t o g r a m a n d was sensitive to T T X a n d ~ - M T confirming t h a t the present dialysis m e t h o d detects neurogenic D A release in the frontal cortex. The time course a n d the m a g n i t u d e o f ~ - M T - i n d u c e d decrement o f D A in the

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frontal dialysates are compatible with the report o[" M o g h a d d a m et al. (1990). In the present study, an acute systemic application o f MK-801 has been shown to enhance extracellular release o f D A and its metabolites in a dose-related fashion in the medial frontal cortex o f the rat. Delayed and much smaller increases in the serotonin metabolite 5H1AA seem to deny the possibility that the

effects on DA metabolism is a non-specific p h e n o m enon. The marked ti~cilitation o f frontal DA release is in line with the previous observations that, in the rat frontal cortex, an i.p. injection o f MK-801 caused an increase in extracellular D A release (Kashiwa el al., 1991 : Wedzony et al., 1993) and an augmentation o f tissue contents o f D O P A C and HVA without changes

MK-801 elicits a tetrodotoxin-sensitive increase in extracellular release of DA

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Fig. 6. Effects of systemic administration of MK-801 on extracellular 5HIAA release of rat medial frontal cortex in the absence (A) or presence (B) of TTX in the perfusates. Each value is the mean with SEM of data obtained from 3-7 animals ( ( ) = n) which is expressed as a percentage of the average (basal level) of three samples collected before the administration of M K-801 (A) or TTX (B). Statistical comparisons were performed among the groups on the overall effects data (AUC 0-180 rain) as described in the Experimental Procedures section. *P < 0.05, **P < 0.01 as compared to saline-injected controls.

in D A levels which indicates acceleration o f D A turnover ( H i r a m a t s u et al., 1989 ; L6scher et al., 1991 ; N i s h i k a w a et al., 1991 ; R a o et al., 1990a ; W h i t t o n et al., 1992b). While the f o r m a t i o n o f D O P A C a n d H V A has been reported to depend u p o n n o t only d o p a minergic b u t noradrenergic n e u r o n a l activity, the direct detection o f increased D A itself in the extra-

cellular space in this study appears to exclude the a s s u m p t i o n t h a t the elevation o f these d e a m i n a t e d metabolites of D A in the dialysates a n d the postm o r t e m tissues of the frontal cortex m a y solely be due to overactivation of noradrenergic neurons. F u r t h e r more, the fact t h a t systemic injection o f MK-801 facilitated D A , but n o t n o r a d r e n a l i n e utilization after

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Table I. Effects of systemic injection of M K-801 on the levels of DA. DOPAC, HVA and 5HIAA in the dialysates from the medial frontal cortex of the rat AUC t%} Treatment Dopamine(DA) Saline MK-801 ().4 mg:kg MK-801 0.8 mg/kg MK-801 1.25 m g k g DOPAC Saline MK-801 0.4 m g k g MK-801 0,8 mg/kg MK-801 1.25 mg/kg HVA Saline MK-801 0.4 mg/kg MK-801 0.8 mg/kg MK-801 1.25 mg/kg 5HIAA Salinc MK-801 0.4 mg:kg MK-801 0.8 mg/kg MK-801 1.25 mg/kg

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The mean with SEM of overall effects data (AUC 0 180 rain) obtained from 3 7 animals ( ( ) = n) was calculated and each value of this table is expressed as a percentage of the theoretical zero overall effect ( A U C - 18,000). AUC was calculated as described m the Experimental Procedures section. *P < 0.05, **P < 0.01 as compared to saline-injected controls,

inhibition of the biosynthesis of these catecholamines (Umino et al., 1990) suggests that the increase in the cortical DA metabolism is mainly due to activation of DA neurons projecting to the prefrontal area. The facilitated extracellular release of DOPAC and HVA is most likely to be concerned with the MK-801induced increase in the impulse traffic in the frontal DA neurons, because (1) the electrical stimulation of the medial forebrain bundle and DA receptor antagonists which augment the firing rates of the DA neurons produce a marked increase in the extracellular levels of these DA metabolites as well as DA (lmperato and Chiara, 1984; Imperato and Chiara, 1985) and (2) DA receptor agonists including apomorphine and selective D1 and D2 agonists which suppress the DA neuronal activity, reduce the DA metabolites liberation (Imperato and Chiara, 1985; Ozaki et al., 1989; Santiago et al., 1993). Indeed, the interruption of the DA nerve impulse by means of TTX infusion totally eliminated the MK-801-induced increment of DA and its metabolites in the cortical dialysates in the present study. These findings strongly suggest that MK-801 may augment the cortical DA release via increasing impulse flow in the mesocortical DA neurons. In accordance with this view, the firing rate of DA cells in the ventral tegmental area, which pro-

jects to the cortical and limbic areas, has previously been found to be increased by i.v. injection of MK801 (French and Ceci, 1990). Together with no interaction between MK-801 and the DA uptake site (Maurice et al., 1991), the TTXreversibility is consonant with the view that MK-801 would modulate frontal dopaminergic transmission by influencingtranssynaptic regulation of the DA neurons projecting to the cortical field. Extrapolating from the facts that MK-801 is one of the most potent antagonists of the NMDA receptor and that other non-competitive and competitive antagonists of the NMDA receptor (given systemically or locally) elevate the levels of DOPAC and HVA, DA utilization or extracellular release of DA and its metabolites in the frontal cortex (Hata et al., 1990: Nishikawa et al., 1991 : Tanii et al., 1990 ; Urn)no et al., 1990), the MK801-induced facilitation of frontal DA release is likely to be related to its blocking action at the NMDA receptor. This hypothesis is further supported by the observation that MK-801 shares preferential effects on cortical to striatal DA metabolism with other NMDA antagonists such as PCP (Bowers and Hoffman, 1984: Deutch et al., 1987: Nishikawa et al., 1991 ), TCP (Urn)no et al., 1990) and ketamine (Rao ct al., 1990b). Moreover, our recent study has demonstrated that intra-frontal cortex infusion of a selective and competitive antagonist of the NMDA receptor, cis-4-phosphonomethyl-2-piperidine carboxylic acid (CGS19755) (Lehmann et al., 1988), also enhances extracellular release of DA and its metabolites in the cortical area in a TTX-sensitive manner (Nishijima et a/., 1994). It cannot be totally excluded that MK-801 might induce frontal hyperdopaminergic activity by primarily blocking nicotinic acetylcholine receptor channels (Amador and Dan), 1991). However, this possibility seems to conflict with an increase in the in cit,o DA release after stimulation of the acetylcholine receptor by nicotine (Toth et al., 1992). The exact neural setup of the regulation by the NMDA receptor over the frontal DA neurons awaits further elucidation. Since electrophysiological studies have revealed that stimulation of the NMDA receptor causes excitation in the postsynaptic membrane in the central nervous system (Collinge and Lester, 1989), hypothetical NMDA receptors located on the prefrontal DA nerve terminals would not be expected to inhibit the release of DA. Consequently, it can be postulated that inhibitory interneurons, such as GABAergic ones, would be inserted between excitatory amino acidergic neurons and DA nerve terminals in the frontal cortex. In this neuronal setup, blockade of the NMDA receptor might cause a dis-

MK-801 elicits a tetrodotoxin-sensitive increase in extracellular release of DA inhibition of the prefrontal D A neurons by interrupting a tonic excitatory drive on the possible inhibitory neurons. MK-801-induced increase in the frontal release of the serotonin metabolite 5 H I A A indicates the plausible control via the N M D A receptor of frontal serotonin neurotransmission. The altered serotonergic tone might be involved in some of the m o t o r symptoms elicited by MK-801 which are similar to the characteristic syndrome induced by serotonin precursor or serotonin receptor agonists (Clineschmidt et al., 1982 ; Hiramatsu et al., 1989) or are attenuated by serotonin-lA receptor ligands (L6scher and Honack, 1992). Although the lack of data concerning the extracellular release of serotonin itself limits the interpretation of the present results, the increased 5 H I A A release appears to be in line with the recent finding that systemic administration of the N M D A antagonist elevated the tissue concentrations of 5 H I A A without affecting the 5HT levels in the rat frontal cortex (L6scher et al., 1991). The potential control by the N M D A receptor is also supported by the N M D A antagonists-induction of enhancement of the serotonin-stimulated phosphoinositide metabolism in the frontal cortex (Gandolfi et al., 1990). Further investigation is needed to clarify not only the mechanisms underlying the s e r o t o n i n - N M D A interaction but the relationship between the MK-801induced changes in D A and serotonin metabolism observed in this study. In conclusion, the present findings demonstrate that the systemic administration of a potent N M D A receptor channel blocker, MK-801, produces a TTX-sensitive increase in extracellular DA, its metabolites and 5 H I A A in the medial frontal cortex. This further supports the view that dopaminergic projections in the frontal cortex may be under a tonic inhibitory control by excitatory amino acidergic transmission through the N M D A receptor (Hata et al., 1990). The excitatory amino a c i d - D A interaction could be implicated in a dopaminergic (neuroleptics-reversible) component of MK-801-induction of abnormal behaviors in experimental animals (Clineschmidt et al., 1982; Criswell et al., 1993 ; Dall'Olio et al., 1992 ; Greenberg and Segal, 1985; Hoffman, 1992; Quagazzel et al., 1993). Excitatory amino acidergic regulation of frontal serotonergic transmission is also proposed.

Acknowledyements--This study was partly supported by

research grants from the Research Grant (2A-7) for Nervous and Mental Disorders from the Ministry of Health and Welfare (Japan), Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Science and Culture (Japan),

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and a Grant from Social Insurance Agency Contract Fund Commissioned to Japan Health Sciences Foundation. REFERENCES

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