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Neuroscience Research 63 (2009) 72–75
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Rapid communication
Enhanced susceptibility to MPTP neurotoxicity in magnesium-deficient C57BL/6N mice Akiko Muroyama a, Makiko Inaka a, Hiroaki Matsushima a, Haruhiko Sugino b, Yoshinori Marunaka b, Yasuhide Mitsumoto a,* a b
Department of Alternative Medicine and Experimental Therapeutics, Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa, Ishikawa 920-1181, Japan Department of Molecular Cell Physiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 May 2008 Received in revised form 23 September 2008 Accepted 30 September 2008 Available online 14 October 2008
We evaluated the effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in C57BL/6N mice fed a magnesium (Mg2+)-deficient diet. On the 3rd week, Mg2+-deficient mice displayed increased anxiety- and depression-like behavior. In the Mg2+-deficient mice, a low does (10 mg/kg) of MPTP treatment decreased dopamine (DA) and its metabolites contents in the striatum, but not in control mice. The same dose of MPTP did not influence these neurochemical markers in the mice fed Mg2+-deficient diet for 1 week which did not exhibit the altered emotional behavior. These results indicate that Mg2+-deficient mice with altered emotional behavior appear to increase the susceptibility to MPTP neurotoxicity in C57BL/6N mice. ß 2008 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
Keywords: MPTP Mouse model Magnesium depletion Emotional behavior Dopamine
Parkinson’s disease (PD) is a chronic and progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta and the concomitant reduction of dopamine at axon terminals in the striatum, and the disorder primarily affects elderly humans (Olanow and Tatton, 1999). Although the primary pathogenesis of PD remains unknown, there has been growing evidences that environmental factors and genetic predisposition, which cause mitochondrial dysfunction and oxidative stress, contribute to pathogenesis of PD (Fukae et al., 2007). Physical and emotional traumas are also considered as factors in the etiology and the pathophysiology of this disorder. For example, when the patients with PD are exposed to severe and long-term stresses, the symptoms are remarkably worsened (Snyder et al., 1985; Smith et al., 2002). On the other hand, a strong positive association between depression caused by stress and subsequent incidence of PD has been reported (Schuurman et al., 2002). However, the bases of these stressinduced impairments in this disease are unknown. Magnesium (Mg2+) plays an important role in neural function in the brain where it acts as the N-methyl-D-aspartate (NMDA) receptor antagonist. Mg2+ blocks the activation of NMDA receptor ion channel in a voltage-dependent manner (Mori et al., 1992;
* Corresponding author. Tel.: +81 76 229 1165x258; fax: +81 76 229 6207. E-mail address: [email protected] (Y. Mitsumoto).
Sobolevskii and Khodorov, 2002). Mg2+-deficiency may be associated with chronic stress (Seelig, 1994) and stress-related disease, such as depression (Rasmussen et al., 1989; Hashizume and Mori, 1990; Murck, 2002). Interestingly, Mg2+-deficiency leads to enhanced depression and anxiety-related behavior in mice (Singewald et al., 2004). These mice could be a useful model for studies of stress-related diseases because the behavioral phenotype can be reversed by known antidepressants and anxiolytic substances (Singewald et al., 2004). Therefore, we investigated whether neurochemical mechanisms underlying emotional behavior or Mg2+ deficiency itself are involved in the stress-induced exacerbation of dopaminergic neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a PD-inducing toxin (Dauer and Przedborski, 2003). Male C57BL/6N mice (8 weeks, Charles River Japan, Atsugi, Japan) were used. They were housed at an ambient temperature of 23 2 8C under a 12 h light/12 h dark cycle (lights on, 7:00 a.m.) with free access to food and water. All procedures were performed in accordance with the Guidelines for Animal Care and Use in Hokuriku University. Mice were placed on the control diet (AIN-93G, Oriental Yeast Co., Ltd., Tokyo Japan) containing Mg2+ at 50 mg/100 g of food for 1 week and then placed on the Mg2+-deficient diet which was commercially prepared with sucrose instead of magnesium oxide in AIN-93G. For quantification of serum Mg2+ levels, animals were deeply anesthetized with ether and blood was collected from the inferior vena cava. Serum Mg2+ concentrations were determined
0168-0102/$ – see front matter ß 2008 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2008.09.009
A. Muroyama et al. / Neuroscience Research 63 (2009) 72–75
colorimetrically using a Magnesium B test kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The behavioral tests were performed in a soundproof room. Prior to all behavioral tests, mice were allowed to habituate to the testing room for at least 1 h. All tests were carried out between 10:00 a.m. and 4:00 p.m. To investigate changes in anxietylike effects, the light/dark test (LDT) was performed as previously described with a minor modification (Crawley and Goodwin, 1980). The LDT apparatus (20.5 41 41 cm each, Sanplatec Corp., Osaka, Japan) consisted of a light compartment and a dark one that were connected by an opening (7 7 cm) located in the center of the partition at floor level. The light compartment was brightly illuminated with a 60 W light source, whereas the other was covered by a black top. The light intensity in the light compartment was 800 lx compared with 40 lx in the dark compartment. At the beginning of the test, each mouse was placed in the dark compartment facing away from the opening. The latency to the first entry into the lit compartment (latency), time spent in the lit compartment (time) and total number of transition (transition) between the two compartments were recorded for 10 min. Depression-like behavior was examined in forced swim test (FST) (Porsolt et al., 1977). Mice were individually placed into vertical Plexiglas cylinders (height 20 cm, diameter 9.0 cm, Sanplatec Corp., Osaka, Japan) containing water column of 9.5 cm height maintained at 23 2 8C. The cylinders were separated by a nontransparent panel to prevent mice from seeing each other. All sessions (6 min each) were recorded by a video camera and scored by a trained observer. The total time of immobility was measured during the last 4 min of the test period. Mice were considered immobile when floating passively in the water, performing only those movements required for keeping their heads above the water level. In the measurement of locomotor activity (LA), mice were individually placed in plastic chamber (40 40 40 cm) equipped with an automated activity monitoring system SCANET MV-20 (Melquest Ltd., Toyama, Japan). The LA was measured for 15 min every 5 min. LDT and FST were employed 24 h and 48 h after the measurement of locomotor activity. MPTP hydrochloride (Sigma–Aldrich, MO, USA) at a dose of 10 mg/kg was dissolved in saline and administered intraperitoneally (i.p.). MPTP was injected 48 h after the FST. The evaluation point was set 3 days after MPTP treatment according to the previous study (Mori et al., 2005). The dopamine (DA) and its metabolites, dihydroxyphenylacetic acid (DAPAC) and homovanillic acid (HVA), contents were assayed using the high performance liquid chromatography (HPLC)–electrochemical detection (ECD) system according to the previously described method (Mori et al., 2005). Mice were sacrificed and their striata were isolated and weighed. Each tissue was homogenized in a 0.1N perchloric acid containing isoproterenol, as an internal standard. The homogenates were centrifuged at 20,000 g for 15 min at 4 8C. The HPLC–ECD system consisted of a reversed phase ODS column (SC-5ODS, Eicom, Kyoto, Japan) and an ECD-100 (Eicom). A graphite working electrode (WE-3G, Eicom) was maintained at 750 mV against an Ag/AgCl reference electrode. The mobile phase consisted of 0.1 M citric acid/0.1 M sodium acetate buffer (pH 3.5) with 190 mg/l sodium-1-octanesulfonate, 5 mg/l EDTA-2Na and 15% (v/v) of methanol. The concentrations of DA, DOPAC and HVA were expressed as mg/g of the wet tissue weight. The turnover was calculated as (DOPAC + HVA)/DA. All results were given as mean S.D. Data analysis of serum Mg2+ concentrations was performed by two-tailed unpaired t-test. Data on body weight and behavioral results from the LDT, FST and LA were analyzed using a one-way analysis of variance (ANOVA) followed by LSD post hoctest. For the estimation of neurochemical results in the experiments with MPTP, one-way ANOVA followed by Tukey test was performed. The analyses were performed with the statistical analysis system StatMate III (ATMS Co., Ltd., Tokyo, Japan). A probability value of less than 5% was considered statistically significant.
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After 1 week of feeding with Mg2+-deficient diet, the body weight of Mg2+-deficient mice did not differ from that of control mice (control mice: 23.6 1.3 g, Mg2+-deficient mice: 22.9 1.0 g) [F(1,18) = 1.75, P = 0.202]. Whereas, Mg2+-deficient mice weighted less than control mice at 3 week (control mice: 25.6 1.5 g, Mg2+deficient mice: 23.5 1.5 g) [F(1,18) = 10.13, P < 0.01]. Serum Mg2+ concentrations were measured in mice at 1 and 3 weeks after being placed on the Mg2+-deficient diet. Mg2+-deficient feeding for 1 and 3 weeks significantly reduced serum Mg2+ concentrations compared with normal diet (normal and Mg2+-deficient diet at 1 week: 1.49 0.07 and 0.69 0.03 mM, n = 3, P < 0.001; 3 week: 1.28 0.10 and 0.45 0.04 mM, n = 3, P < 0.001). We evaluated the effect of Mg2+-deficient diet on emotional behavior using LDT and FST. On the 1st week of feeding with Mg2+-deficient diet (Table 1), all parameters including latency, time and transition in the LDT were not different from that of control mice [latency: F(1,18) = 0.51, P = 0.482; time: F(1,18) = 1.62, P = 0.219; transition: F(1,18) = 3.36, P = 0.084]. Mg2+deficient mice at 2 week exhibited an increase of latency and a reduction of transition (Table 1) [latency: F(1,18) = 6.47, P < 0.05; time: F(1,18) = 3.73, P = 0.69; transition: F(1,18) = 9.55, P < 0.01]. Furthermore, at 3 week of Mg2+-deficient mice displayed an increase of latency and a reduction of time, but not differ in transition from control mice (Table 1) [latency: F(1,17) = 7.79, P < 0.05; time: F(1,17) = 4.76, P < 0.05; transition: F(1,17) = 4.16, P = 0.057]. We evaluated depression-like behavior as measure of immobility time in the FST. During the first 2 weeks of Mg2+-deficient feeding, immobility time did not differ between Mg2+-deficient and control mice (Table 1) [1 week: F(1,18) = 0.64, P = 0.435; 2 week: F(1,18) = 1.13, P = 0.302]. While, Mg2+-deficient mice at 3 week showed increase of immobility time compared with control mice (Table 1) [F(1,18) = 14.90, P < 0.01]. Mg2+deficient mice may be more active or affected by change of neuromuscular function (Topf and Murray, 2003). In order to address to this issue, locomotor activity was measured as a parameter of motor function. At 3 week of Mg2+-deficiency, locomotor activity for 15 min in control and Mg2+-deficient mice were 7065 2365 and 6810 1802 counts, respectively [F(1,18) = 0.07, P = 0.789]. The result indicates that Mg2+-deficiency does not significantly alter locomotor activity. These results clearly confirm the previous study finding that mice receiving low Mg2+ showed depression- and anxiety- related behavior as evaluated by FST and LDT, respectively (Singewald et al., 2004). To Table 1 Effect of Mg2+-deficient diet (Mg2+( )) during 1, 2 and 3 weeks on light/dark test (LDT) and immobility time in forced swim test (FST). n
1 week
2 week
3 week
LDT Latency (s) Control Mg2+( )
10 10
98.0 66.9 120.9 75.6
67.0 50.8 134.1 66.2*
66.8 38.7 123.0 48.0*
Time (s) Control Mg2+( )
10 10
158.4 58.1 128.9 44.7
209.3 61.1 151.0 73.3
242.4 53.8 178.3 71.9*
Transition (n) Control Mg2+( )
10 10
31.1 10.1 23.3 8.9
28.9 7.4 18.4 7.8**
FST Immobility time (s) Control 10 Mg2+( ) 10
195.8 12.6 190.2 18.3
209.2 12.0 214.1 8.3
35.9 9.4 26.7 10.2
190.0 12.8 213.4 14.3**
In the LDT, the latency to the first entry into the lit compartment, time spent in the lit compartment and total number of transition between the two compartments during the 10 min testing period are shown. In the FST, time spent floating during the last 4 min of a 6 min test session was measured and expressed as immobility time. Data are means S.D. n = numbers of animals/experimental group. *P < 0.05; ** P < 0.01 compared to control.
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A. Muroyama et al. / Neuroscience Research 63 (2009) 72–75
Table 2 Effect of MPTP on DA and metabolites contents and DA turnover in the striatum of control and Mg2+-deficient (Mg2+( )) mice. n
DA (mg/g tissue)
DOPAC (mg/g tissue)
HVA (mg/g tissue)
Turnover (DOPAC + HVA)/DA
1 week Control MPTP Mg2+( ) Mg2+( ) + MPTP
4 4 4 4
14.37 1.51 15.45 1.86 15.17 0.12 14.06 1.87
0.97 0.14 1.01 0.08 1.03 0.05 1.00 0.15
1.68 0.12 1.63 0.21 1.62 1.51 1.51 0.22
0.19 0.02 0.17 0.01 0.17 0.01 0.18 0.01
3 week Control MPTP Mg2+( ) Mg2+( ) + MPTP
5 5 5 3
15.91 2.99 14.64 0.59 14.93 2.97 8.01 1.49**
1.99 0.37 1.77 0.33 1.99 0.72 1.41 0.46
1.90 0.29 1.87 0.12 1.98 0.42 1.27 0.26*
0.25 0.01 0.25 0.03 0.26 0.03 0.32 0.03*
At the 1 and 3 weeks of Mg2+ deficiency, MPTP (10 mg/kg) were given to control and Mg2+( ) mice and the evaluation was performed at 3 days after the toxin injection. Striatal DA and its metabolite were extracted and measured by HPLC-ECD. The DA turnover was calculated as (DOPAC + HVA)/DA. Data are means S.D. n = numbers of animals/experimental group. *P < 0.05; **P < 0.01 compared to Mg2+( ) group.
analysis the relationship between the altered emotional behavior and the sensitivity to MPTP, we examined effect of the dopaminergic neurotoxin on DA and its metabolites contents in the striatum from mice at 1 and 3 weeks of Mg2+-deficiency. We chose a low dose (10 mg/ kg) for MPTP treatment used here because single injection of the dose did not affect the dopaminergic marker protein levels such as dopamine transporter and tyrosine hydroxylase in C57BL/6N mice (data not shown). Therefore, the treatment with MPTP does not cause structural damage in nigrostriatal dopaminergic neurons. In mice with Mg2+deficient diet for 1 week, the striatal DA and its metabolites contents and DA turnover after injection of MPTP did not differ to control mice (Table 2). Conversely, Mg2+-deficient mice at 3 week showed that MPTP significantly decreased DA and its metabolite HVA contents (50 and 67% of control mice, respectively, Table 2), but not in control mice. At the same time, DA turnover significantly increased to 130% of control mice (Table 2). The symptoms of patients with PD often are worsened by various stresses (Zigmond and Stricker, 1984; Urakami et al., 1988). When the stress is mild, the symptoms are easily improved by increase of the dose of L-DOPA. However, when the patients with PD are exposed to severe and long-term stress, the symptoms are remarkably aggravated and cannot be easily improved by LDOPA treatment (Urakami et al., 1988). We thought that alteration in central neurotransmission associated with severe emotional stress could be related to the functional impairments in nigrostriatal dopaminergic neurons. Our data show that the decrease of striatal dopamine level by MPTP was only seen in mice expressing depression- and anxiety-related behavior, but not in control mice. Therefore, the brain’s responses to emotional situations influence the susceptibility to the dopmainergic neurotoxin. Interestingly, significant loss of dopaminergic neurons was identified exclusively in the substantia nigra in 1-year-old rats that has been exposed continuously to a low Mg2+ intake (one-fifth of the normal level) over generations (Oyanagi et al., 2006). The recent study showed that a significant effect of Mg2+ for prevention of neurite and neuron pathology in a in vitro PD model using MPP+ (Hashimoto et al., 2008). These findings suggest that both etiologic roles and possible therapeutic applications of Mg2+ in PD. In the central nervous system, Mg2+ plays an important role in neurotransmission. For instance, one of the recycling pathways for synaptic vesicles depends upon presynaptic concentrations of Mg2+ (Koenig and Ikeda, 1996). Mg2+ is also well known as a gating ion within the excitatory NMDA glutamate receptor complex. Coactivation of these receptors by the neurotransmitter glutamate and intracellular depolarization causes Mg2+ displacement from within the NMDA receptor ion channel and allows calcium to enter neurons (Mori et al., 1992; Sobolevskii and Khodorov, 2002). In our study, Mg2+-deficient mice at 1 week show the reduction of blood
Mg2+ concentration and are more sensitive to the convulsant effects of a peripheral injection of NMDA (data not shown) as previously reported from other laboratories (Decollogne et al., 1997; Bardgett et al., 2005). The results suggest that Mg2+ deficiency can lead to activation of NMDA receptor in the brain. However, in these mice, the treatment with MPTP did not affect the striatal dopamine level. This result suggests that neither Mg2+ deficiency nor activation of NMDA receptor directly affect the sensitivity of nigrostriatal dopaminergic neurons to a low dose of MPTP. Experiments are ongoing to explore how Mg2+-deficiency is linked to the functional deficit in the dopaminergic pathway induced by MPTP. In conclusion, Mg2+-deficient mice with altered emotional behavior appear to increase the susceptibility to a low dose of MPTP. We propose that Mg2+-deficiency affecting emotional behavior also disturbs functions of nigrostriatal dopaminergic neurons. Acknowledgements Supported by the Specific Research Fund of Hokuriku University and Otsuka Pharmaceutical Co., Ltd. The author thanks Dr. Atsushi Mori (Otsuka Pharmaceutical Co., Ltd.) for his help with HPLC analysis. References Bardgett, M.E., Schultheis, P.J., McGill, D.L., Richmond, R.E., Wagge, J.R., 2005. Magnesium deficiency impairs fear conditioning in mice. Brain Res. 1038, 100–106. Crawley, J., Goodwin, F.K., 1980. Preliminary report of a simple animal behavior model for the anxiolytic effects of benzodiazepines. Pharmacol. Biochem. Behav. 13, 167–170. Dauer, W., Przedborski, S., 2003. Parkinson’s disease: mechanisms and models. Neuron 39, 889–909. Decollogne, S., Tomas, A., Lecerf, C., Adamowicz, E., Seman, M., 1997. NMDA receptor complex blockade by oral administration of magnesium: comparison with MK801. Pharmacol. Biochem. Behav. 58, 261–268. Fukae, J., Mizuno, Y., Hattori, N., 2007. Mitochondrial dysfunction in Parkinson’s disease. Mitochondrion 7, 58–62. Hashimoto, T., Nishi, K., Nagasao, J., Tsuji, S., Oyanagi, K., 2008. Magnesium exerts both preventive and ameliorating effects in an in vitro rat Parkinson disease model involving 1-methyl-4-phenylpyridinium (MPP+) toxicity in dopaminergic neurons. Brain Res. 1197, 143–151. Hashizume, N., Mori, M., 1990. An analysis of hypermagnesemia and hypomagnesemia. Jpn. J. Med. 29, 368–372. Koenig, J.H., Ikeda, K., 1996. Synaptic vesicles have two distinct recycling pathways. J. Cell Biol. 135, 797–808. Mori, A., Ohashi, S., Nakai, M., Moriizumi, T., Mitsumoto, Y., 2005. Neural mechanisms underlying motor dysfunction as detected by the tail suspension test in MPTP-treated C57BL/6 mice. Neurosci. Res. 51, 265–274. Mori, H., Masaki, H., Yamakura, T., Mishina, M., 1992. Identification by mutagenesis of a Mg(2+)-block site of the NMDA receptor channel. Nature 358, 673–675.
A. Muroyama et al. / Neuroscience Research 63 (2009) 72–75 Murck, H., 2002. Magnesium and affective disorders. Nutr. Neurosci. 5, 375–389. Olanow, C.W., Tatton, W.G., 1999. Etiology and pathogenesis of Parkinson’s disease. Annu. Rev. Neurosci. 22, 123–144. Oyanagi, K., Kawakami, E., Kikuchi-Horie, K., Ohara, K., Ogata, K., Takahama, S., Wada, M., Kihira, T., Yasui, M., 2006. Magnesium deficiency over generations in rats with special references to the pathogenesis of the Parkinsonism-dementia complex and amyotrophic lateral sclerosis of Guam. Neuropathology 26, 115–128. Porsolt, R.D., Bertin, A., Jalfre, M., 1977. Behavioral despair in mice: a primary screening test for antidepressants. Arch. Int. Pharmacodyn. Ther. 229, 327–336. Rasmussen, H.H., Mortensen, P.B., Jensen, I.W., 1989. Depression and magnesium deficiency. Int. J. Psychiatry Med. 19, 57–63. Schuurman, A.G., van den Akker, M., Ensinck, K.T., Metsemakers, J.F., Knottnerus, J.A., Leentjens, A.F., Buntinx, F., 2002. Increased risk of Parkinson’s disease after depression: a retrospective cohort study. Neurology 58, 1501–1504. Seelig, M.S., 1994. Consequences of magnesium deficiency on the enhancement of stress reactions; preventive and therapeutic implications (a review). J. Am. Coll. Nutr. 13, 429–446.
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Singewald, N., Sinner, C., Hetzenauer, A., Sartori, S.B., Murck, H., 2004. Magnesiumdeficient diet alters depression- and anxiety-related behavior in mice—influence of desipramine and Hypericum perforatum extract. Neuropharmacology 47, 1189–1197. Smith, A.D., Castro, S.L., Zigmond, M.J., 2002. Stress-induced Parkinson’s disease: a working hypothesis. Physiol. Behav. 77, 527–531. Snyder, A.M., Stricker, E.M., Zigmond, M.J., 1985. Stress-induced neurological impairments in an animal model of parkinsonism. Ann. Neurol. 18, 544–551. Sobolevskii, A.I., Khodorov, B.I., 2002. Blocker studies of the functional architecture of the NMDA receptor channel. Neurosci. Behav. Physiol. 32, 157–171. Topf, J.M., Murray, P.T., 2003. Hypomagnesemia and hypermagnesemia. Rev. Endocr. Metab. Disord. 4, 195–206. Urakami, K., Masaki, N., Shimoda, K., Nishikawa, S., Takahashi, K., 1988. Increase of striatal dopamine turnover by stress in MPTP-treated mice. Clin. Neuropharmacol. 11, 360–368. Zigmond, M.J., Stricker, E.M., 1984. Parkinson’s disease: studies with an animal model. Life Sci. 35, 5–18.
Contents lists available at ScienceDirect
Neuroscience Research journal homepage: www.elsevier.com/locate/neures
Rapid communication
Enhanced susceptibility to MPTP neurotoxicity in magnesium-deficient C57BL/6N mice Akiko Muroyama a, Makiko Inaka a, Hiroaki Matsushima a, Haruhiko Sugino b, Yoshinori Marunaka b, Yasuhide Mitsumoto a,* a b
Department of Alternative Medicine and Experimental Therapeutics, Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa, Ishikawa 920-1181, Japan Department of Molecular Cell Physiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 May 2008 Received in revised form 23 September 2008 Accepted 30 September 2008 Available online 14 October 2008
We evaluated the effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in C57BL/6N mice fed a magnesium (Mg2+)-deficient diet. On the 3rd week, Mg2+-deficient mice displayed increased anxiety- and depression-like behavior. In the Mg2+-deficient mice, a low does (10 mg/kg) of MPTP treatment decreased dopamine (DA) and its metabolites contents in the striatum, but not in control mice. The same dose of MPTP did not influence these neurochemical markers in the mice fed Mg2+-deficient diet for 1 week which did not exhibit the altered emotional behavior. These results indicate that Mg2+-deficient mice with altered emotional behavior appear to increase the susceptibility to MPTP neurotoxicity in C57BL/6N mice. ß 2008 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
Keywords: MPTP Mouse model Magnesium depletion Emotional behavior Dopamine
Parkinson’s disease (PD) is a chronic and progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta and the concomitant reduction of dopamine at axon terminals in the striatum, and the disorder primarily affects elderly humans (Olanow and Tatton, 1999). Although the primary pathogenesis of PD remains unknown, there has been growing evidences that environmental factors and genetic predisposition, which cause mitochondrial dysfunction and oxidative stress, contribute to pathogenesis of PD (Fukae et al., 2007). Physical and emotional traumas are also considered as factors in the etiology and the pathophysiology of this disorder. For example, when the patients with PD are exposed to severe and long-term stresses, the symptoms are remarkably worsened (Snyder et al., 1985; Smith et al., 2002). On the other hand, a strong positive association between depression caused by stress and subsequent incidence of PD has been reported (Schuurman et al., 2002). However, the bases of these stressinduced impairments in this disease are unknown. Magnesium (Mg2+) plays an important role in neural function in the brain where it acts as the N-methyl-D-aspartate (NMDA) receptor antagonist. Mg2+ blocks the activation of NMDA receptor ion channel in a voltage-dependent manner (Mori et al., 1992;
* Corresponding author. Tel.: +81 76 229 1165x258; fax: +81 76 229 6207. E-mail address: [email protected] (Y. Mitsumoto).
Sobolevskii and Khodorov, 2002). Mg2+-deficiency may be associated with chronic stress (Seelig, 1994) and stress-related disease, such as depression (Rasmussen et al., 1989; Hashizume and Mori, 1990; Murck, 2002). Interestingly, Mg2+-deficiency leads to enhanced depression and anxiety-related behavior in mice (Singewald et al., 2004). These mice could be a useful model for studies of stress-related diseases because the behavioral phenotype can be reversed by known antidepressants and anxiolytic substances (Singewald et al., 2004). Therefore, we investigated whether neurochemical mechanisms underlying emotional behavior or Mg2+ deficiency itself are involved in the stress-induced exacerbation of dopaminergic neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a PD-inducing toxin (Dauer and Przedborski, 2003). Male C57BL/6N mice (8 weeks, Charles River Japan, Atsugi, Japan) were used. They were housed at an ambient temperature of 23 2 8C under a 12 h light/12 h dark cycle (lights on, 7:00 a.m.) with free access to food and water. All procedures were performed in accordance with the Guidelines for Animal Care and Use in Hokuriku University. Mice were placed on the control diet (AIN-93G, Oriental Yeast Co., Ltd., Tokyo Japan) containing Mg2+ at 50 mg/100 g of food for 1 week and then placed on the Mg2+-deficient diet which was commercially prepared with sucrose instead of magnesium oxide in AIN-93G. For quantification of serum Mg2+ levels, animals were deeply anesthetized with ether and blood was collected from the inferior vena cava. Serum Mg2+ concentrations were determined
0168-0102/$ – see front matter ß 2008 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2008.09.009
A. Muroyama et al. / Neuroscience Research 63 (2009) 72–75
colorimetrically using a Magnesium B test kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The behavioral tests were performed in a soundproof room. Prior to all behavioral tests, mice were allowed to habituate to the testing room for at least 1 h. All tests were carried out between 10:00 a.m. and 4:00 p.m. To investigate changes in anxietylike effects, the light/dark test (LDT) was performed as previously described with a minor modification (Crawley and Goodwin, 1980). The LDT apparatus (20.5 41 41 cm each, Sanplatec Corp., Osaka, Japan) consisted of a light compartment and a dark one that were connected by an opening (7 7 cm) located in the center of the partition at floor level. The light compartment was brightly illuminated with a 60 W light source, whereas the other was covered by a black top. The light intensity in the light compartment was 800 lx compared with 40 lx in the dark compartment. At the beginning of the test, each mouse was placed in the dark compartment facing away from the opening. The latency to the first entry into the lit compartment (latency), time spent in the lit compartment (time) and total number of transition (transition) between the two compartments were recorded for 10 min. Depression-like behavior was examined in forced swim test (FST) (Porsolt et al., 1977). Mice were individually placed into vertical Plexiglas cylinders (height 20 cm, diameter 9.0 cm, Sanplatec Corp., Osaka, Japan) containing water column of 9.5 cm height maintained at 23 2 8C. The cylinders were separated by a nontransparent panel to prevent mice from seeing each other. All sessions (6 min each) were recorded by a video camera and scored by a trained observer. The total time of immobility was measured during the last 4 min of the test period. Mice were considered immobile when floating passively in the water, performing only those movements required for keeping their heads above the water level. In the measurement of locomotor activity (LA), mice were individually placed in plastic chamber (40 40 40 cm) equipped with an automated activity monitoring system SCANET MV-20 (Melquest Ltd., Toyama, Japan). The LA was measured for 15 min every 5 min. LDT and FST were employed 24 h and 48 h after the measurement of locomotor activity. MPTP hydrochloride (Sigma–Aldrich, MO, USA) at a dose of 10 mg/kg was dissolved in saline and administered intraperitoneally (i.p.). MPTP was injected 48 h after the FST. The evaluation point was set 3 days after MPTP treatment according to the previous study (Mori et al., 2005). The dopamine (DA) and its metabolites, dihydroxyphenylacetic acid (DAPAC) and homovanillic acid (HVA), contents were assayed using the high performance liquid chromatography (HPLC)–electrochemical detection (ECD) system according to the previously described method (Mori et al., 2005). Mice were sacrificed and their striata were isolated and weighed. Each tissue was homogenized in a 0.1N perchloric acid containing isoproterenol, as an internal standard. The homogenates were centrifuged at 20,000 g for 15 min at 4 8C. The HPLC–ECD system consisted of a reversed phase ODS column (SC-5ODS, Eicom, Kyoto, Japan) and an ECD-100 (Eicom). A graphite working electrode (WE-3G, Eicom) was maintained at 750 mV against an Ag/AgCl reference electrode. The mobile phase consisted of 0.1 M citric acid/0.1 M sodium acetate buffer (pH 3.5) with 190 mg/l sodium-1-octanesulfonate, 5 mg/l EDTA-2Na and 15% (v/v) of methanol. The concentrations of DA, DOPAC and HVA were expressed as mg/g of the wet tissue weight. The turnover was calculated as (DOPAC + HVA)/DA. All results were given as mean S.D. Data analysis of serum Mg2+ concentrations was performed by two-tailed unpaired t-test. Data on body weight and behavioral results from the LDT, FST and LA were analyzed using a one-way analysis of variance (ANOVA) followed by LSD post hoctest. For the estimation of neurochemical results in the experiments with MPTP, one-way ANOVA followed by Tukey test was performed. The analyses were performed with the statistical analysis system StatMate III (ATMS Co., Ltd., Tokyo, Japan). A probability value of less than 5% was considered statistically significant.
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After 1 week of feeding with Mg2+-deficient diet, the body weight of Mg2+-deficient mice did not differ from that of control mice (control mice: 23.6 1.3 g, Mg2+-deficient mice: 22.9 1.0 g) [F(1,18) = 1.75, P = 0.202]. Whereas, Mg2+-deficient mice weighted less than control mice at 3 week (control mice: 25.6 1.5 g, Mg2+deficient mice: 23.5 1.5 g) [F(1,18) = 10.13, P < 0.01]. Serum Mg2+ concentrations were measured in mice at 1 and 3 weeks after being placed on the Mg2+-deficient diet. Mg2+-deficient feeding for 1 and 3 weeks significantly reduced serum Mg2+ concentrations compared with normal diet (normal and Mg2+-deficient diet at 1 week: 1.49 0.07 and 0.69 0.03 mM, n = 3, P < 0.001; 3 week: 1.28 0.10 and 0.45 0.04 mM, n = 3, P < 0.001). We evaluated the effect of Mg2+-deficient diet on emotional behavior using LDT and FST. On the 1st week of feeding with Mg2+-deficient diet (Table 1), all parameters including latency, time and transition in the LDT were not different from that of control mice [latency: F(1,18) = 0.51, P = 0.482; time: F(1,18) = 1.62, P = 0.219; transition: F(1,18) = 3.36, P = 0.084]. Mg2+deficient mice at 2 week exhibited an increase of latency and a reduction of transition (Table 1) [latency: F(1,18) = 6.47, P < 0.05; time: F(1,18) = 3.73, P = 0.69; transition: F(1,18) = 9.55, P < 0.01]. Furthermore, at 3 week of Mg2+-deficient mice displayed an increase of latency and a reduction of time, but not differ in transition from control mice (Table 1) [latency: F(1,17) = 7.79, P < 0.05; time: F(1,17) = 4.76, P < 0.05; transition: F(1,17) = 4.16, P = 0.057]. We evaluated depression-like behavior as measure of immobility time in the FST. During the first 2 weeks of Mg2+-deficient feeding, immobility time did not differ between Mg2+-deficient and control mice (Table 1) [1 week: F(1,18) = 0.64, P = 0.435; 2 week: F(1,18) = 1.13, P = 0.302]. While, Mg2+-deficient mice at 3 week showed increase of immobility time compared with control mice (Table 1) [F(1,18) = 14.90, P < 0.01]. Mg2+deficient mice may be more active or affected by change of neuromuscular function (Topf and Murray, 2003). In order to address to this issue, locomotor activity was measured as a parameter of motor function. At 3 week of Mg2+-deficiency, locomotor activity for 15 min in control and Mg2+-deficient mice were 7065 2365 and 6810 1802 counts, respectively [F(1,18) = 0.07, P = 0.789]. The result indicates that Mg2+-deficiency does not significantly alter locomotor activity. These results clearly confirm the previous study finding that mice receiving low Mg2+ showed depression- and anxiety- related behavior as evaluated by FST and LDT, respectively (Singewald et al., 2004). To Table 1 Effect of Mg2+-deficient diet (Mg2+( )) during 1, 2 and 3 weeks on light/dark test (LDT) and immobility time in forced swim test (FST). n
1 week
2 week
3 week
LDT Latency (s) Control Mg2+( )
10 10
98.0 66.9 120.9 75.6
67.0 50.8 134.1 66.2*
66.8 38.7 123.0 48.0*
Time (s) Control Mg2+( )
10 10
158.4 58.1 128.9 44.7
209.3 61.1 151.0 73.3
242.4 53.8 178.3 71.9*
Transition (n) Control Mg2+( )
10 10
31.1 10.1 23.3 8.9
28.9 7.4 18.4 7.8**
FST Immobility time (s) Control 10 Mg2+( ) 10
195.8 12.6 190.2 18.3
209.2 12.0 214.1 8.3
35.9 9.4 26.7 10.2
190.0 12.8 213.4 14.3**
In the LDT, the latency to the first entry into the lit compartment, time spent in the lit compartment and total number of transition between the two compartments during the 10 min testing period are shown. In the FST, time spent floating during the last 4 min of a 6 min test session was measured and expressed as immobility time. Data are means S.D. n = numbers of animals/experimental group. *P < 0.05; ** P < 0.01 compared to control.
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Table 2 Effect of MPTP on DA and metabolites contents and DA turnover in the striatum of control and Mg2+-deficient (Mg2+( )) mice. n
DA (mg/g tissue)
DOPAC (mg/g tissue)
HVA (mg/g tissue)
Turnover (DOPAC + HVA)/DA
1 week Control MPTP Mg2+( ) Mg2+( ) + MPTP
4 4 4 4
14.37 1.51 15.45 1.86 15.17 0.12 14.06 1.87
0.97 0.14 1.01 0.08 1.03 0.05 1.00 0.15
1.68 0.12 1.63 0.21 1.62 1.51 1.51 0.22
0.19 0.02 0.17 0.01 0.17 0.01 0.18 0.01
3 week Control MPTP Mg2+( ) Mg2+( ) + MPTP
5 5 5 3
15.91 2.99 14.64 0.59 14.93 2.97 8.01 1.49**
1.99 0.37 1.77 0.33 1.99 0.72 1.41 0.46
1.90 0.29 1.87 0.12 1.98 0.42 1.27 0.26*
0.25 0.01 0.25 0.03 0.26 0.03 0.32 0.03*
At the 1 and 3 weeks of Mg2+ deficiency, MPTP (10 mg/kg) were given to control and Mg2+( ) mice and the evaluation was performed at 3 days after the toxin injection. Striatal DA and its metabolite were extracted and measured by HPLC-ECD. The DA turnover was calculated as (DOPAC + HVA)/DA. Data are means S.D. n = numbers of animals/experimental group. *P < 0.05; **P < 0.01 compared to Mg2+( ) group.
analysis the relationship between the altered emotional behavior and the sensitivity to MPTP, we examined effect of the dopaminergic neurotoxin on DA and its metabolites contents in the striatum from mice at 1 and 3 weeks of Mg2+-deficiency. We chose a low dose (10 mg/ kg) for MPTP treatment used here because single injection of the dose did not affect the dopaminergic marker protein levels such as dopamine transporter and tyrosine hydroxylase in C57BL/6N mice (data not shown). Therefore, the treatment with MPTP does not cause structural damage in nigrostriatal dopaminergic neurons. In mice with Mg2+deficient diet for 1 week, the striatal DA and its metabolites contents and DA turnover after injection of MPTP did not differ to control mice (Table 2). Conversely, Mg2+-deficient mice at 3 week showed that MPTP significantly decreased DA and its metabolite HVA contents (50 and 67% of control mice, respectively, Table 2), but not in control mice. At the same time, DA turnover significantly increased to 130% of control mice (Table 2). The symptoms of patients with PD often are worsened by various stresses (Zigmond and Stricker, 1984; Urakami et al., 1988). When the stress is mild, the symptoms are easily improved by increase of the dose of L-DOPA. However, when the patients with PD are exposed to severe and long-term stress, the symptoms are remarkably aggravated and cannot be easily improved by LDOPA treatment (Urakami et al., 1988). We thought that alteration in central neurotransmission associated with severe emotional stress could be related to the functional impairments in nigrostriatal dopaminergic neurons. Our data show that the decrease of striatal dopamine level by MPTP was only seen in mice expressing depression- and anxiety-related behavior, but not in control mice. Therefore, the brain’s responses to emotional situations influence the susceptibility to the dopmainergic neurotoxin. Interestingly, significant loss of dopaminergic neurons was identified exclusively in the substantia nigra in 1-year-old rats that has been exposed continuously to a low Mg2+ intake (one-fifth of the normal level) over generations (Oyanagi et al., 2006). The recent study showed that a significant effect of Mg2+ for prevention of neurite and neuron pathology in a in vitro PD model using MPP+ (Hashimoto et al., 2008). These findings suggest that both etiologic roles and possible therapeutic applications of Mg2+ in PD. In the central nervous system, Mg2+ plays an important role in neurotransmission. For instance, one of the recycling pathways for synaptic vesicles depends upon presynaptic concentrations of Mg2+ (Koenig and Ikeda, 1996). Mg2+ is also well known as a gating ion within the excitatory NMDA glutamate receptor complex. Coactivation of these receptors by the neurotransmitter glutamate and intracellular depolarization causes Mg2+ displacement from within the NMDA receptor ion channel and allows calcium to enter neurons (Mori et al., 1992; Sobolevskii and Khodorov, 2002). In our study, Mg2+-deficient mice at 1 week show the reduction of blood
Mg2+ concentration and are more sensitive to the convulsant effects of a peripheral injection of NMDA (data not shown) as previously reported from other laboratories (Decollogne et al., 1997; Bardgett et al., 2005). The results suggest that Mg2+ deficiency can lead to activation of NMDA receptor in the brain. However, in these mice, the treatment with MPTP did not affect the striatal dopamine level. This result suggests that neither Mg2+ deficiency nor activation of NMDA receptor directly affect the sensitivity of nigrostriatal dopaminergic neurons to a low dose of MPTP. Experiments are ongoing to explore how Mg2+-deficiency is linked to the functional deficit in the dopaminergic pathway induced by MPTP. In conclusion, Mg2+-deficient mice with altered emotional behavior appear to increase the susceptibility to a low dose of MPTP. We propose that Mg2+-deficiency affecting emotional behavior also disturbs functions of nigrostriatal dopaminergic neurons. Acknowledgements Supported by the Specific Research Fund of Hokuriku University and Otsuka Pharmaceutical Co., Ltd. The author thanks Dr. Atsushi Mori (Otsuka Pharmaceutical Co., Ltd.) for his help with HPLC analysis. References Bardgett, M.E., Schultheis, P.J., McGill, D.L., Richmond, R.E., Wagge, J.R., 2005. Magnesium deficiency impairs fear conditioning in mice. Brain Res. 1038, 100–106. Crawley, J., Goodwin, F.K., 1980. Preliminary report of a simple animal behavior model for the anxiolytic effects of benzodiazepines. Pharmacol. Biochem. Behav. 13, 167–170. Dauer, W., Przedborski, S., 2003. Parkinson’s disease: mechanisms and models. Neuron 39, 889–909. Decollogne, S., Tomas, A., Lecerf, C., Adamowicz, E., Seman, M., 1997. NMDA receptor complex blockade by oral administration of magnesium: comparison with MK801. Pharmacol. Biochem. Behav. 58, 261–268. Fukae, J., Mizuno, Y., Hattori, N., 2007. Mitochondrial dysfunction in Parkinson’s disease. Mitochondrion 7, 58–62. Hashimoto, T., Nishi, K., Nagasao, J., Tsuji, S., Oyanagi, K., 2008. Magnesium exerts both preventive and ameliorating effects in an in vitro rat Parkinson disease model involving 1-methyl-4-phenylpyridinium (MPP+) toxicity in dopaminergic neurons. Brain Res. 1197, 143–151. Hashizume, N., Mori, M., 1990. An analysis of hypermagnesemia and hypomagnesemia. Jpn. J. Med. 29, 368–372. Koenig, J.H., Ikeda, K., 1996. Synaptic vesicles have two distinct recycling pathways. J. Cell Biol. 135, 797–808. Mori, A., Ohashi, S., Nakai, M., Moriizumi, T., Mitsumoto, Y., 2005. Neural mechanisms underlying motor dysfunction as detected by the tail suspension test in MPTP-treated C57BL/6 mice. Neurosci. Res. 51, 265–274. Mori, H., Masaki, H., Yamakura, T., Mishina, M., 1992. Identification by mutagenesis of a Mg(2+)-block site of the NMDA receptor channel. Nature 358, 673–675.
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