Biosci. Biotechnol. Biochem., 77 (2), 266–270, 2013

Combined Low Calcium and Lack Magnesium Is a Risk Factor for Motor Deficit in Mice

y Ryoo TANIGUCHI,1 Osamu NAKAGAWASAI,1; Koichi TAN-NO,1 Fumihiro YAMADERA,1 Wataru NEMOTO,1 Shoko SATO,1;2 Fukie YAOITA,1 and Takeshi TADANO1;3

1Department of Pharmacology, Tohoku Pharmaceutical University, Sendai 981-8558, Japan 2Department of Pharmaceutical Sciences, Tohoku Pharmaceutical University, Sendai 981-8558, Japan 3Laboratory of Environmental and Health Sciences, College of Medical Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa 920-1192, Japan

Received August 28, 2012; Accepted October 18, 2012; Online Publication, February 7, 2013 [doi:10.1271/bbb.120671]

The populations of the Kii Peninsula in Japan and of and abnormal deposits have been observed in the brains Guam present high incidences of amyotrophic lateral of patients with PD.1,2) Of particular interest is the sclerosis and Parkinsonism-dementia complex. It is higher incidence of PD and amyotrophic lateral selerosis thought that low levels of calcium (Ca) and magnesium (ALS) among the Auyu and Jakai people on the southern (Mg) in the drinking water are involved in the patho- coastal plain of West New Guinea and the Japanese genesis of these diseases. The present study aimed to test residents of the Kii Peninsula which is attributed to a the hypothesis that catalepsy, behavioral immobility and marked deficiency of Ca and Mg in the soil and water of a Parkinsonian symptom results from functionally these regions.3) Likewise, a survey of Guam also impaired dopaminergic neurons in mice fed low suggested that low Ca and Mg (LCa/Mg) and high amounts of Ca and Mg (LCa/Mg). A group of mice aluminum (Al) and manganese (Mn) in river, soil, and fed a LCa/Mg diet for 6 weeks was compared to a drinking water might be responsible for the higher control group on a standard diet. Cataleptic symptoms incidence of PD in that region.4) Furthermore, a LCa/ such as akinesia and rigidity were measured by the bar Mg diet has been found to accelerate the deposition of test. The anti-parkinsonian drugs dopamine (DA) pre- Mn and Al in the brain.5) Although numerous studies cursor L-3,4-dihydroxy phenylamine (L-DOPA), the have investigated the role of Mg in the etiology of 3,6) selective DA receptor D2 agonist bromocriptine, and clinical and experimental PD, reports on animal the DA releaser amantadine were tested for their effects behavior after LCa/Mg feeding are scarce. The present on induced catalepsy. The mice developed catalepsy study aimed to test the hypothesis that catalepsy can after 3 weeks on the LCa/Mg diet. LCa/Mg diet- result from damage to DA-ergic neurons in LCa/Mg fed induced catalepsy was improved by the administration mice. Accordingly, we examined the effects of DA-ergic of L-DOPA (50–200 mg/kg i.p.) in combination with drugs on the catalepsy induced by LCa/Mg in mice and benserazide (25 mg/kg i.p.), or of bromocriptine (0.25– also intensity tyrosine hydroxylase (TH) immunofluo- 4 mg/kg i.p.) or of amantadine (5–20 mg/kg i.p.). rescence in the SN. Immunohistochemical staining revealed that the inten- sity of tyrosine hydroxylase fluorescence was signifi- Materials and Methods cantly decreased in the substantia nigra at the 6th week of LCa/Mg feeding in comparison with pair-fed con- Animals. Male ddY mice (Japan SLC, Hamamatsu, Japan) weighing trols. These results suggest that catalepsy in LCa/Mg 10 g (post-natal days 20–21) at the beginning of the experiment, were mice results from hypofunction of the dopaminergic used. The animals were housed in plastic cages (31 cm 21 cm neurons. Moreover, our results support the hypothesis 13 cm) under conditions of constant temperature (23 1 C), humidity that LCa/Mg intake is one etiological factor in neuro- (55 5%), and light-dark cycle (light from 9 to 21 h; dark from 21 to degenerative disorders, including Parkinson’s disease. 9 h). The mice had free access to food and water throughout the experimental period. They were divided into three dietary groups for 6 weeks: (i) a low Ca and Mg deficient diet (LCa/Mg) group, (ii) a Key words: calcium; catalepsy; dopamine; magnesium pair-fed (PF) group for LCa/Mg group, and (iii) a control group. The control group had access to a normal diet ad libitum (standard mineral Parkinson’s disease (PD) is characterized by a loss of diet: CLEA Japan, Tokyo). The PF group was also given the standard dopaminergic neurons in the substantia nigra (SN) diet, the amount of food was matched to that of the corresponding accompanied by a reduction in striatal dopamine (DA). deficient diet group (i). The PF groups were included to control for the The etiology of the disease remains unclear, but it is now effects of anorexia. The deficient diet group (i) was given distilled and deionized milli-Q water to drink. All the diet ingredients (CLEA widely accepted that many factors are involved in the Japan) are listed in Table 1. All experiments were performed following development of this neuropathology. Certain metals the Guide for the Care and Use of Laboratory Animals of Tohoku have been suggested to play roles, as imbalanced levels Pharmaceutical University.

y To whom correspondence should be addressed. Tel/Fax: +81-22-727-0123; E-mail: [email protected] Abbreviations: LCa/Mg, low Ca and Mg; DA, dopamine; L-DOPA, L-3,4-dihydroxy phenylamine; PD, Parkinson’s disease; SN, substantia nigra; ALS, amyotrophic lateral selerosis; Al, aluminum; Mn, manganese; TH, tyrosine hydroxylase; PF, pair-fed; i.p., intraperitoneally; ANOVA, analysis of variance; SEM, standard error of the mean; MPPþ, 1-methyl-4-phenylpyridinium; NMDA, N-methyl-D-aspartate Low Calcium and Magnesium as Risk Factor 267 Drugs. All drugs were purchased from Sigma Chemical (St. Louis, previous reports.7,8) The TH antibody (diluted 1:200) was applied to MO). Bromocriptine, L-DOPA, and benserazide were dissolved in each brain slice, which was then incubated at 4 C for 12 h. The 0.5% Tween80. Amantadine was dissolved in saline. Bromocriptine secondary antibody, FITC-labeled anti-rabbit IgG goat serum (diluted (0.25, 1, and 4 mg/kg), L-DOPA (50, 100, and 200 mg/kg) and 1:200), was allowed to react in the dark at room temperature for 3 h. amantadine (5, 10, and 20 mg/kg) were injected 30 min before the The stained sections were mounted in 10% glycerin-PBS and kept at catalepsy test. Benserazide (25 mg/kg) was injected 30 min before 4 C in a dark room until measurements were done. The distribution of L-DOPA. All drug injections were performed intraperitoneally (i.p.). TH was analyzed quantitatively using a modified brain mapping analyzer system (Yamato Scientific, Tokyo). The background value, Catalepsy test. The bar test was used to measure cataleptic including nonspecific fluorescence originating from glutaraldehyde, symptoms, including akinesia and rigidity. Catalepsy was evaluated was subtracted photometrically from the total fluorescence intensities by placing both forepaws of the mouse on a horizontal bar (diameter obtained for the region of interest. The resulting values were compared 0.2 cm) elevated 5 cm from the floor. The degree of catalepsy was to those of the standard, 1 mM quinine sulphate.7) assessed by measuring the time from placement of the forepaws to removal of one of them (descent latency), with a cut-off time of 180 s. Statistical analysis. Significance of differences was determined by The anti-Parkinsonian drugs bromocriptine (a DA receptor D2 Student’s t-test for two-group comparison. Analysis of variance agonist), L-DOPA (a precursor of DA), and amantadine (a DA (ANOVA) was used for multiple comparisons. Post hoc analysis was releaser) were tested at the 6th week in the cataleptic LCa/Mg mice. performed by Fisher’s PLSD test. The criterion of significance was set The duration of catalepsy was measured 0.5, 1, 2 and 24 h after i.p. at p < 0:05. All results were expressed as mean standard error of the administration of a vehicle, bromocriptine (0.25–4 mg/kg) (Fig. 3), mean (SEM). benserazide (25 mg/kg) + L-DOPA (50–200 mg/kg) (Fig. 4), or amantadine (5–20 mg/kg) (Fig. 5). Results Immunohistochemical procedures. Behavioral testing was followed by immunohistochemical analysis of TH. The mice were anesthetized Growth curves and mortality rate with sodium pentobarbital (50 mg/kg i.p.; Dainippon, Osaka, Japan) The mice on the standard mineral and the exper- perfused through the heart with ice-cold phosphate-buffered saline imental diets were housed individually for food intake (PBS, pH 7.4), followed immediately by a fixative containing 4% assessment. As shown by the growth curves in Fig. 1, paraformaldehyde (Sigma) and 0.2% glutaraldehyde (Nacalai Tesque, despite the LCa/Mg diet, the average body weight at the Osaka, Japan) in PBS. The brains were then post-fixed with the same fixative solution at 4 C for 1 h, and placed in 10% sucrose solution at end of the first week had increased steadily to about 4 C for 12 h. The tissue was frozen on dry ice and sliced into 20-mm 14 g, similarly to what was observed for the mice in the sections on a cryostat (Leitz, Stuttgart, Germany). Two to four brain PF group. The LCa/Mg mice showed aphagia from the sections per animal containing the SN were used in analysis. The 2nd week onwards (Table 2). Accordingly, the weight of immunohistochemical staining procedure was performed following these mice started to plateau and to differ significantly from the PF group, which continued to increase (Fig. 1). In the LCa/Mg group, death occurred at 3.3% on the Table 1. Compositions of Experimental Diets 21st d, at 6.6% on the 28th d, and at 8.3% on the 35th– Milk casein 24.5% 42nd d, while no deaths occurred in the PF group. Cornstarch 45.5% Corn oil 6.0% Influence of low Ca and Mg deficiency on the Caster sugar 10.0% catalepsy results Avicel 3.0% A significant increase in the duration of catalepsy was Cellulose powder 2.0% observed from the 3rd to the 6th week in the mice fed Alpha starch 1.0% the LCa/Mg diet. No Catalepsy was observed in the PF Vitamin mix 1.0% Mineral mix 7.0% group (Fig. 2).

Mineral content (mg/100 g) Control LCa/Mg 25 PF (n = 20) CaCO3 1355.40 0.00 LCa/Mg (n = 20) 20 KH2PO4 1730.00 1730.00 NaCl 600.00 600.00 15 ** ** ** FeC6H5O7.5H2O 190.00 190.00 * ** 5ZnO2CO.4H2O 6.00 6.00 10 CuSO4.5H2O 1.26 1.26 Body weight (g) 5 CoCl2.6H2O 0.40 0.40 Ca(IO3)2 1.54 1.54 0 MnSO4.4H2O 15.40 15.40 1 7 14 21 28 35 42 CaHPO4.2H2O 1500.00 0.00 Days MgSO4.7H2O 800.00 0.00 Na2HPO4 0.00 1240.00 Fig. 1. Growth Curves for the LCa/Mg Diet Group. Cornstarch 800.00 3215.40 Vertical bars represent standard errors of the mean (SEM). p < 0:05; p < 0:01 as compared with the PF groups.

Table 2. Effects of LCa/Mg on Food Intake in Mice

Group/Day 1st 7th 14th 21st 28th 35th 42nd Normal diet (n ¼ 20) 1.24 g 3.22 g 3.84 g 4.43 g 4.22 g 4.32 g 4.26 g LCa/Mg (n ¼ 20) 1.5 g 1.95 g 2.84 g 2.37 g 2.56 g 2.50 g 2.44 g 268 R. TANIGUCHI et al.

60 PF (n = 20) 50 LCa/Mg (n = 20) **

40 ** 30 ** 20 **

10

Duration of catalepsy (s) 0 0 1 2 3 4 5 6 Weeks

Fig. 2. Effects of the LCa/Mg Diet on Catalepsy in Mice. Vertical bars represent SEM. p < 0:01 as compared with the PF groups. Fig. 5. Inhibitory Effect of Amantadine on Catalepsy Induced by 6 Weeks of the LCa/Mg Diet in Mice. Vertical bars represent SEM. p < 0:05; p < 0:01 vs. vehicle.

a High

PF b

Low

LCa/Mg c 600 Fig. 3. Inhibitory Effect of Bromocriptine on Catalepsy Induced by 6 Weeks of the LCa/Mg Diet in Mice. 500 Vertical bars represent SEM. p < 0:05; p < 0:01 vs. vehicle. 400 **

300

200

L-DOPA 100 L-DOPA

Fluorescence intensity of TH Fluorescence intensity of 0 L-DOPA PF (n = 10) LCa/Mg (n = 10)

Fig. 6. Distribution and Evaluation of the Immunohistochemical Fluorescence Intensity of TH in the SN of Mice at the 6th Week of Diet Feeding. Representative images for PF mice (a) and LCa/Mg mice (b). (c) Values for the immunofluorescence intensity of TH on the SN. Vertical bars represent SEM. p < 0:01 vs. PF.

in Fig. 6, TH fluorescence intensity was significantly lower in the SN of the mice with catalepsy induced by

Fig. 4. Inhibitory Effect of Combined L-DOPA and Benserazide on the LCa/Mg diet in comparison with the PF group. Thus Catalepsy Induced by 6 Weeks of the LCa/Mg Diet in Mice. the catalepsy induced by LCa/Mg intake appeared to be Vertical bars represent SEM. p < 0:05; p < 0:01 vs. vehicle. related to the decrease in TH levels in the SN. Discussion Effects of antiparkinsonian drugs on catalepsy in the LCa/Mg mice The LCa/Mg diet led to a decrease in daily food The duration of catalepsy improved significantly and intake in the mice, and accordingly to markedly reduced peaked by 1 h after bromocriptine injection (1–4 mg/kg) growth. Several studies have reported reductions in the (Fig. 3) and by 0.5 h after L-DOPA (50–200 mg/kg) levels of serum total protein and albumin in rats fed (Fig. 4) and after amantadine injection (10–20 mg/kg) Mg-deficient diets.9,10) These results suggest that a (Fig. 5) before returning to the control levels. Mg-deficient diet influences protein metabolism. With regard to growth in the mice, the reductions in body TH immunofluorescence intensity in the SN of the weight and food intake might have been due to Mg LCa/Mg mice deficiency. The LCa/Mg group showed prolonged The distribution of TH in each mouse brain section duration of catalepsy as compared to their PF group was determined by microphotometry 6 weeks after the on the bar test. This is the first report to document start of the experiment, and fluorescence intensities were significant cataleptic behavior in LCa/Mg mice. It is categorized into 8 pseudocolor levels (Fig. 6). As seen well known that reduced DA activity causes Parkinso- Low Calcium and Magnesium as Risk Factor 269 nian symptoms such as rigidity and akinesia in humans. important human pathologies, ranging from ALS to This state manifests itself as catalepsy in rodents. The Alzheimer’s disease, PD, depression, epilepsy, trauma, duration of catalepsy was previously found to increase and schizophrenia.28) The neuroprotective effect of Mg with degeneration of the nigrostriatal DA-ergic neuron is mediated via a reduction in pre-synaptic release of the pathway.11,12) Here, the cataleptic behavior observed in neurotransmitter glutamate29) or a blockade of NMDA the LCa/Mg mice was transiently inhibited in a dose- receptors.30) In a variety of experimental models of PD, dependent manner by administration of the anti-Parkin- many NMDA receptor blockers have shown anti- sonian drugs bromocriptine, L-DOPA, and amantadine. cataleptic activity, including dizocilpine, phencyclidine, TH is the rate-limiting enzyme in catecholamine biosyn- memantine.31–34) In the present study, administration of thesis. It functions as an oxidoreductase, and tetrahy- amantadine, a DA releaser and a non-selective NMDA drobiopterin acts as co-factor converting L-tyrosine to receptor antagonist, also alleviated catalepsy in LCa/Mg L-DOPA. TH is also a marker for DA neurons. To mice. This ameliorating effect might be due not only to evaluate DA-ergic hypofunction in the LCa/Mg mice, amantadine’s induction of DA release but also to its we measured TH levels in the SN, and found a NMDA receptor antagonist properties. This suggests significant decrease in the LCa/Mg mice as compared that LCa/Mg-induced catalepsy is caused by DA-ergic to control. Together, these results suggest that LCa/Mg hypofunction via supersensitivity of NMDA receptors. diet-induced catalepsy is due to hypo-DA-ergic func- Further experiments are needed to determine how LCa/ tion. We propose three mechanisms by which LCa/Mg Mg can lead to the degeneration of SN DA-ergic diet feeding might lead to DA-ergic hypofunction. neurons. First, it is well established that Ca and Mg are In conclusion, our results suggest that catalepsy in essential for synaptic transmission and of central LCa/Mg mice results from hypofunctioning of DA-ergic importance to the biochemistry of the cell and its neurons. Moreover, they indicate that a chronic defi- energy metabolism in particular. These elements are also ciency of environmental factors, such as dietary Ca and cofactors in TH activity that is essential for DA Mg, can contribute to the development of neurodege- synthesis.13) In the LCa/Mg group, the fluorescence nerative disorders such as PD. intensity of TH showed a marked decrease in the SN at the 6th week (Fig. 6). Mg has been found to prevent the Acknowledgments death of DA-ergic neurons induced by 1-methyl-4- phenylpyridinium (MPPþ) in an experimental model of This study was supported in part by Grants-in-Aid for PD,14,15) as MPPþ selectively destroys the DA-ergic Scientific Research (22600010) and by the Program for system.15) Thus, Mg deficiency especially might have Strategic Research Foundation at Private Universities been involved to the hypofunction of DA-ergic neurons (2010-2014) from the Ministry of Education, Culture, in the SN of the LCa/Mg mice. Sports, Science, and Technology of Japan. Secondly, it has been reported that an accumulation of pollutant metals such as Mn and Al is caused by References metabolic changes due to a lack of protective factors (e.g., LCa/Mg).5) In this connection, a reduction in 1) Olanow CW and Arendash GW, Curr. Opin. Neurol., 7, 548– blood Ca levels was found to enhance Al absorption.16) 558 (1994). 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