Preventive effect of 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) and zinc, components of the Pacific oyster Crassostrea gigas, on glutamatergic neuron activity in the hippocampus.
The Pacific oyster Crassostrea gigas (Thunberg, 1793) is a marine bivalve that originates from Japan, where it has been farmed since the 1600s. It is the most popular and industrially important oyster in the world, because it grows and spreads easily, and it is environmentally tolerant. The Pacific oyster is enriched with zinc, an essential trace element, and 3,5-dihydroxy-4-methoxybenzyI alcohol (DHMBA), a novel antioxidant (Watanabe et al., 2012a). DHMBA is the major oxygen radical absorbance capacity (ORAC)-positive antioxidant in the ethanol extract of the pressurized, hot-water extract of the Pacific oyster. The Pacific oyster is enriched with antioxidants, and this enrichment is associated with a tolerance to environmental changes (Jo et al., 2008; Watanabe et al., 2012b). Once oysters attach to a substrate, such as a rock, at the fully developed larval stage, they cannot move from that substrate during their lifetime. Therefore, they are more frequently exposed to a variety of natural and anthropogenic stressors than is generally thought, including fluctuations in temperature, salinity, oxygen content, and pollution.
Glutamate is one of the most prominent neurotransmitters in the body, present in over 50% of nervous tissue, and it plays an important role in neuronal excitation (Danbolt, 2001). Multiple abnormal triggers, such as energy deficiency and oxidative stress, can lead to aberration in the neuronal excitation process (Lipton, 2008; Vincent and Mulle, 2009; Mehta et al., 2013). Glutamate excitotoxicity is the pathological process by which neuronal cells are damaged or killed by excess glutamate receptor activation. Such excitotoxic neuronal death has been implicated in neurodegenerative diseases, including stroke, epilepsy, multiple sclerosis, Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, and Huntington's disease, where oxidative stress plays a role in cell damage and cell death (Halliwell, 2006; Quincozes-Santos et at., 2014). N-methyl-D-aspartate (NMDA) receptors, a potential therapeutic target, have been investigated most actively for their role in neurodegenerative pathologies (Gonzalez et al., 2015).
Natural phenolic compounds, such as flavonoids and anthocyanins, have high antioxidant activity (Rice-Evans et al., 1996); some of these compounds are used as nutraceuticals and pharmaceuticals (Dimitrios, 2006). They are mostly extracted and purified from terrestrial plants; marine organisms have not been extensively surveyed (Sakata, 1997). Stress can induce excess glutamatergic neuron activity, followed by oxidative stress. On the basis of the idea that the Pacific oyster is tolerant to stressors, in the present study, we selected DHMBA and zinc as preventive candidates in suppressing glutamatergic neuron activity in the hippocampus. The effect of DHMBA and zinc was assessed in two trials, an in vivo microdialysis experiment and an in vitro brain slice investigation.
Materials and Methods
Synthesis of 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA)
DHMBA was chemically synthesized from methyl gallate by a two-step reaction, as described previously (Watanabe et al., 2012b). Zinc components contained in the Pacific oyster are likely to be absorbed as [Zn.sup.2+] from the gut (Fukada and Kambe, 2011), which, in turn, is transported into the brain via an uknown mechanism (Takeda and Tamano, 2009). Zn[Cl.sub.2] was used to assess the action of zinc components.
Calcium orange AM (Molecular Probes, Inc., Eugene, OR), a membrane-permeable calcium indicator, and ZnAF-2 (Sekisui Medical Co., Ltd., Tokai, Japan), a membrane-permeable zinc indicator, were used in our study. These fluorescent indicators were dissolved in dimethyl sulfoxide (DMSO), then diluted with artificial cerebrospinal fluid (ACSF), which is composed of 124 mmol [1.sup.-1] NaCl, 2.5 mmol [1.sup.-1] KCl, 2.0 mmol [1.sup.-1] [CaCl.sub.2], 1.0 mmol [1.sup.-1] [MgCl.sub.2], 1.25 mmol [1.sup.-1] [NaH.sub.2][PO.sub.4], 26 mmol [1.sup.-1] [NaHCO.sub.3], and 10 mmol [1.sup.-1] D-glucose (pH 7.3). To facilitate cellular uptake of the indicators, Cremophor EL (EMD Millipore Corp., Billerica, MA) was added to the DMSO solutions (final concentration, 0.02%).
Experimental animals and diet
Male Wistar rats (6-wk-old) and male ddY mice (7-wk-old) (Japan SLC, Hamamatsu, Japan) were maintained under standard laboratory conditions (23 [+ or -] 1 [degrees]C; 55% [+ or -] 5% humidity), with free access to water and food (control diet, MF; Oriental Yeast Co., Ltd., Yokohama, Japan). Zinccontent in the animals' drinking water and diet was < 1.0 mg Zn/kg and 52.8 mg Zn/kg, respectively. Lights in the laboratory were automatically turned on at 8:00 h and turned off at 20:00 h. All experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the University of Shizuoka that refer to the American Association for Laboratory Animals Science and the NIH Guide for the Care and Use of Laboratory Animals in the U.S.
In vivo microdialysis
Rats were anesthetized with chloral hydrate (400 mg/kg) and individually placed in a stereotaxic apparatus. The skull was exposed, a burr hole was drilled, and a guide tube was implanted into the right hippocampus according to the three brain map coordinates, the anterior-posterior (AP), -5.6 mm; medial-lateral (ML), +5.2 mm; and dorsal-ventral (DV), +6.6 mm. The guide tube was secured with dental cement and screws. After the surgical operation, each rat was housed individually.
Three or four days after implantation of the guide tube, a microdialysis probe (3-mm membrane; Eicom, Kyoto, Japan) was inserted into the hippocampus and through the guide tube of chloral hydrate-anesthetized rats. The hippocampus was pre-perfused with ACSF (127 mmol [1.sup.-1] NaCl, 2.5 mmol [1.sup.-1] KCl, 1.3 mmol [1.sup.-1] [CaCl.sub.2], 0.9 mmol [1.sup.-1] [MgCl.sub.2], 1.2 mmol [1.sup.-1] [Na.sub.2]H[PO.sub.4], 21 mmol [1.sup.-1] [NaHCO.sub.3], 3.4 mmol [1.sup.-1] D-glucose, pH 7.3) at 4.0 [micro]l/min for 140 min to stabilize the region; perfused for 25 min in the same manner to determine the basal concentrations of glutamate in the extracellular fluid; and perfused with 100 mmol [1.sup.-1] KCl for 5 min to determine the change in extracellular glutamate concentration induced by neuronal depolarization.
In another experiment, the hippocampus was perfused with ACSF for 25 min to determine the basal concentrations of glutamate in the extracellular fluid; then perfused with 1 mmol [1.sup.-1] DHMBA in ACSF or 10 [micro]mol [1.sup.-1] Zn[Cl.sub.2] in ACSF for 25 min; and, finally, perfused with 1 mmol [1.sup.-1] DHMBA in ACSF containing 100 mmol [1.sup.-1] KCl or 10 [micro]mol [1.sup.-1] Zn[Cl.sub.2] in ACSF containing 100 mmol [1.sup.-1] KCl, respectively, for 5 min. Perfusate was collected every 5 min.
The perfusate samples were analyzed for glutamate content by high-performance liquid chromatography (HPLC) (column, CAPCELL PAK C18 UG120A [1 mm X 150 mm]; Shiseido Co., Ltd., Tokyo, Japan); mobile phase, 0.1 mol [1.sup.-1] potassium dihydrogen phosphate, 0.1 mol [1.sup.-1] di-sodium hydrogen phosphate, 10% acetonitrile, 0.5 mmol [1.sup.-1] EDTA-2Na,
3% tetrahydrofuran, and pH 6.0) using the pre-column derivatization technique with o-phthaldialdehyde (OPA) and a fluorescence detector (NANOSPACE SI-2; Shiseido Co., Ltd.).
Brain slice experiment
Mice were anesthetized with diethyl ether and decapitated approximately 7 d after purchase. Each brain was quickly removed and immersed in ice-cold choline-ACSF containing 124 mmol [1.sup.-1] choline chloride, 2.5 mmol [1.sup.-1] KCl, 2.5 mmol [1.sup.-1] MgCL, 1.25 mmol [1.sup.-1] [NaH.sub.2][PO.sub.4], 0.5 mmol [1.sup.-1] CaCL, 26 mmol [1.sup.-1] [NaHCO.sub.3], and 10 mmol [1.sup.-1] glucose (pH 7.3) to suppress excessive neuronal excitation. Horizontal brain slices (400 [micro]m) were prepared using a vibratome ZERO-1 fresh-tissue sectioning system (Dosaka, Kyoto, Japan) in ice-cold choline-ACSF. Slices were then maintained in ACSF (124 mmol [1.sup.-1] NaCl, 2.5 mmol [1.sup.-1] KCl, 2.0 mmol [1.sup.-1] [CaCl.sub.2], 1.0 mmol [1.sup.-1] [MgCl.sub.2], 1.25 mmol [1.sup.-1] [NaH.sub.2][PO.sub.4], 26 mmol [1.sup.-1] [NaHCO.sub.3], and 10 mmol [1.sup.-1] D-glucose (pH 7.3)), 0.1-1.0 [micro]mol [1.sup.-1] Zn[Cl.sub.2] in ACSF, or 0.F1.0 mmol [1.sup.-1] DHMBA in ACSF at 25 [degrees]C for 1 h. All solutions used in the experiments were continuously bubbled with 95% [O.sub.2] and 5% C[O.sub.2].
To assess [Zn.sup.2+] release as an index of glutamate release from neuron terminals, the brain slices immersed in ACSF or 0.1-1.0 mmol [1.sup.-1] DHMBA in ACSF were transferred to a recording chamber filled with 10 [micro]mol [1.sup.-1] ZnAF-2. In another experiment, the brain slices immersed in 0.1-1.0 [micro]mol [1.sup.-1] ZnCL in ACSF were transferred to a chamber filled with ACSF for 15 min to wash out the extracellular ZnCL; then transferred to a recording chamber filled with 10 [micro]mol [1.sup.-1] ZnAF-2. ZnAF-2 fluorescence (excitation, 488 nm; monitoring, 505-530 nm) was measured using a confocal laser-scanning microscopic system (LSM 510; Carl Zeiss AG, Oberkochen, Germany), equipped with an inverted microscope (Axiovert 200M, Carl Zeiss AG), at a rate of 1 Hz through a 10X objective. The region of interest was set in the stratum lucidum, where mossy fiber terminals exist. Basal ZnAF-2 fluorescence was measured for 30 s, and changes in ZnAF-2 fluorescence were measured 150-180 s after the addition of KCl (final concentration, 50 mmol [1.sup.-1]).
To assess intracellular [Ca.sup.2+] levels, the brain slices immersed in ACSF containing DHMBA or Zn[Cl.sub.2] were placed in 5 [micro]mol [1.sup.-1] calcium orange for 30 min, transferred to a chamber filled with ACSF for 15 min to wash out the extracellular calcium orange, then moved to a recording chamber filled with ACSF. Calcium orange fluorescence (excitation, 543 nm; monitoring > 560 nm) was measured in the stratum lucidum with a confocal laser-scanning microscopic system (LSM 510 META; Carl Zeiss AG). Basal calcium orange fluorescence was measured for 30 s, and changes in this fluorescence were measured 150-160 s after the addition of KCl (final concentration, 50 mmol [1.sup.-1]).
Student's t-test was used for comparison of the means of paired and unpaired data. For multiple comparisons, differences between treatments were assessed by one-way ANOVA followed by post hoc testing using Dunnett's test (GraphPad Prism 5; Graph Pad Software, La Jolla, CA). A P-value < 0.05 was considered significant. Data are expressed as means [+ or -] standard error of the mean (SEM). The results of the statistical analysis are described in each figure legend.
Effect of DHMBA and [Zn.sup.2+] on extracellular glutamate levels
The influence of DHMBA and zinc on extracellular glutamate levels was assessed by an in vivo microdialysis experiment. When the hippocampus was perfused with ACSF, the basal concentration of extracellular glutamate was decreased in the presence of DHMBA (1 mmol [1.sup.-1]: ACSF, 100% [+ or -] 0.78%; DHMBA, 78.4% [+ or -] 5.7%) or Zn[Cl.sub.2] (10 [micro]mol [1.sup.-1]: ACSF, 100% [+ or -] 1.2%; Zn, 75.4% [+ or -] 7.7%) in the perfusate (Figs. 1, 2). Significant increases in extracellular glutamate induced with high [K.sup.+] were observed, even in the presence of DHMBA (ACSF, 130% [+ or -] 2.5%; DHMBA, 122% [+ or -] 8.5%) or Zn[Cl.sub.2] (ACSF, 138% [+ or -] 15%; Zn, 102% [+ or -] 16%) in the perfusate. However, the extracellular glutamate concentration tended to be low in the presence of DHMBA or Zn[Cl.sub.2].
Effect of DHMBA and [Zn.sup.2+] on glutamate release from neuron terminals
Extracellular glutamate levels are determined by glutamate release from neuron terminals, and glutamate reuptake by glutamate transporters. All hippocampal mossy fibers contain zinc in presynaptic vesicles, and [Zn.sup.2+] is co-released with glutamate (Frederickson, 1989; Frederickson and Danscher, 1990). Thus, the extracellular [Zn.sup.2+] level was used as an index of glutamate release from neuron terminals. Brain slices were pretreated with DHMBA (0.1-1 mmol [1.sup.-1]) or Zn[Cl.sub.2] (0.1-1 [micro]mol [1.sup.-1]) for 1 h, then stimulated with high [K.sup.+] after washing them out. A high, [K.sup.+]-induced increase in extracellular [Zn.sup.2+] level, which was determined with ZnAF-2, was suppressed with pretreatment with DHMBA (1 mmol [1.sup.-1]) or Zn[Cl.sub.2] (0.1-1 [micro]mol [1.sup.-1]) (Figs. 3, 4).
Effect of DHMBA and [Zn.sup.2+] on intracellular [Ca.sup.2+] level
An increase in intracellular [Ca.sup.2+] in presynaptic terminals triggers glutamate exocytosis, followed by postsynaptic neuron excitation, which induces an increase in intracellular [Ca.sup.2+] in postsynaptic neurons. Thus, the increase in intracellular [Ca.sup.2+] was measured in the brain slices, which were pretreated with DHMBA (1 mmol [1.sup.-1]) or Zn[Cl.sub.2] (100 nmol [1.sup.-1]) for 1 h, then stimulated with high [K.sup.+]. A high, [K.sup.+]-induced increase in intracellular [Ca.sup.2+] level, which was determined with calcium orange, was also suppressed with pretreatment with DHMBA or Zn[Cl.sub.2] (Figs. 5, 6).
Relatively few studies have described antioxidants from oysters. Many papers have focused on antioxidant enzymes or proteins, such as superoxide dismutase (Green et al.,2009) and metallothionein (Tanguy and Moraga, 2001), which are zinc-binding proteins. Zinc has a role in protecting against oxidative stress (Ruttkay-Nedecky et al., 2013), and metallothioneins play a key part in protecting against this stress, even in oysters (Luo et al., 2014). The potential roles of metallothionein as a therapeutic target are reported for cerebral ischemia and retinal diseases (Ito et al., 2013). Zinc strongly induces metallothioneins in the body, including the brain (Singla and Dhawan, 2014). Zinc might be a candidate antioxidant in the form of metallothioneins, and serve to protect the brain from oxidative stress. The content of zinc is approximately 13.2 mg/100 g wet weight of whole oyster meat. Zrt-Irt-like proteins (ZIP) and the zinc transporter (ZnT) family are responsible for the absorption of zinc from the gut in the form of [Zn.sup.2+] and cellular [Zn.sup.2+] uptake (Fukada and Kambe, 2011). Thus, Zn[Cl.sub.2] was used to assess preventive effects against excess glutamatergic neuron activity, which is induced under stressful circumstances. On the other hand, the content of DHMBA, a novel antioxidant, is approximately 6.7 mg/100 g wet weight of whole oyster meat. DHMBA is amphiphilic, and might be a candidate antioxidant to protect the brain from oxidative stress (Watanabe et al., 2012b). In the present study, we hypothesized that DHMBA and zinc preventively suppressed glutamatergic neuron activity in the hippocampus.
The basal concentration of extracellular glutamate in the hippocampus was decreased under hippocampal perfusion with DHMBA (1 mmol [1.sup.-1]) or Zn[Cl.sub.2] (10 [micro]mol [1.sup.-1]), suggesting that DHMBA and [Zn.sup.2+] suppress glutamatergic neuron activity under these (static) conditions, perhaps via suppression of presynaptic activity (Fig. 7). An abnormal increase in extracellular glutamate induced with high [K.sup.+], which is observed in zinc-deficient rats, is suppressed in the presence of 100 [micro]mol [1.sup.-1] Zn[Cl.sub.2] (Takeda et al., 2008a). The increase in extracellular glutamate induced with high [K.sup.+] was not significantly suppressed under hippocampal perfusion with DHMBA (1 mmol [1.sup.-1]) or Zn[Cl.sub.2] ([micro]mol [1.sup.-1]). Thus, we pursued the preventive effect of DHMBA and [Zn.sup.2+] on excess glutamate release from neuron terminals. Brain slices were pretreated with DHMBA (1 mol [1.sup.-1]) or Zn[Cl.sub.2] (100 nmol [1.sup.-1]) for 1 h, then stimulated with high [K.sup.+]. High, [K.sup.+]-induced increases in the extracellular [Zn.sup.2+] level-an index of glutamate release, which was determined with ZnAF-2 (Minami et al., 2006)-were suppressed with pretreatment with DHMBA or Zn[Cl.sub.2]. This finding suggests that DHMBA and [Zn.sup.2+], previously taken up in the hippocampal cells, suppressed high, [K.sup.+]-induced glutamate exocytosis. Glutamate exocytosis from neuron terminals, which is determined with FM4-64, an indicator of presynaptic activity, was enhanced in the hippocampal slices pretreated with 50 mmol 1 pyrithione, a membrane-permeable zinc chelator, which reduced intracellular [Zn.sup.2+] levels (Minami et al., 2006). Glutamate exocytosis at hippocampal mossy fiber boutons is enhanced in zinc-deficient rats (Takeda et al., 2009). In another experiment, synaptic [Zn.sup.2+] release was also reduced in zinc-deficient rats (Takeda et al., 2008b). A high, [K.sup.+]-induced increase in intracellular [Ca.sup.2+] level, which was determined with calcium orange, was also suppressed with pretreatment with DHMBA or Zn[Cl.sub.2]. The suppression may occur in both presynaptic and postsynaptic neurons.
These results suggest that DHMBA and [Zn.sup.2+], previously taken up in hippocampal cells, suppress high, [K.sup.+]-induced glutamate release in the hippocampus, probably via presynaptic suppression of intracellular [Ca.sup.2+] signaling (Fig. 7). The discrepancy noted between the in vivo microdialysis and the in vitro slice experiments might have been the result of a difference in the action mechanism between direct perfusion and pretreatment. In contrast, an excessive increase in intracellular [Zn.sup.2+], which is induced by abnormal excitation of glutamatergic synapses in the hippocampal CA1, leads to cognitive decline (Takeda et al., 2011).
The preventive effect of [Zn.sup.2+] on the suppression of glutamatergic neuron activity was observed in pretreatment with lower concentrations of [Zn.sup.2+] than with DHMBA. On the other hand, cell viability was not affected after incubation with 320 [micro]mol [1.sup.-1] of DHMBA for 5 d, suggesting that DHMBA has low cytotoxicity (Watanabe et al., 2012b). During acute brain slice preparation, neurons are exposed to stress circumstances, such as abnormal excitation and oxidative damage (Suh et al., 2000). In acute brain slice experiments, [Zn.sup.2+] and DHMBA are taken up into cells and may reduce cell damage, potentially via anti-stress (anti-oxidative) action. This action might be involved in the preventive effect of [Zn.sup.2+] and DHMBA. It is possible that appropriate intake of [Zn.sup.2+] and DHMBA is of benefit in reducing oxidative damage associated with excess glutamatergic neuron activity. Our study showed that [Zn.sup.2+] and DHMBA may have a preventive effect in suppressing excess glutamate release in the hippocampus. Further investigation into the mechanism that suppresses glutamatergic neuron activity also is necessary.
We thank Watanabe Oyster Laboratory Co., Ltd., for funding support. We also thank Sekisui Medical Co., Ltd., Tokai, Japan, for donating the membrane-permeable zinc indicator, ZnAF-2, that was used in our study.
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HARUNA TAMANO (1), YUKINA SHAKUSHI (1), MITSUGU WATANABE (2), KAZUMI OHASHI (1), CHIHIRO UEMATSU (1), TADAMUNE OTSUBO (3), KIYOSHI IKEDA (3), AND ATSUSHI TAKEDA (1,*)
(1) Department of Neurophysiology, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan; (2) Watanabe Oyster Laboratory Co. Ltd., 490-3, Shimo-ongata-cho, Hachioji 190-0154, Japan; and (3) Department of Organic Chemistry, School of Pharmaceutical Sciences, Hiroshima International University, Kure 737-0112, Japan
Received 11 May 2015: accepted 28 September 2015.
(*) To whom correspondence should be addressed. E-mail: email@example.com Abbreviations: ACSF. artificial cerebrospinal fluid: DHMBA, 3,5-dihydroxy-4-methoxybenzyl alcohol; DMSO, dimethyl sulfoxide; NMDA, N-methyl-D-aspartate.
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|Author:||Tamano, Haruna; Shakushi, Yukina; Watanabe, Mitsugu; Ohashi, Kazumi; Uematsu, Chihiro; Otsubo, Tadam|
|Publication:||The Biological Bulletin|
|Date:||Dec 1, 2015|
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