Manganese exposure alters the expression of N-methyl-d-aspartate receptor subunit mRNAs and proteins in rat striatum

J BIOCHEM MOLECULAR TOXICOLOGYVolume 24, Number 1, 2010

Manganese Exposure Alters the Expressionof N-Methyl-D-aspartate Receptor Subunit mRNAsand Proteins in Rat StriatumBin Xu, Zhao-Fa Xu, and Yu DengDepartment of Environmental Health, School of Public Health, China Medical University, North 2nd Road 92, Heping ward, Shenyang,Liaoning 110001, People’s Republic of China; E-mail: [email protected]

Received 23 March 2009; revised 13 May 2009; accepted 28 May 2009

ABSTRACT: Manganese is one of the ubiquitousenvironmental pollutants that can induce an indirectexcitotoxicity caused by altered glutamate (Glu)metabolism. The present study has been carried outto investigate the effect of Mn on the expressionof N-methyl-DD-aspartate receptor (NR) subunit mR-NAs and proteins in rat striatum when rats werein manganism. The rats were divided randomlyinto four groups of six males and six females each:control group (group 1) and 8, 40, and 200 μμmol/kgMn-treated groups (groups 2–4). The control grouprats were subcutaneously (s.c.) injected with normalsaline. Manganese-treated rats were s.c. injected withrespectively 8, 40, and 200 μμmol/kg of MnCl2 · 6H2Oin normal saline. The administration of MnCl2 · 6H2Ofor 4 weeks significantly increased Mn concentrationin the striatum. With the increase in administeredMnCl2 dosage, Glu concentration and cell apoptosisrate increased significantly. The relative intensity ofNR2A mRNA decreased significantly in 8 μμmol/kgMn-treated rats. However, relative intensities ofNR1 and NR2B mRNAs decreased significantly in40 μμmol/kg Mn-treated rats. Similarly, the relativeintensity of NR2A protein showed a significant de-crease in 40 μμmol/kg Mn-treated rats whereas those ofNR1 and NR2B decreased significantly in 200 μμmol/kgMn-treated rats. Therefore, the expression of NR2AmRNA and protein were much more sensitive toMn than that of NR1 and NR2B. In conclusion, theresults suggested that Mn induced nerve cell damageby increasing extracellular Glu level and alteredexpression of NR subunit mRNAs and proteins in ratstriatum. C© 2010 Wiley Periodicals, Inc. J Biochem

Correspondence to: Zhao-Fa Xu.Contract Grant Sponsor: The National Natural Science Founda-

tion of China.Contract Grant Number: 30771834.Contract Grant Sponsor: The Research Program of Higher Edu-

cation Foundation of Liaoning Province, People’s Republic of China.Contract Grant Number: 2004C025.

c© 2010 Wiley Periodicals, Inc.

Mol Toxicol 24:1–9, 2010; Published online inWiley InterScience (www.interscience.wiley.com).DOI 10:1002/jbt.20306

KEYWORDS: Manganese; Glutamate; NMDA Receptors;Neurotoxicity

INTRODUCTION

Manganese, an essential nutrient functioning as acritical cofactor for many key enzymes in the body,is required for proper immune function, regulationof metabolism, reproduction, digestion, bone growth,and blood clotting [1]. However, exposure to high con-centrations of Mn is known to result in neurotoxic-ity. Manganese neurotoxicity, or manganism, initiallycharacterized by a psychiatric disorder resemblingschizophrenia, shares neuropathologies with severalclinical disorders, in particular Parkinson’s disease [2].Manganese intoxication occurs from occupational ex-posures [3], administration of total parenteral nutrition[4], and chronic liver failure [5]. Manganese exposurehas also focused on the use of a Mn-containing fueladditive, methylcyclopentadienyl manganese tricar-bonyl, as an antiknock agent in gasoline in Canada andother Western nations [6]. Pathology in manganism ischaracterized by neuronal loss and marked Alzheimertype II astrocytosis, particularly in the globus pallidus,and also in the putamen, caudate nucleus, and sub-stantia nigra pars reticulate [2]. Although the globuspallidus is a primary target in Mn neurotoxicity, datafrom both primates and humans indicate that the stria-tum is also vulnerable [7].

Mutkus et al. [8] reported that Mn-mediated in-hibition of glutamate (Glu) transport in the CHO-K1 cell line DdB7 was pronounced in both the Glu

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2 XU, XU, AND DENG Volume 24, Number 1, 2010

transporter (GLT-1)- and Glu/aspartate transporter(GLAST)-transfected cells. These studies suggest thatMn accumulation in the central nervous system mightcontribute to the dysregulation of Glu homeostasis. In-terestingly, this finding is consistent with other stud-ies reporting that Mn alters the metabolism of cellularGlu [9]. Collectively, these observations suggest thatincreased extracellular Glu induced by decreased as-trocytic Glu uptake, or by enhanced neuronal release,might lead to excitotoxicity in postsynaptic neuronsexpressing Glu receptors [10].

N-Methyl-D-aspartate (NMDA) receptors (NRs)are one of the major classes of ionotropic Glu recep-tors. NMDA receptors are composed of heteromericsubunits and there are three distinct classes of NR sub-units, that is, NR1 with eight splice variants, NR2A-NR2D, and NR3, respectively, that are encoded fromdistinct genes. Native NMDA receptors in neurons areassumed to be composed of NR1 subunit, with at leastone of the NR2 and/or NR3 subunits. The NR1 subunitis assumed to form the ion channel and NR2 subunitsare assumed to have modulatory roles on the chan-nel activities [11]. Different NR2 subunits confer dis-tinct electrophysiological and pharmacological prop-erties on the receptors and couple them with differentsignaling machineries. For instance, NR2A- and NR2B-containing NMDA receptor subtypes have opposingroles in influencing the direction of synaptic plasticity[12]. Moreover, Liu et al. [13] observed that NR2A-and NR2B-containing NMDA receptors had differen-tial roles in supporting neuronal survival and mediat-ing neuronal death and hence had opposing impactson excitotoxic brain damage after acute brain insultssuch as stroke and brain trauma. Cano et al. [14] foundthat chronic treatment of mice with MnCl2 during8 weeks could decrease Glu receptor binding sites,which might result in a protection mechanism againstan excitotoxic process associated with Mn toxicity.Although the inhibitory effect of Mn on NMDA recep-tors have been reported [14,15], little data exist on the invivo effects of Mn subcutaneous injection on the expres-sion of NR subunit mRNAs and proteins, informationthat is critical for more fully evaluating the Mn-inducedneurotoxicity.

We therefore postulated in the present study thatMn induced nerve cell damage by increasing extracel-lular Glu level and altered expression of NR subunitmRNAs and proteins in rat striatum. To test this hy-pothesis, we developed a rat model of manganism toevaluate the effect of manganese following subchronicexposure on nerve cell damage in rat striatum.

MATERIALS AND METHODS

Drugs and Chemicals

Glutamate kit was obtained from NanjingJiancheng Biotech. Co. Ltd. (Nanjing China). AnnexinV-FITC/PI detection and super enhanced chemilumi-nescence (ECL) detection reagents were from NanjingKeyGen Biotech. Co. Ltd. (Nanjing China). TRIZOLreagent, reverse transcription-polymerase chain reac-tion (RT-PCR) kit, and DNA Marker I were from BeijingTransGen Biotech. (Beijing China) Co. Ltd. Goat poly-clonal antibodies developed against a peptide map-ping at the C-terminus of NMDAζ1 (NR1), NMDAε1(NR2A), and NMDAε2 (NR2B) against mouse proteinswere purchased from Santa Cruz Biotechnology, Inc.(Santa Cruz, CA). Mouse monoclonal antibodies de-veloped against β-actin were purchased from SantaCruz. Horseradish peroxidase (HRP)-conjugated anti-goat secondary antibody and HRP-conjugated anti-mouse secondary antibody were purchased from SantaCruz. Other chemicals of analytical grade were ob-tained from local chemical suppliers.

Experimental Design

A total of 24 male and 24 female Wistar ratswere obtained from the Laboratory Animal Center ofChina Medicine University (SPF grade, certificate no.SCXK2008-0005). The rats weighted 180 ± 10 g andwere housed in polypropylene cages under conven-tional conditions at a room temperature of 21 to 24◦C,with a 12-h light: 12-h dark cycle and humidity of30% to 40%. They were maintained on standard ratchow, which was provided by Laboratory Animal Cen-ter of China Medicine University. The rats had freeaccess to water and food throughout the experimen-tal period. Seven days prior to the experiment, ratswere handled daily for 5 min to acclimatize them tohuman contact and minimize their physiological re-sponses to handling. The rats were divided randomlyinto four groups of six males and six females each:control group (group 1) and 8, 40, and 200 μmol/kgMn-treated groups (groups 2–4). The control group ratswere subcutaneously (s.c.) injected with normal saline.Manganese-treated rats were s.c. injected with respec-tively 8, 40, and 200 μmol/kg MnCl2 · 6H2O in normalsaline. The amount of Mn delivered was adjusted forthe molar concentration in the hexahydrate form so asto achieve a precise dose of 200 μmol/kg. The volumeof injection was 5 mL/kg body weight, five times per

J Biochem Molecular Toxicology DOI 10:1002/jbt

Volume 24, Number 1, 2010 MANGANESE ALTERS NMDA RECEPTOR EXPRESSION 3

week, for up to 4 weeks. This dose was selected onthe basis of pilot studies indicating that 200 μmol/kgwas the sufficient dose to induce measurable neuro-logic deficits in exposed rat during the course of sub-chronic exposure by s.c. administration; lower dosesrequired more than 4 weeks to produce measurabledeficits (data not shown). For biochemical analysis, ratswere killed by decapitation. The head was immersed inrunning cold physiological saline for 30 s to clear bloodcontamination and to cool the brain. The striatum wasseparated and placed in a different homogenate bufferfor assay. Tissues were rapidly sonicated in a thermallyregulated sonicator. The animal experiment was car-ried out according to the National Institutes of HealthGuidelines for the Care and Use of Laboratory Animalsand approved by the local authorities. All efforts weremade to minimize the number of animals used andtheir suffering.

Measurement of Manganese Concentrationin Brain

A weighed amount of tissue was wet digested with500 μL of HNO3 s.p. (70% HNO3 for trace metal anal-ysis) in a high-pressure digestion pot at 110◦C. Afterpartial evaporation, samples were cooled down, 500 μLof H2O2 (36.5%–38.0% for trace metal analysis) wasadded, and the solution was totally evaporated. Theprecipitate was dissolved in 5 mL of deionized wa-ter and analysis was performed by HITACHI 180-80atomic absorption spectrophotometer. Concentrationswere measured by a standard calibration curve.

Measurement of Glutamate Concentration

Glutamate was determined by Glu kit (JianchengBiotech. Co. Ltd.). Striatum was homogenized with0.9% normal saline (1:10 w/v). A homogenate of 0.2 mLwas mingled with 0.6 mL of reagent added into eachtube and centrifuged at 3000 rpm for 10 min. The 0.5-mLsupernatant was mingled with other reagents and in-

cubated at 37◦C for 40 min according to the manufac-turer’s recommended protocol. Finally, the absorbanceof the solution was determined at 340 nm. The concen-tration of Glu was expressed in micromoles per gram ofprotein. Protein determination was performed accord-ing to the procedure of Lowry et al. [16], using bovineserum albumin (BSA) as the standard.

Reverse Transcription-PolymeraseChain Reaction

Tissues were homogenized in TRIZOL reagent. Af-ter 5-min room temperature incubation (20◦C), chloro-form was added for phase separation. The upper aque-ous phase was collected and the RNA was precipitatedby mixing with isopropyl alcohol. The RNA pellet waswashed once with 75% ethanol and was air dried. Itwas finally redissolved in RNase-free water. The ab-sorbance of the RNA solution was determined withthe NanoPhotometer (IMPLEN, Munchen, Germany)at 260 and 280 nm, respectively. A260/A280 ratios werebetween 1.6 and 1.8.

Semiquantitative RT-PCR was conducted as de-scribed previously [17], using the housekeeping geneglyceraldehyde-3-phosphate dehydrogenase (G3PDH)as an internal standard. RT-PCR was performed withApplied Biosystems 2720 thermal cycler (Foster City,CA). For the RT-PCR assay, total RNA (5 μg) was di-luted to 1 μg/μL in RNase-free water, mixed with 1 μLof anchored oligo(dT)18 (0.5 μg/μL), 10 mM of dNTPs(1 μL), 4 μL of 5 × RT buffer, 0.5 μL of ribonuclease in-hibitor (50 units/μL), 1 μL of TransScript reverse tran-scriptase, and RNase-free water to a final volume of20 μL in a reaction tube. The reaction was incubated at42◦C for 50 min, followed by 70◦C for 15 min to inacti-vate the reverse transcriptase and to completely dena-ture the template. Gene-specific primer sets were con-structed as described previously (Table 1). One of theprimer sets was added to give a final volume of 50 μL.Reactions were run for the optimal cycles with 55◦Cannealing cycle (1 min), 72◦C extension cycle (1 min),

TABLE 1. Primer Sequences Used for the Amplification of Each Gene in This Studya

Name Oligo Primer Sequence Predicted Size (bp)

NR1 Sense prime 5′-AACCTGCAGAACCGCAAG-3′ 333Anti-sense primer 5′-GCTTGATGAGCAGGTCTATGC-3′

NR2A Sense prime 5′-TCCATTCTTCTGTCATCCTGC-3′ 224Anti-sense primer 5′-AAGACCGTCTCTCACTCTTGC-3′

NR2B Sense prime 5′-TGCACAATTACTCCTCGACG-3′ 222Anti-sense primer 5′-TCCGATTCTTCTTCTGAGCC-3′

G3PDH Sense prime 5′-ATGGTGAAGGTCGGTGTGAAC-3′ 437Anti-sense primer 5′-GCTGACAATCTTGAGGGAGT-3′

a G3PDH was used as a constitutively expressed gene and all the data were normalized to G3PDH expression.



and a 95◦C denaturing cycle (50 s). Control amplifica-tions were done either without RT or without RNA.The amplification from primers, NR1, NR2A, NR2B,and G3PDH, produces a fragment of 333, 224, 222, and437 bp in length, respectively. All glassware and plas-ticware were treated with diethyl pyrocarbonate andautoclaved.

Images of the gels after RT-PCR analyses were dig-itally captured by a gel documentation system. Fluo-rescence intensity of each band was then semiquanti-fied by an image analyzing software (FluorChem v2.0,Alpha Innotech Corporation, CA). The changes of in-tensity of NR1, NR2A, and NR2B mRNAs after Mntreatment were normalized by the intensity obtainedin the internal control bands (G3PDH).

Western Blotting

Protein extraction and immunoblot analysis wereconducted as described previously [18]. Brain sam-ples were homogenized in RIPA buffer (10 mM ofNa2HPO4, pH 7.2, 150 mM of NaCl, 1% sodium de-oxicolate, 1% Nonidet P-40, and 0.1% SDS) contain-ing protease inhibitors (1 mM of phenylmethylsulfonylfluoride, 0.2 mM of 1,10-phenanthroline, 10 μg/mLof pepstatin A, 10 μg/mL of leupeptin, 10 μg/mL ofaprotinin, and 10 mM of benzamidine). Protein con-centrations were determined with the BCA reagentfrom Pierce Biotechnology (Thermo Fisher Scientific,USA). Equal amounts of protein (30 μg) were separatedby 8% polyacrylamide gel electrophoresis and trans-ferred to polyvinylidene difluoride (PVDF) membranes(Millipore, Ternicula, CA). PVDF membranes blockedovernight at 4◦C in TBST containing 5% BSA fraction V.Following this, the membranes were rinsed briefly inTBST and incubated with an indicated concentration ofprimary antibody in TBST for 2 h at room temperature.Specific protein expression was then detected by incu-bating the washed membranes with HRP-conjugatedsecondary antibodies. Protein bands were visualizedwith the ECL Western blotting chemiluminescent de-tection reagents and by autoradiography. The inten-sity of the bands was evaluated semiquantitativelyby densitometry with an image analyzing software(FluorChem v2.0). The changes of intensity of NR1,NR2A, and NR2B proteins after Mn treatment werenormalized by the intensity obtained in the internalcontrol bands (β-actin).

Apoptosis Detection

Striatum (75 mg) was dissected for the preparationof dissociated striatum cells as described by the mod-ified method of Villalba et al. [19]. The tissues werewashed three to five times with phosphate buffered

saline (PBS, pH 7.2–7.4). The striatum was minced in10 mL of PBS and supplemented with 0.125% trypsinfor 10 to 15 min at 37◦C with vigorous shaking. The sub-sequent mechanical dissociation with Pasteur pipettesand nylon mesh screens was performed as described byVillalba at al. Cells were finally suspended in PBS sup-plemented with 0.1% BSA until used. The concentrationof cells was evaluated by viable cell count (trypan bluestained). It was diluted to 1 × 106 cells/mL for flow cy-tometry. Single positive populations were consideredas the early apoptotic cells (Annexin V+/PI−).

Statistical Analysis

The data were statistically analyzed by one-wayanalysis of variance followed by the Student-Newman-Keuls test. The level of significance was set at P < 0.05.The results are presented as means ± SD.

RESULTS

Body Weight and Behavioral Activity

There was no change in body weight of either con-trol or Mn-treated rats (data not shown). After 3 weeks’administration, 200 μmol/kg Mn-treated rats showedsigns of abnormal behavioral activity, including seda-tion, ataxia, enhanced locomotion, grooming, preen-ing, and scratching. However, 8 and 40 μmol/kg Mn-treated rats did not show any signs of abnormal behav-ioral activity at the end of the fourth week.

Manganese and Glutamate Concentrations

There was a significant increase in Mn concen-tration in the striatum of treated rats compared withcontrol rats (Table 2). In 8, 40, and 200 μmol/kg Mn-treated groups, the Mn concentration significantly in-creased 2.2-, 3.8-, and 4.6-fold in striatum, respectively.With the increase in administered MnCl2 dosage, Gluconcentration increased. Glutamate concentrations in40 and 200 μmol/kg Mn-treated rats were signifi-cantly different from those of the controls. In 40 and200 μmol/kg Mn-treated groups, the Glu concentra-tion had increased to 1.6- and 2.5-fold of the controlvalues.

Changes of NMDA Receptor SubunitmRNA Expression After ManganeseTreatment

After 4 weeks of Mn treatment, the RT-PCR re-sults showed that the level of fluorescence intensitiesin the bands that indicated the levels of NR1, NR2A,and NR2B mRNAs were reduced to a different extent



TABLE 2. Changes of Manganese (Mn), Glutamate (Glu), and Apoptosis Rate in Rat Striatum After Mn Treatmenta

Treated Groups Mn (μg/g of wet striatum tissue) Glu (μmol/g of protein) Apoptosis Rate (%)

Control group 0.21 ± 0.08 19.55 ± 3.23 0.43 ± 0.118 μmol/kg Mn-treated group 0.47 ± 0.18∗ 21.95 ± 3.09 26.17 ± 6.45∗∗

40 μmol/kg Mn-treated group 0.80 ± 0.17∗∗ 30.58 ± 5.59∗ 55.06 ± 10.78∗∗

200 μmol/kg Mn-treated group 0.98 ± 0.27∗∗ 48.81 ± 7.23∗∗ 64.27 ± 8.31∗∗

a The values are expressed as mean ± SD for eight animals per group. ∗Significantly different from the control group, P < 0.05. ∗∗Significantly different from thecontrol group, P < 0.01.

compared with those of the control group (Figure 1a).Densitometry evaluation and comparison afternormalization with the housekeeping gene G3PDH hadshown that there was no difference among the expres-sion of NR1, NR2A, and NR2B mRNAs in the controlgroup (Figure 1b). Four weeks after Mn administration,

statistical comparisons revealed that there was a signif-icant decrease in the relative intensities of NR1, NR2A,and NR2B mRNAs compared with those of the con-trol group. The relative intensity of NR2A mRNA de-creased significantly in 8 μmol/kg Mn-treated rats.However, those of NR1 and NR2B mRNAs decreased

FIGURE 1. Changes of N-methyl-D-aspartate receptor (NR) subunit mRNA expression after Mn treatment. (a) The reverse transcription-polymerase chain reaction (RT-PCR) products of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), NR1, NR2A, and NR2B subunits in ratstriatum. (b) Semiquantitative analyses of the expression of NR1, NR2A, and NR2B subunits after RT-PCR experiments are shown. The resultsare expressed as relative intensity in arbitrary units compared with the control values (G3PDH). The data are given as mean ± S.D; n = 4 rats,∗Significantly different from the control group, P < 0.05; ∗∗Significantly different from the control group, P < 0.01.



FIGURE 2. Changes of N-methyl-D-aspartate receptor (NR) subunit protein expression after Mn treatment. (a) The Western blotting productsof β-actin, NR1, NR2A, and NR2B subunits in rat striatum. (b) Semiquantitative analyses of the expression of NR1, NR2A, and NR2B subunitsafter Western blotting experiments. The results are expressed as relative intensity in arbitrary units compared with the control values (β-actin).The data are given as mean ± S.D; n = 4 rats, ∗Significantly different from the control group, P < 0.05; ∗∗Significantly different from the controlgroup, P < 0.01.

significantly in 40 μmol/kg Mn-treated rats. The rela-tive intensity of NR2A mRNA was much more sensitiveto Mn than those of NR1 and NR2B. In 200 μmol/kgMn-treated rats, the relative intensities of NR1, NR2A,and NR2B mRNAs decreased by 7.5%, 10%, and 6.1%,respectively, relative to saline-treated controls.

Changes of NMDA Receptor SubunitProtein Expression After ManganeseTreatment

After 4 weeks of Mn treatment, semiquantitativeanalyses of the results of Western blotting experimentsindicated that the protein levels of NR1, NR2A, andNR2B reduced to different extent compared with thoseof the control group (Figure 2a). Densitometry eval-uation and comparison after normalization with the

housekeeping protein β-actin indicate only a small re-duction in the expression of NR1, NR2A, and NR2Bproteins between controls and the 8 μmol/kg Mn-treated group (Figure 2b). Moreover, this minimal re-duction was not statistically significant. The adminis-tration of 40 μmol/kg of MnCl2 for 4 weeks was enoughto significantly reduce NR2A band density by 34.14%compared with control rats. However, in 200 μmol/kgMn-treated rats, the relative intensities of NR1, NR2A,and NR2B proteins decreased by 33%, 38%, and 31%,respectively, relative to saline-treated controls.

Changes of Striatum Cell Apoptosis AfterManganese Treatment

Percentages of Annexin V-FITC+/PI− positivecells, which are shown in Figure 3 and Table 2, were



FIGURE 3. Changes of striatum cell apoptosis after Mn treatment: (a) control; (b) 8 μmol/kg of Mn; (c) 40 μmol/kg of Mn; (d) 200 μmol/kg ofMn. Single positive populations were considered as the early apoptotic cells (Annexin V+/PI−); n = 8 rats.

considered as the early stage of apoptotic cells. With theincrease in administered MnCl2 dosage, cell apoptosisrate increased significantly. Cell apoptosis rate was0.43% in the control group. However, in 8, 40, and200 μmol/kg Mn-treated groups, this rate respectivelyincreased to 26%, 55%, and 64%.

DISCUSSION

Our study has attempted to evaluate the in vivo ef-fect of Mn administration on the expression of NR sub-unit mRNAs and proteins and Glu levels in rat stria-tum, which is critical for evaluating the Mn-inducedneurotoxicity. In our rat model of manganism, therewas a significant increase in Mn concentration in thestriatum of treated rats compared with control rats. Tofurther confirm irreversible neurodegeneration and fa-cilitate quantification following Mn intoxication, neu-rocyte apoptosis in the striatum was assessed by flowcytometry. The apoptosis rate of the control groupmight be an outcome of spontaneous and rapid apopto-sis of dissociated striatum cells. Furthermore, our find-ings demonstrated that the exposure of MnCl2 causedapoptosis rate to rise up to 64%. These results indicatethat Mn treatment caused appreciable neurotoxicity.

Several mechanisms of Mn neurotoxicity have beenproposed, including the disruption of mitochondrialmetabolism [20], induction of oxidative stress [21], andalteration of iron homeostasis [22]. In this study, thepathogenesis of manganism is considered to involve ex-cessive release of Glu. Our observation of Mn-inducedincreasing Glu concentration in the striatum is consis-tent with the results reported by Zwingmann et al. [9],who observed a 70% to 80% increase in the intracellu-lar abundance of Glu in primary astrocyte and astro-cyte/cortical neuron cocultures treated with 100 μM ofMn for 5 days. The significant increases in the striatumGlu concentrations in Mn-treated rats might be a conse-quence of increased Glu efflux from plasma membraneGlu transporters caused by the significant increases inintracellular Glu concentration. Alternatively, the in-creased extracellular Glu concentration might also re-flect reduced Glu (re)uptake via high-affinity Glu trans-porters, as observed by others in Mn-treated astrocytes[23]. A recent study demonstrated that, on overexpo-sure to Glu, the neuronal cell is apoptosis dominant,which was determined by TUNEL staining, Hoechststaining, and caspase 3 and 9 assays. These resultssuggested that excitotoxicity appeared to be more inti-mately involved through apoptosis [24]. At the cellularlevel, Mn preferentially accumulates in mitochondria,



where it disrupts oxidative phosphorylation and in-creases the generation of reactive oxygen species (ROS)[25]. Excessive production of ROS induces the oxida-tion of membrane polyunsaturated fatty acids, yieldinga multitude of lipid peroxidation products. It has be-come almost dogma that ROS produced in neuronesduring a toxic Glu challenge play a central role in theseprocesses [26]. This Glu toxicity has been clearly at-tributed to a massive influx of Ca2+ through NMDAand non-NMDA channels and a sustained increase in[Ca2+]i, which initiates the excitotoxic processes culmi-nating in a delayed neuronal death [27].

In the present study, RT-PCR and Western blotting,which are considered to have high sensitivity, were em-ployed. The present results thus indicated there werein fact differential patterns of expression of differentNR subunit mRNAs and proteins. The patterns can bedivided into two major trends, namely, (1) the levels(NR1, NR2A, and NR2B) were found to be highest in thecontrol group rats and declined to moderate levels after4 weeks of Mn treatment; This finding indicates that Mntreatment inhibits the expression of NR subunits in ratstriatum. (2) The expression of NR2A mRNA and pro-tein was much more sensitive to Mn than that of NR1and NR2B. The inhibitory effect of Mn on NR subunitproteins has many similarities with the effect of Mn onNR subunit mRNAs. The present results also show thata decrease in NR1, NR2A, and NR2B protein synthesesmay, in turn, decrease the density or functional NMDAchannels in neurons. This may eventually cause a ma-jor upset in NMDA-mediated physiological functionsin the basal ganglia. Similar data have previously beenreported by Guilarte et al. [15]. They found that Mn wasequally potent in inhibiting the NMDA receptor in allbrain regions in the presence of Glu and glycine. More-over, the inhibitory effect of Mn on NR2A was moresignificant than that of NR2B. The result was consid-ered to be related to the differential roles of NR2A- andNR2B-containing NMDA receptors in mediating neu-ronal survival and death [13]. As such, the activation ofNR2A-containing NMDA receptors could trigger dif-ferent signaling events compared with the activationof their NR1/NR2B counterparts, resulting in subunit-specific signaling outcomes. A phosphatidyl-inositol-3-kinase-dependent proneuronal survival signaling hasbeen linked to NR2A-containing NMDA receptor ac-tivation [28], whereas an interference peptide derivedfrom part of the sequence in the NR2B tail has beenshown to provide protection against excitotoxic neu-ronal death by disrupting NR2B C-tail interaction withits binding partner(s) [29]. Therefore, the inhibitory ef-fect of Mn on NR2A is associated with the degree ofimpairment of neurocytes in rat stratum.

In conclusion, the present results showed that Mninduced nerve cell damage by increasing extracellular

Glu level and altered expression of NR subunit mRNAsand proteins in rat striatum. Our understanding of theeffect of Mn on NR subunits is in fact at the very begin-ning. Our present data and our previous report [14,15]provide a body of evidence that the effect of Mn on NRsubunits is useful for the investigation of Mn-inducedneurotoxicity.

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Manganese exposure alters the expression of N-methyl-d-aspartate receptor subunit mRNAs and proteins in rat striatum