Zonisamide regulates basal ganglia transmission via astroglial kynurenine pathway

lable at ScienceDirect

Neuropharmacology xxx (2013) 1e9

Contents lists avai

Neuropharmacology

journal homepage: www.elsevier .com/locate/neuropharm

Zonisamide regulates basal ganglia transmission via astroglialkynurenine pathway

Kouji Fukuyama a,1, Shunske Tanahashi a, Masamitsu Hoshikawa b, Rika Shinagawa b,Motohiro Okada a,*,1

aDepartment of Neuropsychiatry, Division of Neuroscience, Graduate School of Medicine, Mie University, 2-174 Edobashi, Tsu, Mie 514-8507, JapanbDiscovery Research Laboratories I, Ono Pharmaceuticals Co., Ltd., Japan

a r t i c l e i n f o

Article history:Received 25 February 2013Received in revised form1 August 2013Accepted 8 August 2013

Keywords:ZonisamideAstrocyteKynurenine pathwayXanthurenic acidCinnabarinic acidParkinson’s disease

* Corresponding author. Tel.: þ81 59 2315018; fax:E-mail address: [email protected]

1 Kouji Fukuyama and Motohiro Okada contributed

0028-3908/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.neuropharm.2013.08.002

Please cite this article in press as: FukuyamNeuropharmacology (2013), http://dx.doi.or

a b s t r a c t

To clarify the anti-parkinsonian mechanisms of action of zonisamide (ZNS), we determined the effects ofZNS on tripartite synaptic transmission associated with kynurenine (KYN) pathway (KP) in cultured as-trocytes, and transmission in both direct and indirect pathways of basal ganglia using microdialysis. In-teractions between cytokines [interferon-g (IFNg) and tumor-necrosis factor-a (TNFa)] and ZNS onastroglial releases of KPmetabolites, KYN, kynurenic-acid (KYNA), xanthurenic-acid (XTRA), cinnabarinic-acid (CNBA) and quinolinic-acid (QUNA), were determined by extreme liquid-chromatographywithmass-spectrometry. Interaction among metabotropic glutamate-receptor (mGluR), KP metabolites and ZNS onstriato-nigral, striato-pallidal GABAergic and subthalamo-nigral glutamatergic transmission was exam-ined by microdialysis with extreme liquid-chromatography fluorescence resonance-energy transferdetection. Acute and chronic ZNS administration increased astroglial release of KYN, KYNA, XTRA andCNBA, but not QUNA. Chronic IFNg administration increased the release of KYN, KYNA, CNBA and QUNA,but had minimal inhibitory effect on XTRA release. Chronic TNFa administration increased CNBA andQUNA, but not KYN, KYNA or XTRA. ZNS inhibited IFNg-induced elevation of KYN, KYNA and QUNA, butenhanced IFNg-induced that of CNBA. TNFa-induced rises in CNBA and QUNAwere inhibited by ZNS. ZNSinhibited striato-nigral GABAergic, striato-pallidal GABAergic and subthalamo-nigral glutamatergictransmission via activation of groups II and III mGluRs. ZNS enhanced astroglial release of endogenousagonists of group II mGluR, XTRA and group III mGluR, CNBA. Activated endogenous mGluR agonistsinhibited transmission in direct and indirect pathways of basal ganglia. These mechanisms contribute toeffectiveness and well tolerability of ZNS as an adjunct treatment for Parkinson’s disease during L-DOPAmonotherapy.

This article is part of a Special Issue entitled ‘NIDA 40th Anniversary Issue’.� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Parkinson’s disease (PD) is the second most common progres-sive neurodegenerative disorder after Alzheimer’s disease with anestimated incidence of 0.5e1% among people older than 65 years(Toulouse and Sullivan, 2008). PD is a complex multifactorial dis-ease marked by extensive neuropathology in brain with prominentand progressive loss of dopaminergic neurons within substantianigra pars compacta. Both clinical and pre-clinical studies havedemonstrated that activation of pro-inflammatory cytokines, e.g.,interferon-g (IFNg) and tumor-necrosis factor-a (TNFa), and

þ81 59 2315208.(M. Okada).equally to this work.

All rights reserved.

a, K., et al., Zonisamide regug/10.1016/j.neuropharm.2013

dysfunction of synaptic transmission are important factors in thepathomechanism of PD (Boka et al., 1994; Hunot et al., 1996; Dobbset al., 1999; Mogi et al., 2007; Chakrabarty et al., 2011; Zadori et al.,2012). For example, activation of IFNg and TNFa enhances glialkynurenine (KYN) pathway (KP) (Guillemin et al., 2001; Asp et al.,2011). KP synthesizes various endogenous neuroactive metabo-lites. Kynurenic acid (KYNA) is broad-spectrum endogenous AMPA-and NMDA-receptors antagonists (Stone, 2001), whereas quino-linic-acid (QUNA) is endogenous NMDA-agonist (Stone, 2001).Electrophysiological studies of other KP metabolites have so farfailed to determine the direct effects of these metabolites onneuronal activity (Stone, 2001); however, recently xanthurenic acid(XTRA) and cinnabarinic acid (CNBA) have been identified asendogenous agonists of group II (II-mGluR) and group III (III-mGluR) metabotropic glutamate receptors (mGluRs), respectively(Fazio et al., 2012; Copeland et al., 2013). Activation of pre-synaptic

lates basal ganglia transmission via astroglial kynurenine pathway,.08.002

Delta:1_given name

Delta:1_surname

Delta:1_given name

Delta:1_surname

Delta:1_given name

mailto:[email protected]

www.sciencedirect.com/science/journal/00283908

http://www.elsevier.com/locate/neuropharm

http://dx.doi.org/10.1016/j.neuropharm.2013.08.002



K. Fukuyama et al. / Neuropharmacology xxx (2013) 1e92

II-mGluR and III-mGluR reduce neurotransmitter release via inhi-bition of adenylyl cyclase and voltage-sensitive Ca2þ channels,respectively (Cenci, 2007; Duty, 2010). Several pre-clinical studieshave suggested that activation of both II-mGluR and III-mGluR ispotentially important drug targets to provide both symptom reliefand neuroprotection in PD (Duty, 2010; Nicoletti et al., 2011).

Zonisamide (ZNS), 3-sulfamoylmethyl-1,2-benzisoxazole, whichwas developed by Dainippon Pharma (Osaka, Japan: currentlyDainippon-Sumitomo Pharma), is used as an antiepileptic drug inJapan, South Korea, USA and Europe (Seino and Leppik, 2007).Several clinical studies have reported the wide clinical spectrum ofZNS against psychiatric and neurological disorders, including epi-lepsy (Seino and Leppik, 2007), mood-disorders (McElroy et al.,2005), schizophrenia (Nakagawa et al., 2012), essential-tremor(Bermejo, 2007), and its protective effects against ischemic cere-bral damage (Willmore, 2004). Especially, ZNS is effective in PD atdoses lower than the therapeutic doses for epilepsy (Epi-dose:200e600 mg/day) (Murata, 2004; Murata et al., 2007; Seino andLeppik, 2007). Japanese clinical study has demonstrated that ZNS(25 and 50 mg/day) improved motor symptoms of PD patientstreated with L-DOPA, and 50 mg/day ZNS improved L-DOPA-induced dyskinesia (Murata et al., 2007). However, the exactmechanism of action of ZNS when used at the therapeutic dose inPD (PD-dose: 25e50 mg/day) remains to be clarified (Yamamuraet al., 2009). The major mechanisms of antiepileptic actions ofZNS (Rogawski and Porter, 1990) are considered to be inhibition ofvoltage-gated Naþ channel (Rogawski and Porter, 1990), T-typevoltage-sensitive Ca2þ channel (Suzuki et al., 1992) and Ca2þ-induced Ca2þ-releasing system (Yoshida et al., 2005, 2007).Furthermore, candidate mechanisms of anti-parkinsonian effects ofZNS are inhibition of monoamine-oxidase (Okada et al., 1992,Okada et al., 1995), enhancement of dopamine turnover (Okadaet al., 1992, 1995), neuroprotection via activation of glutathione(Asanuma et al., 2010) and inhibition of oxidative stress-inducedexpression of caspase-3 (Yurekli et al., 2013). However, the PD-dose of ZNS does not affect these targets (Okada et al., 1992,1995; Yamamura et al., 2009). We have already demonstratedthat at the PD-dose, ZNS inhibits striato-pallidal GABAergic-trans-mission via activation of striatal d1-receptor (Yamamura et al.,2009). However, the exact mechanisms of the inhibitory effects ofZNS on subthalamo-nigral glutamatergic-transmission remainobscure.

To determine the anti-parkinsonian effects of ZNS at the PD-dose, we examined the effects of ZNS on astroglial KP trans-mission associated with cytokine activation, employing primarycultured astrocytes, and measuring transmission in both direct andindirect pathways of basal ganglia using microdialysis.

2. Materials and methods

Animal care and the experimental procedures described in this report compliedwith the Ethical Guidelines established by the Institutional Animal Care and UseCommittee at Mie University. All studies involving animals are reported in accor-dance with the ARRIVE guidelines for reporting experiments involving animals(McGrath et al., 2010).

2.1. Chemical agents

Zonisamide was provided by Dainippon-Sumitomo Pharma (Osaka, Japan).Synaptobrevin inhibitor, tetanus-toxin (TeNT) (Tanahashi et al., 2012; Yamamuraet al., 2013), and glial-toxin, fluorocitrate (FLC) (Alexander et al., 2011), 3-hydroxy-kynurenine (3H-KYN), XTRA and CNBA were obtained from Sigma (St. Louis, MO).Voltage-sensitive Naþ channel inhibitor, tetrodotoxin (TTX) (Alexander et al., 2011)was obtained fromWako Chemicals (Osaka, Japan). Rat recombinant IFNg and TNFawere purchased from Biolegend (San Diego, CA) and R&D system (Minneapolis, MN),respectively. II-mGluR agonist, LY354740 (Alexander et al., 2011), II-mGluR antago-nist, LY341495 (Alexander et al., 2011), III-mGluR agonist, L-(þ)-2-amino-4-phosphonobutyric acid (L-AP4) (Alexander et al., 2011), and III-mGluR antagonist,

Please cite this article in press as: Fukuyama, K., et al., Zonisamide reguNeuropharmacology (2013), http://dx.doi.org/10.1016/j.neuropharm.2013

(RS)-a-cyclopropyl-4-phosphonophenyl glycine (CPPG) (Alexander et al., 2011) wereobtained from Tocris Bioscience (Bristol, UK).

2.2. Primary astrocyte culture

Astrocytes were prepared using a protocol described previously (Tanahashiet al., 2012; Yamamura et al., 2013) with some modification. Neonatal Sprague-Dawley rats (SLC, Shizuoka, Japan) (n ¼ 16) were sacrificed by decapitation at 0e24 h of age and the cerebral hemispheres were removed under dissecting micro-scope, followed by preparation of cortical astrocyte cultures. Briefly, the corticaltissue was chopped into fine pieces using scissors and then triturated briefly with amicropipette. Suspension was filtered using 70 mm nylon mesh (BD, Franklin Lakes,NJ) and centrifuged. Pellets were re-suspended in 10 mL Dulbecco’s modified Eagle’smedium containing 10% fetal calf serum (fDMEM) (repeated three times). After 14day culture (DIV14), contaminating cells were removed by shaking in standardincubator for 16 h at 200 rpm. On DIV21, astrocytes were removed from flasks bytrypsinization and seeded onto translucent PET membrane (1.0 mm) with 12- or 24-well plates (BD) directly at a density of 105 cells/cm2 for experiments (Tanahashiet al., 2012; Yamamura et al., 2013). During DIV21-DIV28, the culture medium waschanged twice a week, and cultured cells were treated by the test agent (detailedmethods are described under “Treatment of astrocytes and study design”). OnDIV28, the cultured astrocytes were washed out using artificial cerebrospinal fluid(ACSF, in mM: NaCl 130, KCl 5.4, CaCl2 1.8, MgCl2 1, and glucose 5.5, and bufferedwith 20 mM HEPES buffer to pH 7.3) (repeated three times). After the wash-out,astrocytes were incubated in ACSF (100 mL/translucent PET membrane) at 35 �Cfor 60 min in a CO2 incubator. Each collected 100 mL ACSF sample was treated with100 L of ethyl-4-OH-2-quin (20 nM) for internal standards, and filtrated by Vivaspin500-3K (Sartorius, Goettingen, Germany). Each experiment was performed in trip-licate using cultures derived from brains of three different rats. The filtered sampleswere freeze-dried and stored at �80 �C until analysis. The freeze-dried sample wastreatedwith 50 mL acetonitrile containing 5% ammonium (vol/vol) for extreme liquidchromatography (xLC) with mass-spectrum (xLCMS) analysis.

2.3. Treatment of astrocytes and study design

2.3.1. Treatment with IFNg, TNFa and 3H-KYNTo study the effects of IFNg, TNFa and 3H-KYN on KP metabolites, astrocytes

collected at DIV21 were incubated on translucent PET membrane for 7 days (fromDIV21 to DIV28) in fDMEM with or without IFNg (100 U/mL), TNFa (100 U/mL) or3H-KYN (1, 10 and 100 mM) (Tanahashi et al., 2012; Yamamura et al., 2013).

2.3.2. Treatment with TeNT, FLC and TTXTo study the effects of TeNT on KP metabolites, astrocytes collected at DIV27

were incubated on translucent PET membrane for 1 day (from DIV27 to DIV28) infDMEM with or without TeNT (3 mg/mL) (Tanahashi et al., 2012; Yamamura et al.,2013). To study the effects of FLC on KP metabolites, astrocytes collected at DIV28were incubated on translucent PET membrane for 8 h in fDMEMwith or without FLC(1mM) (Tanahashi et al., 2012; Yamamura et al., 2013). To study the effects of TTX onKP metabolites, astrocytes collected at DIV28 were washed out with ACSF, thenincubated on translucent PET membrane for 1 h in ACSF with or without TTX (1 mM)(Tanahashi et al., 2012; Yamamura et al., 2013).

2.3.3. Treatment with ZNSTo study the effects of long-term treatment with ZNS on KP metabolites, as-

trocytes collected at DIV21 were incubated for 7 days (from DIV21 to DIV28) ontranslucent PET membrane in fDMEM with or without ZNS (10, 30, 100 mM)(Tanahashi et al., 2012; Yamamura et al., 2013). To study the acute effects of ZNS onKP metabolites, astrocytes were collected at DIV28, washed out using ACSF, thenincubated on translucent PETmembrane for 2 h in ACSF with or without ZNS (10, 30,100 mM) (Tanahashi et al., 2012; Yamamura et al., 2013).

2.4. Preparation of microdialysis system

Male SpragueeDawley rats, weighing 250e300 g (n ¼ 108), were placed in astereotaxic frame and kept under 1.8% isoflurane anesthesia. Two concentric I-shaped direct insertion type dialysis probes were implanted; D-I-7-02 (0.22 mmdiameter, 2 mm exposed membrane: Eicom) in lateral globus pallidus (LGP:A ¼ �1.6 mm, L ¼ �3.5 mm, V ¼ �7.2 mm, relative to bregma) and D-I-9-01 insubstantia nigra pars reticulata (SNr: A ¼ �5.6 mm, L ¼ �2.3 mm, V ¼ �8.4 mm,relative to bregma) (Paxinos andWatson, 1998). Perfusion experiments commenced18 h after recovery from anesthesia (Tanahashi et al., 2012; Yamamura et al., 2013).The perfusion rate was set at 1 mL/min in all experiments, using modified Ringer’ssolution (MRS) composed of (in mM) 145 Naþ, 2.7 Kþ, 1.2 Ca2þ, 1.0 Mg2þ, and 154.4Cl�, buffered to pH 7.4 with 2 mM phosphate buffer and 1.1 mM Tris buffer(Tanahashi et al., 2012; Yamamura et al., 2013). Perfusion commenced using MRSalone. Extracellular levels of L-glutamate and GABA were measured at 6 h after thestart of perfusion. After collection, each dialysate sample was injected immediatelyinto xLCMS. When the coefficient of variation of the level of each neurotransmitterwas less than 5% over 60 min (stabilization period), control data were obtained over


Fig. 1. Concentration-dependent effects of long-term 3H-KYN administration on astroglial release of KP metabolites. Concentration-dependent effects of 7-day exposure to 3H-KYN(0, 1, 10 and 100 mM) on extracellular levels of (A) KYN, (B) KYNA, (C) XTRA, (D) CNBA and (E) QUNA. Data are mean � SEM (n ¼ 12). *p < 0.05; **p < 0.01 vs ZNS-free (0 mM) by one-way ANOVAwith Tukey’s multiple comparison. One-way ANOVA indicated significant concentration-dependent effects of 7-day administration of 3H-KYN on extracellular levels ofKYN [F(3,44) ¼ 3.1, p < 0.05], KYNA [F(3,44) ¼ 4.5, p < 0.01], XTRA [F(3,44) ¼ 10.9, p < 0.01] and CNBA [F(3,44) ¼ 12.2, p < 0.01] and QUNA [F(3,44) ¼ 10.6, p < 0.01].

K. Fukuyama et al. / Neuropharmacology xxx (2013) 1e9 3

another 60-min period. The location of the dialysis probes was verified at the end ofeach experiment with 100 mm brain slices (Vibratome 1000, Technical ProductsInternational Inc., St. Louis, MO).

2.5. Determination of KP metabolite, L-glutamate and GABA levels

KYNA, XTRA, CNBA and QUNA levels were determined by xLC (3185PU, Jasco,Tokyo) with MS (Acquity SQ detector, Waters, Milford, MA). Twenty microliters offiltrated samples were injected by auto-sampler (xLC3059AS, Jasco). The concen-trations of KP metabolites were determined by xLCMS equipped with Hypercarbcolumn (particle 3 mm, 150 � 2.1 mm, Thermo, Waltham, MA) at 35 �C, and themobile phase was set at 500 mL/min. A linear gradient elution program was per-formed over 10 minwith mobile phase A (5 mM ammonium acetate buffer, pH 11.0)and B (acetonitrile). Nitrogen flows of desolvation and cone were set at 800 and 5 L/h, respectively, and desolvation temperature was set al 450 �C. The cone voltages fordetermination of KYNA (m/z¼ 190.2), XTRA (m/z ¼ 206.2), CNBA (m/z¼ 301.22) andQUNA (m/z ¼ 166.1) were 42, 50, 38 and �25 V, respectively.

L-KYN, L-glutamate and GABA levels were determined by xLC (xLC3185PU,Jasco) with fluorescence resonance energy transfer (FRET) detection (xLC3120FP,Jasco) after derivatization with isobutyryl-Lcysteine and o-phthalaldehyde. Deriv-ative reagent solutions were prepared by dissolving isobutyryl-L-cysteine (2 mg)and o-phthalaldehyde (1 mg) in 0.1 mL ethanol followed by the addition of 0.9 mL0.2 M sodium borate buffer (pH 9.0). Automated pre-column derivative was carriedout by drawing up 5 mL aliquot sample, standard or blank solution and 5 mL ofderivative reagent solution, and holding in reaction vials for 5 min before injection.Derivatized samples (5 mL) were injected by auto-sampler (xLC3059AS, Jasco).Analytical column (YMC Triat C18, particle 1.8 mm, 50 � 2.1 mm, YMC, Kyoto, Japan)was maintained at 45 �C and flow rate was set at 500 mL/min. A linear gradientelution program was performed over 10 min with mobile phase A (0.05 M citratebuffer, pH 5.0) and B (0.05 M citrate buffer containing 30% acetonitrile and 30%methanol, pH 3.5). The excitation/emission wavelengths of fluorescence detectorwere set at 280/455 nm.


2.6. Statistical analysis

Values were expressed as mean � SEM. The concentration-dependent effects ofZNS and 3H-KYN on extracellular levels of KP metabolites from astrocytes werecompared using one-way analysis of variance (ANOVA) with Tukey’s multiplecomparison. The interaction between ZNS and cytokines (IFNg and TNFa) onextracellular levels of KP metabolites was compared using two-way ANOVA withTukey’s multiple comparison. The effects of ZNS and mGluR agents on extracellularlevels of L-glutamate and GABA in SNr and LGP were compared using repeatedANOVA with Tukey’s multiple comparison. Interactions among ZNS, KP metabolitesand mGluR antagonists on extracellular levels of GABA and L-glutamate in SNr andLGP were compared using multivariate analysis of variance (MANOVA) with Tukey’smultiple comparison. A p value less than 0.05 was considered statistically.

3. Results

3.1. Effects of ZNS on astroglial release of KP metabolites

3.1.1. Concentration-dependent effects of long-term treatment with3H-KYN on astroglial release of KP metabolites

Tryptophan and KYN levels in the culture medium (fDMEM)were 75.6 � 10.8 mM and 8.5 � 0.9 nM, respectively, whereas thelevels of other KP metabolites, KYNA, 3H-KYN, XTRA, CNBA orQUNA, in the culture medium (fDMEM) were below the detectionlimit.

KYN and KYNAwere detected in agent-free fDMEM of astrocytecultures (non-treatment condition) (Fig. 1A and B). However, XTRA,CNBA and QUNA under non-treatment condition were not detect-able. Incubation of astrocytes in fDMEM containing 3H-KYN for 7


Fig. 2. Concentration-dependent effects of acute ZNS administration on astroglialrelease of KP metabolites. After incubation in 3H-KYN-free fDMEM, interaction amongacute administration of ZNS for 2 h (0, 10, 30 and 100 mM: B), 3 mg/mL TeNT (C) and1 mM FLC (-) on extracellular levels of (A) KYN and (B) KYNA. Data are mean � SEM ofKP metabolite levels (n ¼ 12). *p < 0.05; **p < 0.01 vs ZNS-free (0 mM) by one-wayANOVA with Tukey’s multiple comparison. One-way ANOVA indicated significantconcentration-dependent effects of acute administration of ZNS on extracellular levelsof KYN [F(3,44) ¼ 6.1, p < 0.01] and KYNA [F(3,44) ¼ 12.1, p < 0.01]. XTRA, CNBA andQUNA were not detected after incubation in 3H-KYN-free fDMEM.


days resulted in measurable levels of XTRA, CNBA and QUNA(Fig. 1CeE). Long-term administration of 3H-KYN concentration-dependently increased extracellular levels of XTRA, CNBA andQUNA, but decreased those of KYN and KYNA (Fig. 1AeE). Inparticular, 1 mM 3H-KYN increased the extracellular levels of XTRA,CNBA and QUNA, but did not those of KYN or KYNA (Fig. 1AeE);however, at both 10 and 100 mM, 3H-KYN decreased the levels ofKYN and KYNA (Fig. 1A and B).

3.1.2. Effects of TTX, TeNT, FLC, and ZNS on astroglial release of KPmetabolites

Astrocytes can release a variety of transmitters in response tostimuli that induce increases in intracellular Ca2þ levels. Thisrelease is regulated by exocytotic mechanism via N-ethylmaleimide-sensitive fusion protein attachment protein receptor(SNARE) complex (Montana et al., 2006), but is not sensitive to TTX(Tanahashi et al., 2012; Yamamura et al., 2013). Therefore, to studyastroglial release mechanisms of KP metabolites, the present studydetermined the effects of TeNT (synaptobrevin inhibitor), FLC (glialaconitase inhibitor) and TTX on extracellular levels of KPmetabolites.

Extracellular KYN level under non-treatment condition wasdecreased by 1 mM FLC, but not affected by 1 mM TTX (data notshown) or 3 mg/mL TeNT (Fig. 2A), whereas under the same con-dition, extracellular KYNA level was decreased by TeNT and FLC(Fig. 2BeE), but not affected by TTX (data not shown). Incubation infDMEM containing 1 mM 3H-KYN for 7 days decreased extracellularlevels of XTRA, CNBA and QUNA by FLC and TeNT, but not by TTX(Fig. 3AeC). We reported previously that acidic KP metabolites(KYNA, XTRA, CNBA and QUNA) are released from astrocytes byastroglial regulation system associated with synaptobrevin (TeNT)and aconitase (FLC) (Tanahashi et al., 2012; Yamamura et al., 2013).Thus, extracellular acidic KP metabolites levels were of astroglialexocytosis origin (basal release); while that of KYN was notexocytotic (Tanahashi et al., 2012; Yamamura et al., 2013).

After incubation in 3H-KYN-free fDMEM, acute ZNS adminis-tration concentration-dependently increased extracellular levels ofKYN and KYNA (Fig. 2A and B), but not those of XTRA, CNBA or


QUNA. However, the addition of ZNS at 30 and 100 mM, but not10 mM, increased extracellular levels of KYN and KYNA (Fig. 2A andB). The stimulatory effects of ZNS on the extracellular levels ofKYNA were prevented by TeNT and FLC (Fig. 2B), whereas ZNS-induced elevation of KYN level was prevented by FLC, but notaffected by TeNT (Fig. 2A).

After incubation in fDMEM containing 1 mM 3H-KYN, acute ZNSadministration concentration-dependently increased extracellularlevels of XTRA and CNBA, but did not affect that of QUNA (Fig. 3Aand B). Extracellular XTRA level was increased by 100 mM ZNS, byunaffected by 10 nor 30 mM ZNS (Fig. 3A). Extracellular CNBA levelwas increased by 30 and 100 mM ZNS, but not by 10 mM ZNS(Fig. 3B). The stimulatory effects of ZNS on the extracellular levels ofXTRA and CNBA were abrogated by TeNT and FLC (Fig. 3A and B).

3.1.3. Effects of long-term administration of ZNS on astroglialrelease of cytokine-induced KP metabolites

After incubation in fDMEM containing ZNS (10, 30 and 100 mM)for 7 days, ZNS increased extracellular levels of KYN and KYNA(Fig. 4A and B). Furthermore, incubation in fDMEM containing IFNg(100 U/mL) for 7 days increased KYN and KYNA levels; whereasincubation in fDMEM containing TNFa (100U/mL) for 7 days did not(Fig. 4A and B). ZNS concentration-dependently inhibited IFNg-induced elevation of KYN, and KYNA (Fig. 4A and B).

Incubation in fDMEM containing 1 mM 3H-KYN and ZNS (10, 30and 100 mM) for 7 days increased extracellular levels of XTRA andCNBA but not that of QUNA (Fig. 5AeC), while incubation in fDMEMcontaining 1 mM 3H-KYN and IFNg (100 U/mL) for 7 days, increasedCNBA and QUNA levels, but decreased that of XTRA (Fig. 5AeC).Incubation in fDMEM containing 1 mM 3H-KYN and TNFa (100 U/mL) for 7 days, increased CNBA and QUNA levels, and decreasedthat of XTRA (Fig. 5AeC). ZNS concentration-dependently inhibitedIFNg-induced rise in QUNA, as well TNFa-induced rises in CNBAand QUNA. Surprisingly, ZNS concentration-dependently enhancedIFNg-induced CNBA release (Fig. 5AeC).

3.2. Effects of ZNS on basal ganglial transmission associated withmGluR using microdialysis

In vitro study using primary cultured astrocytes demonstratedthat ZNS increased endogenous II-mGluR agonist, XTRA, and III-mGluR agonist, CNBA. These results suggest that ZNS enhancesthe transmission associated with II-mGluR and III-mGluR throughincreased levels of XTRA and CNBA, respectively. In this regard,evidence suggests that II-mGluR is expressed in striato-pallidalGABAergic terminals (Nicoletti et al., 2011), while III-mGluR isexpressed in striato-nigral GABAergic, striato-pallidal GABAergicand subthalamo-nigral glutamatergic terminals (Duty, 2010). Theextracellular L-glutamate level in SNr was 1.4 � 0.2 mM. Theextracellular levels of GABA in SNr and LGP were 0.06 � 0.01 mMand 0.09 � 0.02 mM, respectively. Contrary to amino acids, theextracellular levels of XTRA and CNBA in SNr and LGP were notdetectable. Therefore, to study the effects of ZNS on basal ganglialtransmission associated with mGluR, we examined the interactionbetween ZNS and mGluR agents (II-mGluR agonist, LY354740, II-mGluR antagonist, LY341495, III-mGluR agonist, L-AP4, and III-mGluR antagonist, CPPG, XTRA and CNBA) on the extracellularlevels of L-glutamate in SNr and GABA in LGP using in vivomicrodialysis.

3.2.1. Effects of perfusion with mGluR and KP metabolites into SNron subthalamo-nigral L-glutamate release

After confirming culture stabilization, the nigral perfusion me-dium was switched from MRS to MRS containing 30 mM LY354740(II-mGluR agonist), 30 mM L-AP4 (III-mGluR agonist), 100 mM


Fig. 3. Concentration-dependent effects of acute ZNS administration on astroglial release of KP metabolites, after 7-day incubation in 3H-KYN-containing fDMEM. After 7-dayincubation in 1 mM 3H-KYN-containing fDMEM, the interaction among acute administration of ZNS for 2 h (0, 10, 30 and 100 mM: B), 3 mg/mL TeNT (C), and 1 mM FLC (-)on extracellular levels of (A) XTRA, (B) CNBA, and (C) QUNA. Data are mean � SEM of KP metabolite levels (n ¼ 12). *p < 0.05; **p < 0.01 vs ZNS-free (0 mM) by one-way ANOVAwithTukey’s multiple comparison. One-way ANOVA indicated significant concentration-dependent effects of acute administration of ZNS on extracellular levels of XTRA [F(3,44) ¼ 13.7,p < 0.01] and CNBA [F(3,44) ¼ 10.4, p < 0.01] but not extracellular QUNA level. XTRA, CNBA and QUNA were not detected in 3H-KYN-free fDMEM, but were detected after 7 days ofincubation in 1 mM 3H-KYN-containing fDMEM.


LY341495 (II-mGluR antagonist) or 100 mM CPPG (III-mGluRantagonist). Nigral perfusion with 30 mM L-AP4 and LY354740decreased nigral extracellular L-glutamate level, whereas neither100 mM CPPG nor LY341495 had any effect (Fig. 6A).

In a similar experiment, nigral perfusion mediumwas switchedfrom MRS containing with or without 100 mM LY341495 (II-mGluRantagonist) to MRS containing 10 or 100 mMXTRA. Nigral perfusionwith 100 mM XTRA, but not 10 mM XTRA, decreased nigral extra-cellular L-glutamate level (Fig. 6B). Nigral pre-perfusion withLY341495 inhibited 100 mM XTRA-induced L-glutamate reduction

Fig. 4. Concentration-dependent effects of long-term ZNS administration on astroglialrelease of KP metabolites. After incubation in 3H-KYN-free fDMEM, interaction among7-day administration of ZNS (0, 10, 30 and 100 mM: open columns), IFNg (100 U/mL:closed columns), and TNFa (100 U/mL: stripped columns) on extracellular levels of (A)KYN and (B) KYNA. Data are mean � SEM of KP metabolite levels (n ¼ 12). *p < 0.05;**p < 0.01 vs ZNS free (0 mM), and #p < 0.05; ##p < 0.01 vs control by two-way ANOVAwith Tukey’s multiple comparison. Two-way ANOVA indicated significant interactionbetween 7-day administration of ZNS and IFNg on extracellular levels of KYN[FZNS(3,88) ¼ 3.2, p < 0.05; FIFN(1,88) ¼ 44.9, p < 0.01; FZNS*IFN(3,88) ¼ 4.5, p < 0.01]and KYNA [FZNS(3,88) ¼ 66.2, p < 0.01; FIFN(1,88) ¼ 9.9, p < 0.01; FZNS*IFN(3,88) ¼ 0.4,p > 0.5]. Two-way ANOVA also indicated significant interaction between 7-dayadministration of ZNS and TNFa on extracellular levels of KYN [FZNS(3,88) ¼ 11.6,p < 0.01; FIFN(1,88) ¼ 8.4, p < 0.01; FZNS*IFN(3,88) ¼ 0.1, p > 0.5] and KYNA[FZNS(3,88) ¼ 61.8, p < 0.01; FTNF(1,88) ¼ 3.1, p > 0.05; FZNS*TNF(3,88) ¼ 0.2, p > 0.5].XTRA, CNBA and QUNA were not detected 3H-KYN-free fDMEM.


(Fig. 6B). Similarly, a switch of nigral perfusion medium from MRScontaining with or without 100 mM CPPG (III-mGluR antagonist) toMRS containing 10 or 100 mM CNBA. Nigral perfusion with CNBAconcentration-dependently decreased nigral extracellular L-gluta-mate level, whereas pre-perfusion with 100 mM CPPG inhibited100 mM CNBA-induced L-glutamate reduction (Fig. 6C).

3.2.2. Effects of perfusion with mGluR and KP metabolites into LGPand SNr on GABA release from striato-pallidum and striato-nigra

After confirming culture stabilization, the pallidal perfusionmediumwas switched fromMRS toMRS containing 30 mML-AP4 or100 mM CPPG. Pallidal perfusion with 30 mM L-AP4, but not with100 mM CPPG, altered pallidal extracellular GABA level (Fig. 7A).

In a similar experiment, the nigral perfusion medium wasswitched from MRS to MRS containing 30 mM L-AP4 or 100 mMCPPG. Nigral perfusion with 30 mM L-AP4, but not with 100 mMCPPG, altered nigral extracellular GABA level (Fig. 7B). Similarly,pallidal perfusion with 10 or 100 mM CNBA concentration-dependently decreased pallidal extracellular GABA level, and pre-perfusion with 100 mM CPPG inhibited 100 mM CNBA-inducedGABA reduction (Fig. 7C).

After confirming culture stabilization, the nigral perfusion me-diumwas switched fromMRSwith or without 100 mMCPPG toMRSwith 10 or 100 mM CNBA. Nigral perfusion with CNBAconcentration-dependently decreased nigral extracellular GABAlevel, whereas pre-perfusion with 100 mM CPPG inhibited CNBA-induced GABA reduction (Fig. 7D).

3.2.3. Interaction between mGluR and ZNS on releases of GABA andL-glutamate in basal ganglia

After confirming culture stabilization, the nigral perfusion me-diumwas switched fromMRS with or without 100 mM LY341495 toMRSwith 100 mMZNS. Nigral perfusionwith 100 mMZNS decreasedextracellular L-glutamate level in SNr (Fig. 8A), and such reductionwas inhibited by nigral pre-perfusion with 100 mM LY341495(Fig. 8A). In a similar experiment, nigral perfusionwith 100 mMZNSdecreased extracellular GABA level in SNr (Fig. 8B), and such ZNS-induced reductions was inhibited by pre-perfusion with 100 mMCPPG in SNr (Fig. 8B).

Pallidal perfusion with 100 mM ZNS decreased pallidal extra-cellular GABA level (Fig. 8C), and such reduction was inhibited bypallidal pre-perfusion with 100 mM CPPG (Fig. 8A).


Fig. 5. Concentration-dependent effects of 7-day ZNS administration on astroglial release of KP metabolites, after 7-day incubation in 3H-KYN-containing fDMEM. After 7-dayincubation in 1 mM 3H-KYN-containing fDMEM, interaction among 7-day administration of ZNS (0, 10, 30 and 100 mM: open columns), IFNg (100 U/mL: closed columns) andTNFa (100 U/mL: stripped columns) on extracellular levels of (A) XTRA, (B) CNBA, and (C) QUNA. *p < 0.05; **p < 0.01 vs ZNS-free (0 mM), and #p < 0.05; ##p < 0.01 vs control bytwo-way ANOVA with Tukey’s multiple comparison. Two-way ANOVA indicated significant interaction between 7-day administration of ZNS and IFNg on extracellular levels ofXTRA [FZNS(3,88) ¼ 30.4, p < 0.01; FIFN(1,88) ¼ 4.2, p < 0.05; FZNS*IFN(3,88) ¼ 3.7, p < 0.05], CNBA [FZNS(3,88) ¼ 7.2, p < 0.01; FIFN(1,88) ¼ 36.4, p < 0.01; FZNS*IFN(3,88) ¼ 1.9, p > 0.1]and QUNA [FZNS(3,88) ¼ 30.4, p < 0.01; FIFN(1,88) ¼ 4.2, p < 0.05; FZNS*IFN(3,88) ¼ 3.7, p < 0.05]. Two-way ANOVA also indicated significant interaction between 7-day administrationof ZNS and TNFa on extracellular levels of XTRA [FZNS(3,88) ¼ 22.0, p < 0.01; FTNF(1,88) ¼ 0.6, p > 0.1; FZNS*TNF(3,88) ¼ 1.4, p > 0.1], CNBA [FZNS(3,88) ¼ 0.6, p > 0.5; FTNF(1,88) ¼ 89.0,p < 0.01; FZNS*TNF(3,88) ¼ 3.4, p < 0.05] and QUNA [FZNS(3,88) ¼ 19.7, p < 0.01; FTNF(1,88) ¼ 25.1, p < 0.01; FZNS*TNF(3,88) ¼ 14.5, p < 0.01]. XTRA, CNBA and QUNAwere not detectedafter incubation in 3H-KYN-free fDMEM, but were detectable after incubation in fDMEM containing 1 mM 3H-KYN for 7 days.


4. Discussion

In the central nervous system (CNS), KP is shown to be fullypresent in microglia (Guillemin et al., 2003), whereas indoleamine2,3-dioxygenase (which is involved in the synthesis of KYN fromtryptophan), kynurenine aminotransferase (which is involved inthe synthesis of KYNA from KYN), kynureninase (which is involvedin the synthesis of 3H-ANTA from 3H-KYN), and 3-hydroxy-anthranilate-3,4-dioxygenase (which is involved in the synthesis ofQUNA precursor from 3H-ANTA) are expressed in astrocytes, butnot kynurenine 3-monooxygenase (which is involved in the syn-thesis of 3H-KYN fromKYN) (Guillemin et al., 2001, 2003). Thus, themajor products of KP in astrocytes are considered to be KYNA anddo not synthesize QUNA (Guillemin et al., 2003). The present studyalso demonstrated detectable extracellular levels of KYN and KYNA(KYNA-branch) under non-treatment conditions, but not those ofXTRA, CNBA and QUNA (QUNA-branch). However, the released

Fig. 6. Interaction among perfusion with KP metabolites and mGluR agents on L-glutamate(C), 30 mM LY354740 (,) and 100 mM LY341495 (-) on extracellular L-glutamate level in SLY341495 into SNr. (B) Effects of nigral perfusion with 10 mM XTRA (:), 100 mM XTRA (-) aEffects of nigral perfusion with 10 mM CNBA (:), 100 mM CNBA (-) and 100 mM CNBA þ 100perfusion with XTRA or CNBA into SNr, closed horizontal bars indicate pre-perfusion with Lafter administration of target agents (min). *p < 0.05; **p < 0.01 vs pre-treatment periodindicated significant effects of perfusion into SNr of 30 mM L-AP4 (III-mGluR agonist) [F(9significant interaction between perfusion with XTRA and LY341495 [Fagents (2,15) ¼ 5.2, p <

CNBA and CPPG [Fagents (2,15) ¼ 4.4, p < 0.05; Ftime (9,135) ¼ 89.5, p < 0.01; Fagents � time (


levels of XTRA, CNBA and QUNA became detectable after incubationof astrocytes in fDMEM containing 1 mM 3H-KYN. These resultssuggest that exposure of astrocytes, which can take 3H-KYN(Eastman et al., 1992), to 3H-KYN from neighboring microglia orextra-brain transported (with transport across the blood brainbarrier), can induce the synthesis of KP metabolites with QUNA-branch, including XTRA, CNBA and QUNA (Guillemin et al., 2003).

Evidence suggests that induction of IFNg and TNFa playsimportant roles in the pathophysiology of PD. For example, clinicaland pre-clinical studies demonstrated hyperactivation of IFNg andTNFa in the brain of PD compared with healthy control (Boka et al.,1994; Hunot et al., 1996; Dobbs et al., 1999; Mogi et al., 2007;Chakrabarty et al., 2011). Both IFNg and TNFa enhance toxin-driven nigro-striatal degeneration (Frank-Cannon et al., 2009;Chakrabarty et al., 2011). Up-regulation of QUNA productionprobably induces oxidative stress, which occurs in the early stagesof PD (Zinger et al., 2011). While there is no clinical evidence for

release in rat SNr. (A) Effects of nigral perfusion with 30 mM L-AP4 (B), 100 mM CPPGNr. Stripped horizontal bar indicates time of perfusion with L-AP4, CPPG, LY354740 andnd 100 mM XTRA þ 100 mM LY341495 (,) on extracellular L-glutamate level in SNr. (C)mM CPPG (,) on extracellular L-glutamate level in SN. Stripped horizontal bars indicateY341495 or CPPG. Ordinate: mean � SEM L-glutamate level (mM, n ¼ 6), abscissa: timeby repeated ANOVA or MANOVA with Tukey’s multiple comparison. Repeated ANOVA,45) ¼ 196.4, p < 0.01] and 30 mM LY354740 (II-mGluR agonist). MANOVA indicated0.05; Ftime (9,135) ¼ 13.1, p < 0.01; Fagents � time (18,135) ¼ 32.3, p < 0.01] and between18,135) ¼ 25.7, p < 0.01] on nigral extracellular L-glutamate level.


Fig. 7. Interaction among perfusion with KP metabolites and mGluR agents on GABArelease in LGP and SNr. Effects of perfusion with 30 mM L-AP4 (B) and 100 mM CPPG(C) into LGP and SNr on extracellular GABA levels in (A) LGP and (B) SNr. Strippedhorizontal bars: perfusion with L-AP4 and CPPG. (C and D) Effects of perfusion with10 mM CNBA (:), 100 mM CNBA (-) and 100 mM CNBA þ 100 mM CPPG (,) into (C)LGP and (D) SNr on extracellular GABA levels in LGP and SNr. Stripped horizontal bars:perfusion with CNBA, closed horizontal bars: perfusion with CPPG. Data aremean � SEM of GABA levels (n ¼ 6). *p < 0.05; **p < 0.01 vs pre-treatment period byrepeated ANOVA or MANOVA with Tukey’s multiple comparison. Repeated ANOVAindicated significant effects of perfusionwith 30 mM L-AP4 (III-mGluR agonist) into LGPand SNr on respective pallidal [F(9,45) ¼ 38.4, p < 0.01] and nigral [F(9,45) ¼ 38.6,p < 0.01] extracellular GABA levels. MANOVA indicated significant interaction betweenperfusion with CNBA and CPPG (III-mGluR antagonist) into LGP and SNr on respectivepallidal [Fagents (2,15) ¼ 4.9, p < 0.05; Ftime (9135) ¼ 60.4, p < 0.01; Fagents � time

(18,135) ¼ 53.2, p < 0.01] and nigral [Fagents (2,15) ¼ 6.8, p < 0.01; Ftime (9,135) ¼ 68.1,p < 0.01; Fagents � time (18,135) ¼ 32.5, p < 0.01] extracellular GABA levels.

Fig. 8. Interaction among ZNS and mGluR antagonists on release of L-glutamate from SNr anCPPG þ 100 mM ZNS (C), and 100 mM LY341495 þ 100 mM ZNS (-) on extracellular L-gCPPG þ 100 mM ZNS into SNr and LGP on extracellular GABA levels in (B) SNr, and (C) LGP,Data mean � SEM of L-glutamate and GABA levels (n ¼ 6). *p < 0.05; **p < 0.01 vs pre-tsignificant interaction between perfusion with ZNS and mGluR antagonists on extracellularFdrug*time(18,135) ¼ 60.5, p < 0.01], GABA in SNr [Fdrug(1,10) ¼ 5.1, p < 0.05; Ftime(9,90) ¼Ftime(9,90) ¼ 24.9, p < 0.01; Fdrug*time(9,90) ¼ 7.5, p < 0.01].



high levels of QUNA in patients with PD, high brain 3H-KYN levels[which are accompanied by equally high levels of QUNA in otherneurodegenerative diseases (Schwarcz et al., 2012)], have beendescribed in PD (Ogawa et al., 1992). In the present study, long-termexposure to 3H-KYN and IFNg increased the levels of CNBA andQUNA, but decreased those of XTRA. In contrast to IFNg, long-termexposure to 3H-KYN and TNFa increased CNBA and QUNA levels,without affecting those of XTRA. These results suggest thatenhanced QUNA and reduced XTRA induced by activation of IFNgand/or TNFa play important roles in PD patho-mechanisms.

In the present study, both acute and chronic ZNS administrationenhanced inhibitory astroglial transmission rather than excitatorytransmission, based on the findings of ZNS-induced increase inKYNA (endogenous NMDA/AMPA antagonist) (Stone, 2001), XTRA(endogenous II-mGluR agonist) (Copeland et al., 2013) and CNBA(endogenous III-mGluR agonist) (Fazio et al., 2012) withoutaffecting QUNA (endogenous NMDA agonist) (Stone, 2001).Furthermore, long-term exposure to ZNS concentration-dependently inhibited IFNg-induced rise in QUNA levels, butenhanced IFNg-induced rise in CNBA levels. Additionally, ZNSconcentration-dependently inhibited TNFa-induced elevation ofCNBA and QUNA. Therefore, ZNS enhanced the protection providedby KYNA, XTRA and CNBA, and reduced the harmful effects ofQUNA. The concentration-dependent inhibitory effects of ZNS onIFNg- and TNFa-induced up-regulation of QUNA productionhighlight the therapeutic potential of ZNS in PD. However, thestimulatory effects of ZNS on other KP metabolites with QUNA-branch, XTRA and CNBA should be explored, since XTRA andCNBA are endogenous agonists of II-mGluR and III-mGluR,respectively (Fazio et al., 2012; Copeland et al., 2013). Several pre-clinical studies suggested activation of both II-mGluR and III-mGluR as potentially important drug targets for providing bothsymptom relief and neuroprotection in PD (Duty, 2010; Nicolettiet al., 2011). II-mGluR is expressed in striato-pallidal GABAergicterminals (Nicoletti et al., 2011), and III-mGluR is expressed instriato-nigral GABAergic, striato-pallidal GABAergic andsubthalamo-nigral glutamatergic terminals (Duty, 2010).

In the present microdialysis study, local administration of ZNS(100 mM) and endogenous II-mGluR agonist, XTRA (100 mM),decreased nigral L-glutamate release, and these actions wereinhibited by II-mGluR antagonist. Local administration of ZNS andendogenous III-mGluR agonist, CNBA (10 and 100 mM), decreasedGABA release in LGP, SNr and L-glutamate in SNr, and these actionswere inhibited by III-mGluR antagonist. Therefore, ZNS probably

d GABA from SNr and LGP. (A) Effects of nigral perfusion with 100 mM ZNS (B), 100 mMlutamate level in SNr. (B and C) Effects of perfusion with 100 mM ZNS and 100 mMrespectively. Stripped horizontal bars: ZNS, closed horizontal bars: LY341495 or CPPG.reatment period by MANOVA with Tukey’s multiple comparison. MANOVA indicatedlevels of L-glutamate in SNr [Fdrug(2,15) ¼ 4.2, p < 0.05; Ftime(9,135) ¼ 68.9, p < 0.01;31.8, p < 0.01; Fdrug*time(9,90) ¼ 10.1, p < 0.01] and LGP [Fdrug(1,10) ¼ 5.3, p < 0.05;



reduces transmission in both direct and indirect pathways viaactivation of II-mGluR and III-mGluR. Thus, the present studyindicated the two mechanisms of ZNS on basal ganglial trans-missions. The first is the stimulatory effects of ZNS on astroglialreleases of endogenous II-mGluR and III-mGluR agonistic KP me-tabolites, XTRA and CNBA. The other is that ZNS enhances the basalganglial functions of pre-synaptic II-mGluR and III-mGluR. Theeffective concentration of XTRA and CNBA on respective II-mGluRand III-mGluR were considered to be micromolar range (Fazioet al., 2012; Taleb et al., 2012; Copeland et al., 2013). Unfortu-nately, the present microdialysis study could not detect the extra-cellular levels of XTRA and CNBA in basal ganglia of rat (notParkinson’s disease model). These results suggest that the majoraction of ZNS on basal ganglial transmission of L-glutamate andGABA associated with II-mGluR and III-mGluRwere direct effects ofZNS on mGluRs rather than indirect effects of ZNS on mGluRs viaenhanced astroglial releases of XTRA and CNBA. However, the ab-normality of QUNA-branch of KP contributes to patho-mechanismsof Parkinson’s disease (Ogawa et al., 1992). We shall determine theeffects of ZNS on astroglial releases of XTRA and CNBA in basalganglia of genetic Parkinson’s animal model using microdialysis.

Reduced striato-pallidal GABAergic- and subthalamo-nigralglutamatergic-transmission through the indirect pathway im-proves the imbalance between direct and indirect pathways in PD;however, reduced striato-nigral GABAergic-transmission in thedirect pathway should have a negative effect in the treatment of PD.Clinical studies have demonstrated that ZNS prevented L-DOPA-induced dyskinesia (Murata et al., 2007). Although various path-ways within the basal ganglia contribute to L-DOPA-induceddyskinesia, the main pathomechanism of L-DOPA-induced dyski-nesia is striato-nigral GABAergic hyperactivation (Cenci, 2007).Therefore, reduced striato-nigral GABAergic-transmission by ZNSprobably contributes to prevention of L-DOPA-induced dyskinesiavia inhibitory compensation of hyperactivation GABAergic-transmission in the direct pathway (Cenci, 2007). In other words,the stimulatory effect of ZNS on II-mGluR activity in direct pathwayplays important roles in the prevention of L-DOPA-induceddyskinesia.

Clinical studies have demonstrated the effectiveness of andtolerance to ZNS as an adjunct treatment in PD patients treatedwith L-DOPA, including improvement of the “wearing-off”, “on-off”phenomena, freezing and disabling dyskinesia (Murata et al., 2007).Based on the clinical evidence, the major therapeutic target of ZNSwould be the adjunct effectiveness on PD patients treated with L-DOPA. Therefore, to explore the mechanisms of adjunct effective-ness of ZNS in the treatment with PD patients as well as neuro-protective mechanisms of ZNS, further pre-clinical study is neededregarding interaction between L-DOPA, ZNS and mGluR on basalganglial transmission.

The antiepileptic and anti-parkinsonian effects of ZNS aremediated through various pharmacodynamics targets. Especially,the major mechanism of the antiepileptic effects of ZNS is consid-ered to be inhibition of neuronal hyperactivity via inhibition ofvoltage-gated Naþ-channel, T-type voltage-sensitive Ca2þ-channel,and Ca2þ-induced Ca2þ-releasing system (Rogawski and Porter,1990; Suzuki et al., 1992; Yoshida et al., 2005; Seino and Leppik,2007; Yoshida et al., 2007). Recent studies described severalpossible neuroprotective mechanisms for ZNS against progressivedopaminergic neurodegeneration, e.g., ZNS prevents caspase-3activation and oxidative stress via activation of glutathione(Asanuma et al., 2010; Yurekli et al., 2013). Furthermore, inhibitionof monoamine oxidase B and activation of d1-receptor of ZNSprobably improve extrapyramidal symptoms of PD (Okada et al.,1995; Yamamura et al., 2009). While these effects of ZNS (withthe exception of enhancement of d1-receptor) are observed at high


doses (Epi-dose); complete lack of efficacy has not been excluded atlower doses (PD-dose). Taken together with the above previousstudies, the present findings suggest the involvement of the com-plex actions of ZNS in mediating its anti-parkinsonian effects.

Declaration

All authors declare no conflict of interest.

Acknowledgments

This study was supported by a Grant-in-Aid for ScientificResearch from the Japanese Ministry of Education, Science andCulture (23659564 and 22390224), and a Grant from the JapanEpilepsy Research Foundation.

References

Alexander, S.P., Mathie, A., Peters, J.A., 2011. Guide to Receptors and Channels(GRAC), 5th edition. Br. J. Pharmacol. 164 (Suppl. 1), S1eS324.

Asanuma, M., Miyazaki, I., Diaz-Corrales, F.J., Kimoto, N., Kikkawa, Y., Takeshima, M.,Miyoshi, K., Murata, M., 2010. Neuroprotective effects of zonisamide targetastrocyte. Ann. Neurol. 67, 239e249.

Asp, L., Johansson, A.S., Mann, A., Owe-Larsson, B., Urbanska, E.M., Kocki, T.,Kegel, M., Engberg, G., Lundkvist, G.B., Karlsson, H., 2011. Effects of pro-inflammatory cytokines on expression of kynurenine pathway enzymes inhuman dermal fibroblasts. J. Inflamm. (Lond) 8, 25.

Bermejo, P.E., 2007. Zonisamide in patients with essential tremor and Parkinson’sdisease. Mov Disord. 22, 2137e2138.

Boka, G., Anglade, P., Wallach, D., Javoy-Agid, F., Agid, Y., Hirsch, E.C., 1994. Immu-nocytochemical analysis of tumor necrosis factor and its receptors in Parkin-son’s disease. Neurosci. Lett. 172, 151e154.

Cenci, M.A., 2007. Dopamine dysregulation of movement control in L-DOPA-induced dyskinesia. Trends Neurosci. 30, 236e243.

Chakrabarty, P., Ceballos-Diaz, C., Lin, W.L., Beccard, A., Jansen-West, K.,McFarland, N.R., Janus, C., Dickson, D., Das, P., Golde, T.E., 2011. Interferon-gamma induces progressive nigrostriatal degeneration and basal gangliacalcification. Nat. Neurosci. 14, 694e696.

Copeland, C.S., Neale, S.A., Salt, T.E., 2013. Actions of Xanthurenic Acid, a putativeendogenous Group II metabotropic glutamate receptor agonist, on sensorytransmission in the thalamus. Neuropharmacology 66, 133e142.

Dobbs, R.J., Charlett, A., Purkiss, A.G., Dobbs, S.M., Weller, C., Peterson, D.W., 1999.Association of circulating TNF-alpha and IL-6 with ageing and parkinsonism.Acta Neurol. Scand. 100, 34e41.

Duty, S., 2010. Therapeutic potential of targeting group III metabotropic glutamatereceptors in the treatment of Parkinson’s disease. Br. J. Pharmacol. 161, 271e287.

Eastman, C.L., Guilarte, T.R., Lever, J.R., 1992. Uptake of 3-hydroxykynureninemeasured in rat brain slices and in a neuronal cell line. Brain Res. 584, 110e116.

Fazio, F., Lionetto, L., Molinaro, G., Bertrand, H.O., Acher, F., Ngomba, R.T.,Notartomaso, S., Curini, M., Rosati, O., Scarselli, P., Di Marco, R., Battaglia, G.,Bruno, V., Simmaco, M., Pin, J.P., Nicoletti, F., Goudet, C., 2012. Cinnabarinic acid,an endogenous metabolite of the kynurenine pathway, activates type 4metabotropic glutamate receptors. Mol. Pharmacol. 81, 643e656.

Frank-Cannon, T.C., Alto, L.T., McAlpine, F.E., Tansey, M.G., 2009. Does neuro-inflammation fan the flame in neurodegenerative diseases? Mol. Neurodegener.4, 47.

Guillemin, G.J., Kerr, S.J., Smythe, G.A., Smith, D.G., Kapoor, V., Armati, P.J.,Croitoru, J., Brew, B.J., 2001. Kynurenine pathway metabolism in human astro-cytes: a paradox for neuronal protection. J. Neurochem. 78, 842e853.

Guillemin, G.J., Smith, D.G., Smythe, G.A., Armati, P.J., Brew, B.J., 2003. Expression ofthe kynurenine pathway enzymes in human microglia and macrophages. Adv.Exp. Med. Biol. 527, 105e112.

Hunot, S., Boissiere, F., Faucheux, B., Brugg, B., Mouatt-Prigent, A., Agid, Y.,Hirsch, E.C., 1996. Nitric oxide synthase and neuronal vulnerability in Parkin-son’s disease. Neuroscience 72, 355e363.

McElroy, S.L., Suppes, T., Keck Jr., P.E., Black, D., Frye, M.A., Altshuler, L.L., Nolen, W.A.,Kupka, R.W., Leverich, G.S., Walden, J., Grunze, H., Post, R.M., 2005. Open-labeladjunctive zonisamide in the treatment of bipolar disorders: a prospective trial.J. Clin. Psychiatry 66, 617e624.

McGrath, J.C., Drummond, G.B., McLachlan, E.M., Kilkenny, C., Wainwright, C.L.,2010. Guidelines for reporting experiments involving animals: the ARRIVEguidelines. Br. J. Pharmacol. 160, 1573e1576.

Mogi, M., Kondo, T., Mizuno, Y., Nagatsu, T., 2007. p53 protein, interferon-gamma,and NF-kappaB levels are elevated in the parkinsonian brain. Neurosci. Lett.414, 94e97.

Montana, V., Malarkey, E.B., Verderio, C., Matteoli, M., Parpura, V., 2006. Vesiculartransmitter release from astrocytes. Glia 54, 700e715.

Murata, M., 2004. Novel therapeutic effects of the anti-convulsant, zonisamide, onParkinson’s disease. Curr. Pharm. Des. 10, 687e693.


http://refhub.elsevier.com/S0028-3908(13)00359-6/sref1

















































































Murata, M., Hasegawa, K., Kanazawa, I., 2007. Zonisamide improves motorfunction in Parkinson disease: a randomized, double-blind study. Neurology68, 45e50.

Nakagawa, M., Yamamura, S., Motomura, E., Shiroyama, T., Tanii, H., Okada, M., 2012.Combination therapy of zonisamide with aripiprazole on ECT- andbenzodiazepine-resistant periodic catatonia. J. Neuropsychiatry Clin. Neurosci.24, E9.

Nicoletti, F., Bockaert, J., Collingridge, G.L., Conn, P.J., Ferraguti, F., Schoepp, D.D.,Wroblewski, J.T., Pin, J.P., 2011. Metabotropic glutamate receptors: from theworkbench to the bedside. Neuropharmacology 60, 1017e1041.

Ogawa, T., Matson, W.R., Beal, M.F., Myers, R.H., Bird, E.D., Milbury, P., Saso, S., 1992.Kynurenine pathway abnormalities in Parkinson’s disease. Neurology 42, 1702e1706.

Okada, M., Kaneko, S., Hirano, T., Ishida, M., Kondo, T., Otani, K., Fukushima, Y., 1992.Effects of zonisamide on extracellular levels of monoamine and its metabolite,and on Ca2þ dependent dopamine release. Epilepsy Res. 13, 113e119.

Okada, M., Kaneko, S., Hirano, T., Mizuno, K., Kondo, T., Otani, K., Fukushima, Y.,1995. Effects of zonisamide on dopaminergic system. Epilepsy Res. 22, 193e205.

Paxinos, G., Watson, C., 1998. The Rat Brain: in Stereotoxic Coordinates. AcademicPress, San Dieg.

Rogawski, M.A., Porter, R.J., 1990. Antiepileptic drugs: pharmacological mechanismsand clinical efficacy with consideration of promising developmental stagecompounds. Pharmacol. Rev. 42, 223e286.

Schwarcz, R., Bruno, J.P., Muchowski, P.J., Wu, H.Q., 2012. Kynurenines in themammalian brain: when physiology meets pathology. Nat. Rev. Neurosci. 13,465e477.

Seino, M., Leppik, I., 2007. Zonisamide. In: Engel Jr., J., Pedley, T.A. (Eds.), Epilepsy: aComprehensive Text Book, vol. 2. Lippincott Williams & Wilkins, Philadelphia,pp. 1695e1701.

Stone, T.W., 2001. Kynurenines in the CNS: from endogenous obscurity to thera-peutic importance. Prog. Neurobiol. 64, 185e218.

Suzuki, S., Kawakami, K., Nishimura, S., Watanabe, Y., Yagi, K., Seino, M.,Miyamoto, K., 1992. Zonisamide blocks T-type calcium channel in culturedneurons of rat cerebral cortex. Epilepsy Res. 12, 21e27.


Taleb, O., Maammar, M., Brumaru, D., Bourguignon, J.J., Schmitt, M., Klein, C.,Kemmel, V., Maitre, M., Mensah-Nyagan, A.G., 2012. Xanthurenic acid binds toneuronal G-protein-coupled receptors that secondarily activate cationic chan-nels in the cell line NCB-20. PloS One 7, e48553.

Tanahashi, S., Yamamura, S., Nakagawa, M., Motomura, E., Okada, M., 2012. Cloza-pine, but not haloperidol, enhances glial D-serine and L-glutamate release in ratfrontal cortex and primary cultured astrocytes. Br. J. Pharmacol. 165, 1543e1555.

Toulouse, A., Sullivan, A.M., 2008. Progress in Parkinson’s disease-where do westand? Prog. Neurobiol. 85, 376e392.

Willmore, L.J., 2004. Zonisamide overview of the United States experience. Seizure13 (Suppl. 1), S57eS58.

Yamamura, S., Hoshikawa, M., Kato, D., Saito, H., Suzuki, N., Niwa, O., Okada, M.,2013. ONO-2506 inhibits spike-wave discharges in a genetic animal modelwithout affecting traditional convulsive tests via gliotransmission regulation.Br. J. Pharmacol., 1088e1100.

Yamamura, S., Ohoyama, K., Nagase, H., Okada, M., 2009. Zonisamide enhances deltareceptor-associated neurotransmitter release in striato-pallidal pathway.Neuropharmacology 57, 322e331.

Yoshida, S., Okada, M., Zhu, G., Kaneko, S., 2005. Effects of zonisamide on neuro-transmitter exocytosis associated with ryanodine receptors. Epilepsy Res. 67,153e162.

Yoshida, S., Okada, M., Zhu, G., Kaneko, S., 2007. Carbamazepine prevents break-down of neurotransmitter release induced by hyperactivation of ryanodinereceptor. Neuropharmacology 52, 1538e1546.

Yurekli, V.A., Gurler, S., Naziroglu, M., Uguz, A.C., Koyuncuoglu, H.R., 2013. Zonisa-mide attenuates MPPþ-induced oxidative toxicity through modulation of Ca2þ

signaling and caspase-3 activity in neuronal PC12 cells. Cell Mol. Neurobiol. 33,205e212.

Zadori, D., Klivenyi, P., Toldi, J., Fulop, F., Vecsei, L., 2012. Kynurenines in Parkinson’sdisease: therapeutic perspectives. J. Neural Transm. 119, 275e283.

Zinger, A., Barcia, C., Herrero, M.T., Guillemin, G.J., 2011. The involvement of neu-roinflammation and kynurenine pathway in Parkinson’s disease. ParkinsonsDis. 2011, 716859.

























































































Documents

Zonisamide regulates basal ganglia transmission via astroglial kynurenine pathway