Glutathione peroxidase-1 overexpressing transgenic mice are protected from cocaine-induced drug dependence

Accepted Manuscript Glutathione peroxidase-1 overexpressing transgenic mice are protected from cocaineinduced drug dependence Huynh Nhu Mai, Yoon Hee Chung, Eun-Joo Shin, Dae-Joong Kim, Naveen Sharma, Yu Jeung Lee, Ji Hoon Jeong, Seung-Yeol Nah, Choon-Gon Jang, Hyoung-Chun Kim PII:

S0197-0186(18)30603-X

DOI:

https://doi.org/10.1016/j.neuint.2019.01.018

Reference:

NCI 4386

To appear in:

Neurochemistry International

Received Date: 4 November 2018 Revised Date:

13 January 2019

Accepted Date: 22 January 2019

Please cite this article as: Mai, H.N., Chung, Y.H., Shin, E.-J., Kim, D.-J., Sharma, N., Lee, Y.J., Jeong, J.H., Nah, S.-Y., Jang, C.-G., Kim, H.-C., Glutathione peroxidase-1 overexpressing transgenic mice are protected from cocaine-induced drug dependence, Neurochemistry International (2019), doi: https:// doi.org/10.1016/j.neuint.2019.01.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Glutathione peroxidase-1 overexpressing transgenic mice are protected from cocaine-

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induced drug dependence

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Huynh Nhu Maia, 1, Yoon Hee Chungb, 1, Eun-Joo Shina,*, Dae-Joong Kimc, Naveen Sharmaa,

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Yu Jeung Leed, Ji Hoon Jeonge, Seung-Yeol Nahf, Choon-Gon Jangg, Hyoung-Chun Kima, *

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a

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University, Chunchon 24341, Republic of Korea

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b

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Neuropsychopharmacology and Toxicology Program, College of Pharmacy, Kangwon National

Department of Anatomy, College of Medicine, Chung-Ang University, Seoul 06974, Republic

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of Korea

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Chunchon 24341, Republic of Korea

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Republic of Korea

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Republic of Korea

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Medicine, Konkuk University, Seoul 05029, Republic of Korea

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Republic of Korea

Department of Anatomy and Cell Biology, Medical School, Kangwon National University,

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Clinical Pharmacy, College of Pharmacy, Kangwon National University, Chunchon 24341,

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Department of Pharmacology, College of Medicine, Chung-Ang University, Seoul 06974,

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Ginsentology Research Laboratory and Department of Physiology, College of Veterinary

Department of Pharmacology, School of Pharmacy, Sungkyunkwan University, Suwon 16419,

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*

Corresponding author.

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The first two authors contributed equally to this work

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*Corresponding

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Neuropsychopharmacology and Toxicology Program, College of Pharmacy, Kangwon National

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University, Chunchon 24341, Republic of Korea Tel.: +82 33 250 6917; fax: +82 33 259 5631,

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E-mail address: [email protected] (H. C. Kim) or [email protected] (E.J. Shin).

Professor

Hyoung-Chun

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Kim

Eun-Joo

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author:

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Shin,

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Abstract

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Converging evidence has demonstrated that oxidative burdens are associated with drug

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dependence induced by psychostimulants. Here, we investigated whether oxidative stress directly

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mediates conditioned place preference and behavioral sensitization (drug dependence) induced

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by cocaine and whether glutathione peroxidase-1 (GPx-1), a major GPx, modulates cocaine-

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induced psychotoxic changes in mice. Cocaine-induced drug dependence was followed by

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increases in c-Fos-immunoreactivity (c-Fos-IR) in the nucleus accumbens. Simultaneously,

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cocaine significantly increased oxidative parameters and nuclear factor κB (NFκB) activity (i.e.

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nuclear translocation and DNA binding activity) in the striatum (including nucleus accumbens).

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Genetic depletion of GPx-1 made mice susceptible to drug dependence induced by cocaine in

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mice, while genetic overexpression of GPx-1 protected the mice from drug dependence.

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Pyrrolidine dithiocarbamate (PDTC), a NFκB inhibitor, significantly attenuated the sensitivity

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induced by the genetic depletion of GPx-1 in mice. However, PDTC did not exhibit any additive

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effects against the protection afforded by the genetic overexpression of GPx-1. Our results

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suggest that drug dependence induced by cocaine requires oxidative stress and NFκB activation,

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and that the GPx-1 gene is a potential protective factor against cocaine-induced drug dependence

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through positive modulation of NFκB.

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Keywords: Cocaine drug dependence; Oxidative stress; Nuclear factor κB; Striatal complex;

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Glutathione peroxidase-1 knockout mice; Glutathione peroxidase-1 overexpressing transgenic

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mice

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1. Introduction

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We have recently demonstrated that the major antioxidant enzyme glutathione

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peroxidase-1 (GPx-1) gene possesses protective potentials against memory impairments and drug

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dependence induced by methamphetamine (MA) (Mai et al., 2018c, 2018d; Shin et al., 2017;

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Tran et al., 2017, 2018b), suggesting that oxidative stress mediates MA-induced psychotoxic

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response. Indeed, Walker et al. (2014) demonstrated that both the cocaine-dependent and MA-

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dependent participants in the clinical trial showed a significantly decreased total antioxidant

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capacity (TAC) compared with the controls. Although respective human data are lacking, it is

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known that cocaine abuse leads to elevated oxidative stress and thereby enhanced

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proinflammatory status in drug-abusing patients (Fox et al., 2012).

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Consistently, evidence associating the harmful outcome of cocaine toxicity with oxidative

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stress has accumulated during the last decade, and it is believed to play a major role in the

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negative consequences of cocaine intake. Moreover, cocaine exposure has also been linked to

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reduced non-enzymatic antioxidant levels, including reduced glutathione (GSH), resulting in

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elevated reactive oxygen species (ROS), and damaged brain function (Muriach et al., 2010).

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Furthermore, Uys et al. (2011) demonstrated that increased oxidative stress following cocaine

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exposure contributes to cocaine-induced behaviors via modulation of glutathione redox status.

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This demonstration prompt to us to investigate the role of GPx-1 in the current experimental

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condition.

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Accumulating evidence suggests that nuclear factor kappaB (NFκB) transcription factor is a 4

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critical molecular target for drug dependence (Ang et al., 2001; Nennig and Schank, 2017; Zhang

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et al., 2011). In particular, it is well-recognized that functional activity of NFκB is upregulated by

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repeated cocaine treatment (Ang et al., 2001; Russo et al., 2009). Similarly, morphine and other

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µ-opioid receptor agonists increase NFκB function in vitro (Sawaya et al., 2009; Wang et al.,

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2004), and regulate NFκB phosphorylation (Zhang et al., 2011). Consistently, pyrrolidine

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dithiocarbamate (PDTC), a NFκB inhibitor attenuated drug dependence induced by cocaine or

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morphine (Russo et al., 2009; Zhang et al., 2011).

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In this study, we investigated whether conditioned place preference (CPP) and behavioral

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sensitization (drug dependence) induced by cocaine produce oxidative burdens, whether

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oxidative stress responsible transcription factor NFκB plays a role as a mediator of the drug

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dependence induced by cocaine, and whether GPx-1 gene modulates this drug dependence.

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Here, we propose that cocaine-induced drug dependence leads to oxidative damage, and that

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GPx-1 gene acts as a protective modulator against cocaine-induced drug dependence by

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inhibiting modulation of NFκB signaling.

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2. Materials and methods

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2.1. Animal

The handling of animals was performed according to the National Institutes of Health

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(NIH) Public Health Service Policy on Humane Care and Use of Laboratory Animals (2015

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Edition; grants.nih.gov/grants/olaw/references/PHSPolicyLabAnimals.pdf) and Institute for 5

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Laboratory Animal Research Guidelines for the Care and Use of Laboratory Animals (8th

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Edition; grants.nih.gov/grants/olaw/Guide-for-the-care-and-use-of-laboratory-animals.pdf). Mice

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were preserved in a 12 h/12 h light/dark cycle and fed ad libitum. GPx-1 knockout (KO) mice

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were derived from C57BL/6 × 129/SVJ mice as previously described (Ho et al., 1997; Mai et al.,

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2018b). The breeding pairs of GPx-1 knockout (KO) mice were kindly provided by Professor Ye-

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Shih Ho (Wayne State University, Detroit, MI, USA). The coding sequence of the GPx-1 gene

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was disrupted by insertion of a neomycin resistance gene cassette (neo) in the exon 2 in this

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mouse strain. Breeding pairs of GPx-1 overexpressing transgenic (GPx-1 TG) mice were kindly

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provided by Professor Xin Gen Lei (Department of Animal Science, Cornell University, Ithaca,

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New York, U.S.A.). The GPx-1 TG mice were derived from B6C3 (C57BL/6 × C3H) hybrid

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mice, and have three copies of the GPx-1 transgene (Cheng et al., 1997; Xiong et al., 2004).

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Characterization using DNA template extracted from mouse tail was performed by

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polymerase chain reaction (PCR) analysis. Primer sequences (Bioneer Corporation, Daejeon,

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Republic of Korea) used for characterization were obtained from previous studies (Mai et al.,

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2016; Shin et al., 2018).

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2.2. Drugs

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Cocaine (15 mg/kg, i.p.; Macfarlan Smith Ltd., Edinburgh, UK) was liquefied in saline

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before use. In order to examine the role of NFκB, mice received pyrrolidine dithiocarbamate

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(PDTC; 50 mg/kg, i.p.; Sigma-Aldrich, St. Louis, MO, USA), a selective NFκB inhibitor, 1 h

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prior to every cocaine administration. PDTC was liquefied in saline before use (Shin et al., 6

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2004a). The doses of the drugs were established in our previous studies (Shin et al., 2011; Shin et

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al., 2018).

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2.3. Conditioned place preference (CPP)

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The CPP apparatus and procedure were described before (Shin et al., 2009, 2011). For the

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control group, mice were administrated saline before placing in both compartments. While, mice

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received cocaine (15 mg/kg, i.p.) before entering the white compartment (Fig. 1). The differences

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between the time spent in the posttest (day 3) and pretest (day 15) periods were calculated and

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presented as seconds, indicating the place preference. The period of time for CPP was between

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0900 and 1700 hr.

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2.4. Behavioral sensitization

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Under typical experimental conditions (Fig. 1), mice received cocaine seven times (on

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day 4, 6, 8, 10, 12, 14, and 16) followed by a withdrawal period for 7 days, before receiving the

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final dose of cocaine (8th dose, day 23). Every locomotor activity (LOC) was recorded for a

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monitoring period of 30 min using an automated video-tracking system (Noldus Information

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Technology, Wageningen, The Netherlands). An IBM computer operated eight test boxes (40 ×

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40 × 30 cm high). Mice were sacrificed 2 h after the behavioral sensitization measurement (Mai

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et al., 2018c; Shin et al., 2009).

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2.5. ROS formation measurement 7

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ROS formation was determined by the measurement of the conversion from 2′,7′-

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dichlorofluorescin diacetate (DCFH-DA) to dichlorofluorescin (DCF) (Nguyen et al., 2018).

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Striatal homogenates (including nucleus accumbens) were mixed with 2 mL of phosphate-

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buffered saline (PBS) and 10 nmol of DCFH-DA in methanol. After incubation for 3 h at 37 °C,

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the mixture was measured at 480 nm excitation and 525 nm emission (Dang et al., 2018b).

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2.6. 4-Hydroxynonenal (HNE) measurement

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The assessment of the level of lipid peroxidation followed the instructions of the

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manufacturer, which was described before [OxiSelect™ HNE adduct ELISA kit (Cell Biolabs,

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Inc., San Diego, CA, USA)] (Dang et al., 2018a; Nguyen et al., 2018). The mixture was

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measured at 450 nm (Molecular Devices Inc.) and calculated according to the standard curve

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(Dang et al., 2018a; Nguyen et al., 2018).

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2.7. Protein carbonyl measurement

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Protein carbonyl determination based on the amount of protein carbonyl groups, which

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were assessed spectrophotometrically with 2,4-dinitrophenylhydrazine (DNPH)-labeling

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procedure (Nguyen et al., 2018; Oliver et al., 1987), and measured at 370 nm. The results were

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divided according to the protein concentration using BCA protein assay reagent (Thermo

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Scientific, Rockford, IL, USA), and expressed as nmol/mg protein.

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2.8. Cytosolic and nuclear NFκB

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The procedure of cytosolic and nuclear extraction using NE-PER nuclear and

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cytoplasmic extraction kit (Thermo Scientific, Rockford, IL) was carried out as previously

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described (Tran et al., 2012; Tran et al., 2018a). Primary antibody NFκB p65 subunit antibody

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(65 kDa; 1:1000; sc-372, Santa Cruz Biotechnology, Santa Cruz, CA) was used to detect the

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NFκB. The intensity of NFκB in the cytosolic and nuclear fractions were normalized to the

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intensity of matrix p84 antibody (ab487, 1:2000; Abcam, Cambridge, MA) or β-actin antibody

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(1:300,000; Sigma-Aldrich), respectively.

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2.9. DNA binding activity assay

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The extraction of nuclear fraction was carried out using a nuclear extraction kit (#40410;

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Active Motif, Carlsbad, California) according to the manufacturer’s instructions (Shin et al.,

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2018).

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The DNA binding activities of NFκB p65 were determined by using TransAM® NFκB

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transcription factor ELISA kit (Active Motif) according to the manufacturer’s instructions.

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Briefly, the wells coated with oligonucleotides containing the NFκB consensus binding site (5’ -

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GGGACTTTCC-3’) and 20 µg of nuclear protein extract were incubated at room temperature for

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1 h. Primary antibody against NFκB p65 subunit and horseradish peroxidase (HRP)-conjugated

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secondary anti-rabbit IgG were added to the mixture and incubated for 1 h. The mixture was

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measured at 450 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, U.S.A.)

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(Shin et al., 2018).

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2.10. Western Blot analysis

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After homogenizing with lysis buffer, containing 200 mM Tris–HCl (pH 6.8), 10% SDS,

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5 mM ethylene glycol tetra-acetic acid (EGTA), 5 mM ethylenediaminetetraacetic acid (EDTA),

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10% glycerol, and protease inhibitor cocktail (Sigma-Alrich), striatal lysate was centrifuged for

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30 min at 13,000 × g. The supernatant used for analyzing proteins by western blot as previously

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described (Mai et al., 2018a). The primary antibodies and HRP-conjugated secondary antibody

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used were β-actin (42 kDa, 1:300000 dilution; Sigma-Alrich), or GPx-1 (23 kDa, 1:1000; R&D

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Systems, Minneapolis, MN, USA); and anti-mouse IgG (1:5000; Sigma-Alrich), or anti-goat IgG

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(1:5000; Sigma-Alrich), respectively. Finally, the membranes were detected using an enhanced

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chemiluminescence system (ECL Plus®, GE Healthcare, Arlington Heights, IL, USA). The

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intensity of the bands was measured by PhotoCapt MW (version 10.01 for Windows; Vilber

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Lourmat, Marne la Vallee, France) and normalized to the intensity of the housekeeping gene (β-

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actin) (Mai et al., 2018e; Shin et al., 2014).

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2.11. Immunocytochemistry

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The procedures of perfusion and brain storage were carried as previously described (Mai

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et al., 2018c). Smaples of brains were sectioned into 35-µm and blocked with 0.3% hydrogen

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peroxide/PBS and 0.4% Triton X-100 plus 1% normal serum/PBS for 30 min and 20 min,

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respectively. c-Fos (1:5000; ABE457, Merck Millipore, Billerica, MA, U.S.A.) was used as a

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primary antibody. After incubating with c-Fos antibody for 24 h, the sections were immersed in 10

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biotinylated secondary antibodies (1:1000; Vector Laboratories, Burlingame, CA, USA) for 1 h.

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Next, sections were incubated with avidin–biotin-peroxidase complex (Vector Laboratories) for

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1 h and detected by 3,3′-diaminobenzidine (DAB; Sigma-Aldrich). An Olympus microscope

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(BX51, Olympus, Tokyo, Japan) with a digital microscope camera (DP72, Olympus) and an IBM

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PC (Armonk, NY, USA) at 200 × magnification was used to take the digital images. ImageJ

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version 1.47v software (NIH) was employed to analyze the positive c-Fos-immunoreactivity

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cells (Mai et al., 2018c; Shin et al., 2018).

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2.12. Statistical analysis

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ANOVA followed by Fisher’s LSD pairwise comparisons were applied to analyze the data. P-values of less than 0.05 were considered statistically significant.

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3.1. Cocaine-induced increases in GPx-1 expression in the striatum of mice

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3. Results

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According to Fig. 1 of the experimental design, we sacrificed mice to investigate the

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changes in GPx-1 expression in the striatum of the wild type (WT) mice. GPx-1 expression was

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significantly increased in the striatum (p = 0.002 vs. saline) of WT mice after cocaine treatment

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[t (10) = -4.148, p = 0.002 (Fig. 2 A)]. Cocaine treatment significantly increased GPx-1

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expression in the striatum (p = 0.021 vs. saline) of non-TG mice [F (3, 20) = 10.308, p = 2.5 x

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10-5 (Fig. 2B)]. The basal level of GPx-1 expression was significantly higher in GPx-1 TG (p = 11

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0.035) than in non-TG mice. Cocaine-induced increases in GPx-1 expression were more

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pronounced in GPx-1 TG (p = 0.007) than in non-TG mice.

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3.2. Cocaine-induced change in oxidative parameters in the striatum of wild type (WT), GPx-1

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knockout (GPx-1KO), non-transgenic (non-TG) and GPx-1 overexpressing transgenic (GPx-1

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TG) mice.

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As shown in Fig.3, we examined the levels of reactive oxygen species (ROS), 4-

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hydroxynonenal (HNE), and protein carbonyl after cocaine treatment in the striatum of WT,

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GPx-1 KO, non-TG, and GPx-1 TG mice. Cocaine-treated WT mice showed a significant

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increase in ROS formation (p = 0.045 vs. corresponding saline). This increase was more

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pronounced in cocaine-treated GPx-1 KO mice (p = 3.4 x 10-5 vs. corresponding saline; p =

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0.001 vs. cocaine-treated WT mice) [F(3, 20) = 14.186, p = 3.4 x 10

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cocaine-treated non-TG also showed a significant increase in ROS formation (p = 0.013 vs.

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corresponding saline). Genetic overexpression of GPx-1 significantly attenuated (p = 0.037)

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ROS formation of cocaine-treated non-TG mice [F(3, 20) = 4.899, p = 0.01 (Fig. 3B)]. This

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phenomenon, in the case of ROS, was consistently comparable to that of HNE or protein

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carbonyl (Fig. 3C-F).

(Fig. 3A)]. In contrast,

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3.3. Cocaine-induced change in NFκB activity in the striatum of wild type (WT), GPx-1 knockout

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(GPx-1KO), non-transgenic (non-TG) and GPx-1 overexpressing transgenic (GPx-1 TG) mice.

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As shown in Fig. 4, we examined the cytosolic and nuclear expression levels of NFκB 12

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and its DNA binding activity in the striatum of WT, GPx-1 KO, non-TG, and GPx-1 TG mice.

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Cocaine treatment significantly decreased cytosolic NFκB expression in WT (p = 0.016). This

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decrease was more pronounced (p = 9.7 x 10-6) in cocaine-treated GPx-1 KO than in cocaine-

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treated WT mice [F(3, 20) = 15.281, p = 2.1 x 10-5 (Fig. 4A)]. In addition, cocaine-treatment

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significantly decreased cytosolic NFκB expression (p = 5.4 x 10-5 vs. saline) in non-TG.

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Cytosolic NFκB expression was higher (p = 0.001) in cocaine-treated GPx-1 TG than in cocaine-

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treated non-TG mice [F(3, 20) = 11.075, p = 1.68 x 10-4 (Fig. 4B)].

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In contrast, cocaine treatment significantly increased NFκB nuclear translocation [F(3,

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20) = 47.367, p = 2.84 x 10-9 (Fig. 4C)] and DNA binding activity [F(3, 20) = 22.209, p = 1.41 x

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10-6 (Fig. 4E)] in the striatum of GPx-1 KO (both nuclear translocation and DNA binding

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activity; p = 1.4 x 10-2 and p = 0.01 vs. saline/GPx-1 KO) and this increment in GPx-1 KO mice

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was higher (p = 1.5 x 10-5 and p = 1.44 x 10-4 vs. cocaine/WT) than in WT mice. In contrast,

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cocaine-induced increases in NFκB nuclear translocation [F(3, 20) = 14.837, p = 2.57 x 10-5 (Fig.

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4D)] and DNA binding activity [F(3, 20) = 12.672, p = 7.266 x 10-5 (Fig. 4F)] were more evident

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in non-TG (p = 2.97 x 10-4 and p = 0.001 vs. cocaine/GPx-1 TG) than in GPx-1 TG, indicating

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that genetic overexpression of GPx-1 significantly attenuated NFκB nuclear translocation and

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DNA binding activity induced by cocaine.

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3.4. Effects of pyrrolidine dithiocarbamate (PDTC, a NFκB inhibitor) on cocaine-induced

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increases in c-Fos-immunoreactivity (c-Fos-IR) in the nucleus accumbens (NAc) of wild type

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(WT), GPx-1 knockout (GPx-1 KO), non-transgenic (non-TG) and GPx-1 overexpressing

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transgenic (GPx-1 TG) mice.

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We then examined c-Fos-IR to investigate cocaine-induced neural stimulation in the NAc

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of GPx-1 KO and GPx-1 TG mice (Fig. 5). A very little c-Fos-IR was observed in the absence of

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cocaine, and c-Fos-IR was significantly increased (p = 1.15 x 10-5 vs. corresponding saline) after

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the final cocaine treatment in WT mice [F(7, 40) = 49.967, p = 8.6 x 10-18 (Fig. 5A)]. Cocaine-

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induced increments in c-Fos-IR were more pronounced in GPx-1 KO (p = 8.12 x 10-11 vs.

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cocaine/WT) than in WT mice. These increases were significantly reversed by PDTC, a NFκB

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inhibitor in WT mice (p = 0.038 vs. cocaine/WT) and GPx-1 KO mice (p = 6.12 x 10-7 vs.

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cocaine/GPx-1 KO). In addition, a cocaine-induced significant increase (p = 6.19 x 10-10) in c-

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Fos-IR was observed in non-TG mice. PDTC treatment significantly attenuated (p = 0.006) this

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increase in non-TG mice. Genetic overexpression of GPx-1 significantly attenuated (p = 10-5 vs.

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cocaine/non-TG) cocaine-induced c-Fos-IR. However, PDTC treatment did not show any

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additive effects on the attenuation by genetic overexpression of GPx-1 [F(7, 40) = 17.296, p =

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2.68 x 10-10 (Fig. 5B)].

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3.5. Effects of PDTC, a NFκB inhibitor, on cocaine-induced conditioned place preference (CPP)

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and behavioral sensitization in of wild type (WT), GPx-1 knockout (GPx-1KO), non-transgenic

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(non-TG) and GPx-1 overexpressing transgenic (GPx-1 TG) mice.

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CPP and behavioral sensitization induced by cocaine were shown in Figs. 6 and 7. In the

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absence of cocaine, CPP was not induced in the mice. Cocaine-induced CPP (p = 9.21 x 10-6 vs.

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Saline-treated WT) was observed in WT mice. This CPP was more evident in GPx-1 KO (p =

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0.006) than in WT. In addition, cocaine-induced CPP (p = 6.96 x 10-10 vs. Saline-treated non-TG)

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was also significantly increased in non-TG mice. However, this increase was significantly 14

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inhibited by genetic overexpression of GPx-1 (p = 0.001 vs. cocaine-treated non-TG). PDTC

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treatment significantly attenuated cocaine-induced CPP (WT: p = 0.049 vs. Saline/cocaine; GPx-

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1 KO: p = 0.001 vs. Saline/cocaine; Non-TG: p = 0.006 vs. Saline/cocaine). However, PDTC did

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not show any additive effect against attenuation offered by genetic overexpression of GPx-1,

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indicating that NFκB is a critical mediator for the protective potential of GPx-1.

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The changes in locomotor activity over time are presented in supplementary Fig. S1.

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Notably, although the first challenge of cocaine significantly increased locomotor activity in WT,

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GPx-1 KO, non-TG and GPx-1 TG mice, PDTC did not affect the hyperlocomotion induced by

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cocaine (Supplementary Fig.S2). The profile of behavioral sensitization is comparable to that of

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CPP. Cocaine-induced behavioral sensitization was significantly observed in WT (p = 3.03 x 10-

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mice (p = 0.003 vs. cocaine-treated WT mice). Genetic overexpression of GPx-1 significantly

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attenuated the behavioral sensitization (p = 6.44 x 10-8 vs. cocaine-treated non-TG mice). Similar

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to CPP, PDTC treatment significantly attenuated cocaine-induced behavioral sensitization (WT:

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p = 8.07 x 10-8 vs. Saline/cocaine; GPx-1 KO: p = 2.5 x 10-5 vs. Saline/cocaine; Non-TG: p = 7.5

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x 10-5 vs. Saline/cocaine). However, PDTC did not show any additional effects against protective

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activity mediated by genetic overexpression of GPx-1.

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vs. saline-treated WT mice). The behavioral sensitization is more pronounced in GPx-1 KO

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4. Discussion

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Cocaine treatment was found to significantly induce GPx-1 expression, oxidative

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parameters, NFκB activity, c-Fos-IR in the striatal complex of mice. Importantly, oxidative

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parameters, NFκB activity, and c-Fos-IR were more pronounced in GPx-1 KO than in WT mice, and 15

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were attenuated by PDTC, a NFκB inhibitor. In addition, PDTC did not demonstrated any additional

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positive effects against the protective potentials mediated by the genetic overexpression of GPx-1,

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suggesting that NFκB is a critical mediator of the protective potential of GPx-1 in cocaine-induced

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CPP and behavioral sensitization. Here, we suggest that cocaine treatment facilitates NFκB nuclear

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translocation and DNA binding activity in striatal tissues in response to oxidative stress, and that

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cocaine-induced oxidative stress activates NFκB induction, which could possibly be attenuated by

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the compensative induction of GPx-1.

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In our study, repeated cocaine treatment resulted in a significant increase in superoxide

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dismutase (SOD) activity in the striatum of wild type mice, but did not involve in a concomitant

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increase in GPx activity (Supplementary Fig. S3). Increased SOD activity may lead to an

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accumulation of H2O2, which in the absence of simultaneous increases in the activity of GPx, could

12

induce a Fenton reaction, leading to the formation of lipid peroxidation/protein oxidation, resulting

13

in irreversible cellular damage (Shin et al., 2014). Our observation of increased ROS/lipid

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peroxidation/protein oxidation products implies that GPx activity, rather than increased SOD,

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modulates these endpoints. However, the underlying mechanism of cocaine-induced decrease in

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glutathione-S-transferase (Supplementary Fig. S3E-F) remains to be elucidated.

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It is well-known that dopamine plays a crucial role in cocaine-induced CPP and

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behavioral sensitization. The significant increase of dopamine release due to the binding of

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cocaine to the transporter sites of monoamines results in the inhibition of their uptake in the

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presynaptic neuron (Foley, 2005; Vitcheva et al., 2015). Therefore, we speculate that cocaine-

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induced increase in extracellular dopamine will be more pronounced in GPx-1 KO than in WT

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mice. In addition, this increase in extracellular dopamine might be attenuated by GPx-1

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overexpression or PDTC, although evidence for this is lacking. Similarly, we cannot rule out the

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possibility that cocaine-induced increase in c-Fos-immunoreactivity in the NAc of GPx-1 KO

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(Fig. 5A) might be comparable to increase in extracellular dopamine levels, and this

3

phenomenon may be less pronounced in GPx-1 overexpression (Fig. 5B) or PDTC treatment in

4

mice than GPx-1 depletion in mice.

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Lubos et al. (2011) indicated that GPx-1 overexpression itself has been shown to initially

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and significantly suppress the activation of NFκB pathway. Therefore, cocaine-induced CPP and

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behavioral sensitization cannot be further attenuated by PDTC in GPx-1 TG mice (Figs. 6-7).

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Similarly, we have demonstrated this phenomenon in our previous reports (Shin et al., 2018;

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Tran et al., 2017).

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Most previous studies have emphasized the putative role of oxidative stress as a crucial

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mediator for cocaine-induced neuronal cell death in brain reward systems (Kovacic, 2005). In

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line with current result, previous studies suggest that oxidative stress in the brain reward system

13

is associated with cocaine psychomotor responses (Numa et al., 2008; Uys et al., 2011). Previous

14

investigations

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tetramethylpiperidine-1-oxyl (TEMPOL) can attenuate oxidative stress in the prefrontal cortex

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(PFC) and NAc as well as the development of behavioral sensitization induced by cocaine

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(Numa et al., 2008).

that

the

superoxide

scavenger

4-hydroxy-2,2,6,6-

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have

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Consistently, TEMPOL is also involved in regulating cocaine reward as measured by the

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CPP model in rats. Substantial behavioral changes in the TEMPOL-administered animals

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correlated with attenuation of cocaine-induced oxidative damage in their NAc and PFC (Beiser

21

et al., 2017).

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Jang et al. (2015) demonstrated that ROS produced by cocaine exposure is localized in 17

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the nucleus accumbal neurons. In addition, they observed that intra-NAc infusion of TEMPOL

2

decreases ROS formation in the accumbal neurons, and cocaine self-administration in rats,

3

suggesting that cocaine drug intoxication is associated with generation of superoxide in neurons

4

of the NAc. Because the basal activity of SOD in NAc was lowest in the brain of rats (Carvalho

5

et al., 2001), it is plausible that nucleus accumbal oxidative burdens mediate cocaine drug

6

dependence. In addition, it is recognized that alteration of enzymatic oxidants may result in

7

accumulations of hydrogen peroxide/peroxide (Shin et al., 2017). Recently, we demonstrated,

8

for the first time, that hydrogen peroxide scavenger GPx-1 plays a protective role in memory

9

impairments and behavioral sensitization induced by MA (Mai et al., 2018c; Mai et al., 2018d;

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Shin et al., 2017; Tran et al., 2018b). Our reports support current finding that GPx-1 gene

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attenuates cocaine-induced CPP and behavioral sensitization via antioxidant potential by

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modulation of NFκB in mice.

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NFκB binding sites were identified in the promotor regions of genes encoding for

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oxidative/inflammatory cytokines (i.e., IL-1, IL-6, and TNFα). It is interesting that the

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expression of these cytokines is dependent on activated NFκB and, in turn, these cytokines can

16

stimulate this transcription factor (Lee et al., 2001). Thus, it may be possible that cocaine-

17

mediated cellular effects use NFκB to amplify their own signals.

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It has also been shown that the activation of NFκB can enhance the ability of NFκB to

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bind DNA and induce gene transcription (Natoli et al., 2005; Wan and Lenardo, 2009). ROS are

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critical intermediates for various NFκB-activating signals (Schreck et al., 1992). Since it has

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been shown that the neurotoxic effects of drugs of abuse are often associated with oxidative

22

stress among other mechanisms (Herrera et al., 2003; Numa et al., 2008), NFκB could be

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involved in drug-induced neuronal dysfunction and neurotoxicity. 18

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Cocaine can decrease glutathione-dependent system and enhance NFκB and activator

2

protein-1 (AP-1) binding activities as well as stimulate gene expression regulated by these

3

transcription factors. The AP-1 protein c-Fos encodes nuclear transcription of genes involved in

4

the transmission of inter- and intracellular information through multiple signaling transduction

5

pathways. c-Fos may function in coupled short-term changes in cellular phenotype by

6

modulating the expression of specific target genes. It is well-established that dopamine receptor

7

stimulation or psychostimulation (i.e., MA and cocaine) increases c-Fos expression, eventually

8

resulting in oxidative damage and abnormal behavior. We observed that behavioral responses

9

induced by MA (Mai et al., 2018c; Shin et al., 2009) are comparable to those induced by cocaine

10

(current finding). NAc belongs to striatal complex and NAc is recognized as the most sensitive

11

region in response to the neuronal stimulation. For example, c-Fos-IR was more pronounced in

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NAc than in other striatal areas (Gerth et al., 2017; Mai et al., 2018c; Shin et al., 2004b; et al.,

13

2013). Consistently, behavioral responses induced by MA or cocaine paralleled those of c-Fos-IR

14

in NAc. Enhanced c-Fos-IR prevented by the overexpression of GPx-1 gene, reflecting that the

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protective potential of GPx-1 (antiperoxidative system) in CPP and behavioral sensitization

16

induced by cocaine.

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The nucleus accumbens (NAc) is a part of the mesolimbic reward circuit, and is a major

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target of drugs of abuse, including opiates and psychostimulants (Boileau et al., 2003; Salgado

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and Kaplitt, 2015). Previous exposure to cocaine increases preference for the drug and increases

20

self-administration of the drug (Ahmed and Koob, 1998; Ahmed et al., 2003; Ferrario et al.,

21

2005; Renthal et al., 2007; Shippenberg and Heidbreder, 1995). Drug regimens that increase the

22

frequency of motivated behaviors also cause long-lasting structural changes within the NAc.

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Chronic cocaine treatment increased the NFκB activity in the striatal complex (including NAc),

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while inhibition of NFκB activity blocked the cocaine-induced behavioral response. Here, we

2

observed that NFκB inhibition significantly ameliorates CPP and behavioral sensitization

3

induced by cocaine in GPx-1 KO mice. However, NFκB inhibition did not show any additive

4

effects in response to protective potentials conveyed by genetic overexpression of GPx-1,

5

suggesting that NFκB is a neuropsychoprotective target for GPx-1 gene in response to cocaine

6

drug dependence.

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GPx mRNA was found in all regions of basal ganglia (Kunikowska and Jenner, 2002).

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This wide distribution of GPx mRNA throughout rat basal ganglia correlates well with that of its

9

protein in both human (Damier et al., 1993) and murine brain tissue (Trepanier et al., 1996), and

10

enzyme activity in rodent brain tissue (Brannan et al., 1980). GPx has been found in glial cells,

11

in particular astrocytes (Pearce et al., 1997). The striatal area appeared to express higher levels of

12

GPx mRNA than other basal ganglial areas (Kunikowska and Jenner, 2002), suggesting that the

13

striatal area may be relatively vulnerable to oxidative damage, and thus dependent upon the high

14

antioxidant capacity provided by GPx. Importantly, disruption in astrocyte-mediated modulation

15

of neural function has been implicated in the abusive process (Verkhratsky and Parpura, 2016).

16

In addition, Scofield et al. (2016) provide a novel evidence of astrocyte dysfunction within the

17

NAc of cocaine-experienced animals. Therefore, role of GPx-1 in cocaine-caused astrocytic

18

alteration in NAc remains to be further elucidated.

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In summary, in this study, we demonstrate that the GPx-1 gene protects against CPP and

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behavioral sensitization induced by cocaine by inhibiting oxidative burdens via modulation of

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NFκB signaling. Finally, we propose that the GPx-1 gene offers a potential therapeutic target

22

against drug dependence induced by cocaine, although precise mechanism remains to be further

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characterized. 20

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1 2

Conflict of interest

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The authors have no conflicts of interest to declare

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Acknowledgements

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This study was supported by a grant (14182MFDS979) from the Korea Food and Drug

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Administration, by Basic Science Research Program through the National Research Foundation

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of Korea (NRF) funded by the Ministry of Science and ICT (#NRF-2017R1A2B1003346 and

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#NRF-2016R1A1A1A05005201), Republic of Korea. Huynh Nhu Mai, and Naveen Sharma

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were supported by the BK21 PLUS program, National Research Foundation of Korea, Republic of

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Korea. The English in this manuscript has been checked by native speakers of English

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(https://app.editage.co.kr/orders/download-files/CAUNE_431P1).

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Figure legends

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Fig. 1. Experimental schedule for understanding on the role of glutathione peroxidase-1 (GPx-1)

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in response to conditioned place preference (CPP) and behavioral sensitization (BS) induced by

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cocaine in mice. For the CPP paradigm, mice received cocaine (15 mg/kg, i.p.) 7 times every

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other day. For the BS paradigm, mice received final (8th) cocaine (15 mg/kg, i.p.) after the

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withdrawal period (7 d) post-CPP paradigm. A NFκB inhibitor, PDTC (50 mg/kg, i.p.) was

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administered 1 h prior to each cocaine injection. AD = adaptation. COC = cocaine. Sal = saline.

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LOC = locomotor activity.

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Fig. 2. Cocaine-induced changes in GPx-1 expression in the striatum of wild type (WT), non-

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transgenic (non-TG) and GPx-1 overexpressing transgenic (GPx-1 TG) mice. Sal = Saline. Each

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value is the mean ± S.E.M of six animals. *p<0.05, **p<0.01 vs. corresponding saline. #p < 0.01

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vs. cocaine/non-TG mice. &p < 0.05 vs. corresponding non-TG saline [t-test (A) or two-way

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ANOVA (B). Post-hoc Fisher’s LSD pairwise comparisons were followed (B)].

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Fig. 3. Cocaine-induced changesa in reactive oxygen species (ROS)(A, B), 4-hydroxy-2-nonenal

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(HNE) (C, D), and protein carbonyl (E, F) levels in the striatum of wild type (WT), GPx-1

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knockout (GPx-1 KO), non-transgenic (non-TG) and GPx-1 overexpressing transgenic (GPx-1

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TG) mice. Sal = Saline. Each value is the mean ± S.E.M of six animals. *p < 0.05, **p < 0.01 vs.

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corresponding saline, #p < 0.05, ##p < 0.01 vs. cocaine/WT or cocaine/non-TG (two-way ANOVA

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followed by Fisher’s LSD pairwise comparisons).

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Fig. 4. Cocaine-induced changes in cytosolic (A, B) and nuclear NFκB expression (C, D), and

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NFκB DNA binding activity (E, F) in the striatum of wild type (WT), GPx-1 knockout (GPx-1

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KO), non-transgenic (non-TG) and GPx-1 overexpressing transgenic (GPx-1 TG) mice. Sal =

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Saline. Each value is the mean ± S.E.M of six animals. *p < 0.05, **p < 0.01 vs. corresponding

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saline, #p < 0.05, ##p < 0.01 vs. cocaine/WT or cocaine/non-TG (two-way ANOVA followed by

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Fisher’s LSD pairwise comparisons).

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Fig. 5. Effects of pyrrolidine dithiocarbamate (PDTC, a NFκB inhibitor) on cocaine-induced c-

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Fos-IR in the nucleus accumbens of wild type (WT), GPx-1 knockout (GPx-1 KO), non-

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transgenic (non-TG) and GPx-1 overexpressing transgenic (GPx-1 TG) mice. Each value is the

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mean ± SEM of six mice. *p<0.05, **p<0.01 vs. corresponding saline. #p < 0.05, ##p < 0.01 vs.

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cocaine/WT or cocaine/non-TG. ∫p < 0.01 vs. cocaine/GPx-1 KO (three-way ANOVA followed

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by Fisher’s LSD pair-wise comparisons). Scale bar = 200 µm.

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Fig. 6. Effects of pyrrolidine dithiocarbamate (PDTC, a NFκB inhibitor) on cocaine-induced

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conditioned place preference (CPP) in wild type (WT), GPx-1 knockout (GPx-1 KO) (A), non-

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transgenic (non-TG) and GPx-1 overexpressing transgenic (GPx-1 TG) (B) mice. Each value is

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the mean ± SEM of ten mice. *p < 0.01 vs. corresponding saline. #p < 0.05,

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cocaine/WT or cocaine/non-TG. ∫p < 0.01 vs. cocaine/GPx-1 KO (three-way ANOVA followed

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by Fisher’s LSD pair-wise comparisons).

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##

p < 0.01 vs.

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Fig. 7. Effects of pyrrolidine dithiocarbamate (PDTC, a NFκB inhibitor) on cocaine-induced

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behavioral sensitization in wild type (WT), GPx-1 knockout (GPx-1 KO) (A), non-transgenic 33

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(non-TG) and GPx-1 overexpressing transgenic (GPx-1 TG) (B) mice. Each value is the mean ±

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SEM of ten mice. *p < 0.05, **p < 0.01 vs. corresponding saline. #p < 0.01 vs. cocaine/WT or

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cocaine/non-TG. ∫p < 0.01 vs. cocaine/GPx-1 KO (three-way ANOVA followed by Fisher’s LSD

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pair-wise comparisons).

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Highlight Treatment with cocaine resulted in a compensative induction of GPx-1

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GPx-1 gene attenuated cocaine-induced oxidative potentials in the striatum

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NFkB is a critical target for the GPx-1 activity against cocaine drug dependence

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