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Neuroscience 145 (2007) 621–630

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OCAINE INCREASES ENDOPLASMIC RETICULUM STRESS PROTEIN
XPRESSION IN STRIATAL NEURONS
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. H. SHIN,a S. BIAN,a Y.-B. SHIM,b M. A. RAHMAN,b

. T. CHUNG,c J. Y. KIM,d J. Q. WANGe

ND E. S. CHOEa*

Department of Biology and Center for Innovative BioPhysio Sensorechnology, Pusan National University, 30 Jangjeon-dong, Kumjeong-u, C6-311, Pusan 609-735, Korea

Department of Chemistry and Center for Innovative BioPhysio Sensorechnology, Pusan National University, Pusan 609-735, Korea

Department of Life Science and Biotechnology, Dong-Eui University,usan 614-714, Korea

Department of Physiology, Yeungnam University College of Medi-ine, Daegu 705-717, Korea

Department of Basic Medical Science, University of Missouri–Kansasity, School of Medicine, Kansas City, MO 64108, USA

bstract—Cocaine administration upregulates the levels ofxtracellular glutamate and dopamine in the striatum. Activa-ion of the receptors alters calcium homeostasis in striataleurons leading to the expression of the endoplasmic retic-lum (ER) stress proteins. It was therefore hypothesized thatocaine upregulates the expression of the ER stress pro-eins, immunoglobulin heavy chain binding protein (BiP),re1� and perk via glutamate and dopamine receptor activa-ion. A novel glutamate microbiosensor and Western immu-oblot analyses were mainly performed to test the hypothe-is in the rat dorsal striatum. The results showed that i.p.njection of repeated cocaine (20 mg/kg) for nine consecutiveays significantly increased extracellular glutamate levelshile acute cocaine injection did not. However, the immuno-

eactivities (IR) of the ER stress proteins in the dorsal stria-um were significantly increased by either acute or repeatedocaine injections as compared with saline controls. Intrastri-tal injection (i.s.) of the selective group I metabotropic gluta-ate receptor (mGluR) antagonist N-phenyl-7-(hydroxyimino)

yclopropa[b]chromen-1a-carboxamide (PHCCC; 25 nmol) orhe mGluR5 subtype antagonist 2-methyl-6-(phenylethynyl)pyri-ine hydrochloride (MPEP; 2 and 25 nmol) significantly de-reased repeated cocaine-induced increases in the IR of the ERtress proteins in the injected dorsal striatum. Similarly, theelective D1 antagonist (R)-(�)-7-chloro-8-hydroxy-3-methyl--phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochlorideSCH23390; 0.1 mg/kg, i.p.) or the N-methyl-D-aspartate an-

Corresponding author. Tel: �82-51-510-2272; fax: �82-51-581-2962E. S. Choe).-mail address: [email protected] (E. S. Choe).bbreviations: AsOx, ascorbate oxidase; BiP, immunoglobulin heavyhain binding protein; CP, conductive polymer; ER, endoplasmic re-iculum; GluOx, glutamate oxidase; IR, immunoreactivity; i.s., intra-triatal injection; mGluR, metabotropic glutamate receptor; MK801,izocilpine/(5S,10R)-(�)-5-methyl-10,11-dihydro-5H-ibenzo[a,d]yclohepten-5,10-imine maleate; MPEP, 2-methyl-6-(phenylethynyl)yridine hydrochloride; NMDA, N-methyl-D-aspartate; PHCCC,-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide;CH23390, (R)-(�)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-

letrahydro-1H-3-benzazepine hydrochloride; SDS, sodium dodecylulfate; UPR, unfolded protein response.

306-4522/07$30.00�0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reseroi:10.1016/j.neuroscience.2006.12.013

621

agonist dizocilpine/(5S,10R)-(�)-5-methyl-10,11-dihydro-5H-benzo[a,d]cyclohepten-5,10-imine maleate (MK801; 2 nmol,.s.) decreased acute or repeated cocaine-induced the IR ofhe ER stress proteins in the dorsal striatum. These datauggest that cocaine upregulates expression of the ER stressroteins in striatal neurons via a mechanism involving acti-ation of glutamate and dopamine receptors. © 2006 IBRO.ublished by Elsevier Ltd. All rights reserved.

ey words: drugs of abuse, BiP, Ire1�, perk, glutamate, mi-robiosensor.

ocaine administration is known to upregulate the levels ofxtracellular glutamate and dopamine in the striatum. Ac-ivation of the receptors by cocaine disrupts calcium ho-eostasis that may express the endoplasmic reticulum

ER) stress proteins in the striatum. Since ER stress haseen considered to be one of the most important routeshat are involved in neuronal cell apoptosis (Cadet et al.,003; Jayanthi et al., 2004), it is interesting to understandutative mechanisms underlying cocaine-mediated ERtress protein expression in the striatum.

ER is the site of synthesis, folding and assembly ofecretory proteins, which is controlled in part by immuno-lobulin heavy chain binding protein (BiP), an ER Hsp70amily member. BiP, the first identified component of theR quality control apparatus, was found by virtue of itsssociation with the unassembled, non-transported heavyhains produced in pre-B cell lines (Haas and Wabl, 1983).isturbance of normal cellular physiology can affect theiosynthesis of proteins in the ER, resulting in the accu-ulation of unfolded proteins. Cells under stress activate a

ignaling cascade termed the unfolded protein responseUPR) to prevent the formation of insoluble protein aggre-ates (Kaufman, 1999). Prolonged UPR activation leads topoptotic cell death (Patil and Walter, 2001). The UPRathway transmits information about protein folding status

n the ER lumen to the cytoplasm and the nucleus. ThreeR-transmembrane proteins have been identified as the

ransducers of the UPR: Ire1, perk and ATF6. Ire1 anderk have cytoplasmic serine/threonine kinase domains. Inase of Ire1 RNase domain is also present in the cytoplas-ic domain. Under the ER stress the luminal domains areomodimerized and subsequently autophosphorylated inhe cytoplasmic domains (Cox et al., 1993; Mori et al.,993; Harding et al., 1999).

The present study, therefore, was performed to test theypothesis that cocaine upregulates expression of the ERtress proteins in the striatum since cocaine increases

evels of glutamate (Hotsenpiller and Wolf, 2003; McFar-
and et al., 2003; Smith et al., 1995) and dopamineved.

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E. H. Shin et al. / Neuroscience 145 (2007) 621–630622

Jocham et al., 2006; Izawa et al., 2006), which furtheregulates calcium homeostasis. A novel electrochemicallutamate microbiosensor and Western blotting analysesere mainly applied to test the hypothesis in the dorsaltriatum.

EXPERIMENTAL PROCEDURES

nimals

dult male Sprague–Dawley rats (200–250 g) were obtained fromyo-Chang Science Co. (Daegu, Korea). Rats were individuallyoused in a controlled environment during all experimental treat-ents. Food and water were provided ad libitum and rats wereaintained on a 12-h light/dark cycle. On the day of the experi-ent injection was made in a quiet room to minimize stress. Allnimal use procedures were approved by the Institutional Animalare and Use Committee and were accomplished in accordanceith the provisions of the NIH “Guide for the Care and Use ofaboratory Animals.”

xperimental design

ive separate experiments were conducted in this study. The firstxperiment was designed to investigate whether acute or re-eated cocaine alters extracellular glutamate release using alutamate microbiosensor developed in our research group (seeelow). Rats were randomly divided into four groups (n�3–4 perroup). Each rat received i.p. injection of acute saline, acuteocaine, repeated saline or repeated cocaine (20 mg/kg). Forepeated cocaine, rats received cocaine (20 mg/kg, i.p.) once dailyor nine consecutive days. The second experiment was designedo investigate whether acute or repeated cocaine induces expres-ion of the ER stress proteins using Western blotting analysis.ats were randomly divided into four groups (n�4–5 per group).ach rat received i.p. injection of acute saline, acute cocaine,

epeated saline or repeated cocaine. Parallel to the sensor exper-ment, each rat received i.p. injection of cocaine once daily for nineonsecutive days for repeated cocaine. The third and fourth ex-eriments were designed to investigate whether group I metabo-ropic glutamate receptors (mGluRs) regulate expression of theR stress proteins by repeated cocaine using Western blot anal-sis. Rats were randomly divided into four groups (n�4–5 perroup) for each experiment. The selective group I mGluR antag-nist N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-car-oxamide (PHCCC; 25 nmol) (Tocris Cookson, Ballwin, MO, USA)r the selective mGluR5 subtype antagonist 2-methyl-6-(phenyl-thynyl)pyridine hydrochloride (MPEP; 2 and 25 nmol) (Tocrisookson) was infused into the center of the dorsal striatum 0.5

BiP), 1 (perk) or 4 (Ire1�) h after each or final cocaine injectioni.p.) depending on the maximum induction of the proteins asevealed by the second experiment. The fifth experiment wasesigned to investigate whether D1 or N-methyl-D-aspartateNMDA) receptor activation by acute or repeated cocaine regu-ates expression of the ER stress proteins using Western blotnalysis. Rats were randomly divided into four groups (n�4–5er group) for acute or repeated experiment. The selective1 receptor antagonist (R)-(�)-7-chloro-8-hydroxy-3-methyl--phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochlorideSCH23390; 0.1 mg/kg, i.p.) (Tocris Cookson) or the selectiveMDA antagonist dizocilpine/(5S,10R)-(�)-5-methyl-10,11-di-ydro-5H-ibenzo[a,d]cyclohepten-5,10-imine maleate (MK801;nmol) (Tocris Cookson) was infused into the center of the

orsal striatum 0.5 (BiP), 1 (perk) or 4 (Ire1�) h after each ornal cocaine injection (i.p.) as conducted by the third and fourthxperiments. The doses of antagonists were determined fromur previous studies (Wang and McGinty, 1995; Choe and
ang, 2001, 2002; Choe et al., 2005). Throughout the exper- p
ment, cocaine (Belgopia, Louvain-La-Neuve, Belgium) wasissolved in physiological saline (0.9% sodium chloride).

reparation of a glutamate microbiosensor

he glutamate microbiosensor was prepared as described previ-usly (Rahman et al., 2005). Briefly, a micropipette was made byulling a glass capillary (inner diameter 1.1–1.2 mm, length5 mm) (Chase Scientific Glass Inc., Rockwood, TN, USA) with aicroelectrode puller. The tip of the micropipette was broken to

he desired inner diameter (150 �m) with a diamond cutter. At-wire (Pt 99.999%, 100 �m in diameter) (Johnson Matthey Inc.,est Chester, PA, USA) of about 2 mm long was connected withcopper wire for electrical contact using electroconductive silver

aste (Dotite Electroconductives, Type D-500) (Fujikura Kaseio., Tokyo, Japan). This copper wire connected Pt electrode was

ntroduced into the micropipette and advanced until it protrudedbout 0.2–0.3 mm from hollow opening of the micropipette. Then,he tip of the micropipette was heated gently in a butane flamentil the glass collapsed and was sealed the Pt electrode. Thether end of the micropipette was fixed using epoxy resin. Theotal length of the Pt microelectrode was 40 mm. The Pt micro-lectrode was coated with a conductive polymer (CP) through thelectropolymerization of 5,2=:5=,2�-terthiophene-3=-containing car-oxylic acid containing monomer (Lee and Shim, 2001) by cycling

he potential three times at a fast scan rate. The CP-coated Pticroelectrode was then immersed in a 0.1 M phosphate buffer

olution (pH�7.0) containing10 mM 1-ethyl-3 (3-(dimethylamino)-ropyl) carbodimide to activate the carboxylic acid groups of theP. Then, glutamate oxidase (GluOx, EC. 1.4.3.11, 10.8 unitsg�l) (Sigma-Aldrich, St. Louis, MO, USA) and ascorbate oxidase

AsOx, EC. 1.10. 3. 3, 100 units mg�l) (Sigma-Aldrich) wereo-immobilized onto the CP layer through the formation of cova-ent bonds between the carboxylic acid groups of the CP and themine groups of the GluOx and AsOx.

The working mechanism of glutamate microbiosensor ishown in Fig. 1. The GluOx/CP modified Pt microelectrode wassed as a working electrode for the detection of glutamate. Am-erometric responses were recorded using a Potentiostat (KST-2, Kosentech, Busan, Korea) with two electrode configurations:glutamate microbiosensor and a micro-Ag/AgCl (in saturated

Cl) electrode as working and reference/counterelectrodes, re-pectively. The experiments were carried out by applying a po-ential of �0.40 V at GlOx/CP/Pt microbiosensor to oxidize the

2O2 generated from the enzymatic reaction. All the microbiosen-ors were calibrated before and after measurements.

urgery and glutamate microbiosensor detection

n the day of the experiment, rats were anesthetized with 8%hloral hydrate (6 ml/kg, i.p.) and placed in a Stoelting stereotaxicpparatus. Under aseptic conditions, the enzyme-coated elec-rode and the reference electrode of glutamate microbiosensorere implanted at the coordinates of 1 mm anterior to bregma,.5 mm right to midline and 4 mm below the surface of the skull.he sensor tip was inserted unilaterally into the central part of theight dorsal striatum. The output of electrodes was connected tohe electrochemical detector, Potentiostat. The electrochemicalignal can be read and stored by the workstation. The measure-ent of glutamate levels in the dorsal striatum can be performed

mmediately after implantation of the microbiosensor electrodes.efore the measurement, one sensor among the same batch wasalibrated with a series of glutamate standard solutions. In thistudy, 30 min time point was selected based on the study deliv-red from time course after repeated cocaine injections in whichlutamate levels at 30 min were significantly increased as com-
ared with acute and saline injections (Rahman et al., 2005).

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ntrastriatal drug infusion

ats were anesthetized with 8% chloral hydrate (6 ml/kg, i.p.) andlaced in a Stoelting stereotaxic apparatus. Under aseptic condi-ions, a 23-guage stainless steel guide cannula (inner diameter:.29 mm, 10 mm in length) was implanted at the coordinates ofmm anterior to bregma, 2.5 mm right to midline and 4 mm below

he surface of skull. The guide cannula was sealed with a stainlessteel wire of the same length. Rats were allowed 3 days’ recoveryrom surgery. On the day of the experiment, the inner steel wireas replaced by a 30-gauge stainless steel injection cannula

inner diameter: 0.15 mm) with a length of 12.5 mm that protruded.5 mm beyond the guide cannula. Through the injection can-ula, PHCCC, MPEP or MK801 was infused unilaterally into theentral part of the right dorsal striatum in a volume of 1 �l at aate of 0.2 �l/min in freely moving rats. Progress of injectionas monitored by observing movement of a small air bubble

hrough a length of precalibrated PE-10 tubing inserted be-ween the injection cannula and a 2.5 �l Hamilton microsyringeHamilton, Reno, NV, USA). After completion of injection, thenjector was left in place for an additional 5 min to reduce anyossible backflow of the solution along with the injection tract.ll injections were made in the home cage.

estern blotting

ats were deeply anesthetized with 8% chloral hydrate (6 ml/kg,.p.) and decapitated at 0.5, 1, 2 or 4 h after final injection. Brainsere removed, frozen in isopentane at �70 °C and stored in aeep freezer. Sections were serially cut in a cryostat and injectedight dorsal striatum was removed with a steel borer (inner diam-ter: 2 mm). All tissue samples were lysed in sodium dodecylulfate (SDS) sample buffer for 5 min at 95 °C. The samples werehen sonicated for 30 s on ice and centrifuged for 10 min at2,000�g with an Eppendorf tabletop centrifuge (Eppendorf,amburg, Germany). The supernatant was resolved using 10%DS–polyacrylamide gel electrophoresis (SDS-PAGE) and theeparated proteins were transferred to a nitrocellulose membrane.he membrane was blocked with blocking buffer (5% skim milk in

ig. 1. Schematic diagram illustrating a glutamate microbiosensor syevels evoked by saline or cocaine administration. Dorsal striatum cauas performed at the same time points measured with the microbiose

BST). The membrane was probed with each primary antiserum w

gainst BiP (1:1000), Ire1� (1:1000), perk (1:1000) or actin (1:000) overnight at 4 °C on a shaker. BiP antiserum was kindlyrovided by Dr. L. M. Hendershot (Department of Tumor Celliology, St. Jude Children’s Research Hospital, Memphis, TN,SA). Perk and Ire1� antisera were purchased from Santa Cruziotechnology (Santa Cruz, CA, USA), and actin antiserum wasurchased from Sigma-Aldrich. The membrane was then incu-ated with appropriate secondary antisera for 1 h. Immunoreac-ive proteins were detected by enhanced chemiluminescence re-gents (ECL; Amersham Pharmacia Biotech, Piscataway, NJ,SA) on X-ray films.

mmunohistochemistry

arallel to the Western blot analysis, rats were deeply anesthe-ized with 8% chloral hydrate (6 ml/kg, i.p.), and transcardiallyerfused with 4% paraformaldehyde at 4 °C. Brains were removednd post-fixed in 10% sucrose/4% paraformaldehyde for 2 h at°C and then placed in 20% sucrose/phosphate-buffered saline

PBS) at 4 °C overnight. Using a cryostat microtome, 30 �mrozen sections were cut. Two sections per BiP antiserum perrain were collected at striatal levels and processed for immuno-istochemistry with the method as described previously (Choe etl., 2004). Briefly, sections were incubated with BiP antiserum for0 h at 4 °C on a shaker. Sections were then incubated in goatnti-rabbit secondary antiserum (Vector Laboratories, Burlin-ame, CA, USA) for 1 h, followed by avidin–biotin–peroxidaseeagents (Elite Vectastain kit, Vector Laboratories) for 1 h at roomemperature. Diaminobenzidine was used as the chromogen andiCl2 was added to enhance reaction product.

uantitation of immunoreactivity (IR)

mmunoreactive protein bands on films were semi-quantified us-ng an imaging digital camera and NIH Image 1.62 software.riefly, film background was measured and saved as a “blankeld” to correct uneven illumination. The upper limit of the densitylice option was set to eliminate any background, and this value

its working model for the detection of altered extracellular glutamatemen (CPu) was removed to prepare for Western blot analysis, which

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tatistics

tatistical significance on the glutamate levels or the number ofmmunoreactive pixels per measured area between groups wasetermined using a one-way ANOVA on ranked data followedy a Tukey’s HSD (honestly significant difference) test in SASCary, NC, USA). Statistically significant level was taken as�0.05.

RESULTS

epeated cocaine, but not acute, increasedlutamate responses in the dorsal striatum

glutamate microbiosensor was applied to investigatehether acute or repeated cocaine elevates extracellu-

ar glutamate levels in the striatum. Fig. 2A showedurrent-time plots of glutamate responses obtainedpon the addition of varying amounts of standard gluta-

ig. 2. Alterations of extracellular glutamate levels by cocaine usingpon the addition of varying amounts of standard glutamate solution ins. glutamate concentration detected by the glutamate microbiosensohe final injection of saline, acute cocaine (20 mg/kg, i.p.) or repeated cesponse produced by the final injection of saline, acute or repeated coorsal striatum. * P�0.05 as compared with repeated saline and acut

ate solutions in the 0.1 M phosphate buffer solution at a

H 7.2. The linearity of the calibration curve for theurrent vs. glutamate concentration was qualifiedr�0.99) as presented in Fig. 2B. As shown in Fig. 2C,mperometric response produced at 30 min after re-eated cocaine is significantly increased as comparedith both saline and acute cocaine injections. Glutamateoncentrations in acute saline or repeated saline, acuteocaine and repeated cocaine were determined to be.3, 0.35 and 1.0 �M, respectively from the calibrationlot after in vivo measurement (Fig. 2B). Semiquantita-

ive analysis shown in Fig. 2D confirmed that rats treatedith daily cocaine injection for 9 days caused signifi-antly altered current response in the dorsal striatum asompared with repeated saline and acute cocaine at 30in time point.

cute or repeated cocaine increased BiP, Ire1�nd perk IR in the dorsal striatum

his experiment was designed to investigate whether

mate microbiosensor in the striatum. (A) Current-time plots obtainedM phosphate buffer solution at pH 7.2. (B) Calibration plots of currentrent-time plots detected by glutamate microbiosensor at 30 min after0 mg/kg, i.p., 9 days). (D) Semi-quantitative analysis on the glutamateepeated cocaine for 9 days significantly increased the response in thee groups.

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ig. 3. Western immunoblot analysis for the effect of cocaine on BiP (A), Ire1� (B), perk (C) and actin (D) IR in striatal neurons at different time points.emi-quantitative analysis confirms that both acute and repeated cocaine injections caused increases in the IR of BiP, Ire1� and perk in striataleurons (n�3–4 per group). L1, saline; L2, 0.5 h; L3, 1 h; L4, 2 h; L5, 4 h. * P�0.05 as compared with saline groups. Immunohistochemical

ocalization of BiP IR (E) induced by 30 min after the final injection of either acute or repeated cocaine was enhanced in the cytoplasmicompartments in striatal neurons arrow as compared with saline (a, saline; b, cocaine). Inset c was magnified from the neurons around arrow
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tress proteins in the dorsal striatum. The changes ofiP, Ire1� and perk IR in the dorsal striatum were mon-

tored at 0.5, 1, 2 and 4 h after both acute and repeatedocaine injections. The results showed that acute orepeated cocaine significantly increased the IR of theroteins as compared with actin controls. The BiP IRFig. 3A) was significantly increased at 1 h and wasrolonged up to 2 h after acute cocaine injection,hereas it was increased at 0.5 h and reached a peak ath after repeated cocaine injections. BiP IR induced by

cute or repeated cocaine was enhanced in the cyto-lasmic compartments of striatal neurons as comparedith saline injection (Fig. 3E). Similarly, perk IR (Fig. 3C)

ig. 4. Western blot analysis of the effect of the mGluR5 antagonist PiP (A), Ire1� (B) and perk (C) IR by cocaine in striatal neurons. Semignificantly attenuated repeated cocaine-induced BiP, Ire1� and perkroups; # P�0.05 as compared with 0.5 (A, BiP; E, Actin), 1 (C, perk)

as also significantly increased at 0.5 h and was pro- s

onged up to 2 h after acute cocaine injection, whereast was observed to be prolonged up to 1 h by repeatedocaine. However, Ire1� IR (Fig. 3B) was significantlyncreased at 2 and 4 h after acute and repeated cocainenjections, respectively. Control actin IR was not alteredt all time points as shown in Fig. 3D.

HCCC or MPEP decreased repeatedocaine-stimulated BiP, Ire1� and perk IRn the dorsal striatum

ince repeated cocaine increased glutamate responsesnd expression of the ER stress proteins in the dorsal

PHC; 25 nmol) and the mGluR5 antagonist MPEP (MPE 25 nmol) onative analysis confirms that pretreatment of either PHCCC or MPEPiatal neurons (n�3–4 per group). * P�0.05 as compared with saline, Ire1�) h groups.

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nfused intrastriatally to determine whether group IGluRs regulate BiP, Ire1� and perk induction by re-eated cocaine. Like saline or vehicle, PHCCC infusionlone did not alter the basal levels of BiP, Ire1� and perk

R (data not shown). As shown in Fig. 4, repeated co-aine injections significantly increased IR of the ERtress proteins as compared with the controls. In theresence of PHCCC, repeated cocaine induced much

ess BiP (Fig. 4A), Ire1� (Fig. 4B) and perk (Fig. 4C) IR.emi-quantitation confirmed that repeated cocaine in-reased IR of all the ER stress proteins in the dorsaltriatum, which was blocked by pretreatment withHCCC.

ig. 5. The effect of the D1 dopamine antagonist SCH23390 (0.1ocaine-induced BiP (A), Ire1� (B) and perk (C) IR in striatal neuronsK801 significantly decreased acute or repeated cocaine induction of
2, cocaine; L3, SCH23390�cocaine; L4, MK801�cocaine. * P�0.05 as compaocaine groups at 0.5 (A, BiP; E, Actin), 1 (C, perk) and 4 (B, Ire1�) h groups
A separate study was carried out to evaluate themportance of mGluR5 in the regulation of the ER stressrotein expression by repeated cocaine. Like saline orehicle, MPEP alone at the two doses (2 and 25 nmol,

ntrastriatal injection (i.s.)) had no significant effects onhe basal levels of BiP, Ire1� and perk IR in the dorsaltriatum (data not shown). Pretreatment of MPEP at a

ower dose (2 nmol) attenuated BiP, Ire1� and perk IRy repeated cocaine in the dorsal striatum. Similarly,PEP at a higher dose (25 nmol) also significantlyttenuated the IR of all three ER stress proteins inducedy repeated cocaine (Fig. 4).

or the NMDA antagonist MK801 (2 nmol) on acute or repeatedantitative analysis confirms that pretreatment of either SCH23390 orER stress proteins in striatal neurons (n�3–4 per group). L1, saline;

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CH23390 or MK801 decreased acute or repeatedocaine-stimulated BiP, Ire1� and perk IR in theorsal striatum

ince cocaine increases dopamine and glutamate levelsnd the ER stress protein expression in the dorsal stria-um, the D1antagonist SCH23390 (0.1 mg/kg, i.p.) or theMDA antagonist MK801 (2 nmol, i.s.) was infused toetermine the involvement of dopamine or NMDA recep-ors in the regulation of BiP, Ire1� and perk induction byither acute or repeated cocaine. The results demon-trated that SCH23390 or MK801 significantly decreasedcute or repeated cocaine induction of the IR of the ERtress proteins in the dorsal striatum (Fig. 5), whereasCH23390 or MK801 had no significant effects on theasal levels of BiP, Ire1� and perk IR in the dorsal striatumdata not shown).

DISCUSSION

he present data demonstrated that cocaine has the ca-ability to induce expression of the ER stress proteins andlutamate and dopamine receptors participate in the me-iation of the expression of those proteins induced bycute or repeated cocaine in the dorsal striatum.

epeated cocaine-regulated glutamate releasen the dorsal striatum

n this study, the glutamate response was significantlyncreased in the dorsal striatum by i.p. injection of re-eated, but not by acute cocaine. The altered glutamateoncentration by repeated cocaine was determined to behreefold greater than that of acute cocaine, suggesting anncrease in extracellular glutamate release in the dorsaltriatum by repeated cocaine injections. Our data are con-istent with previous studies from our laboratory and otherrain regions such as the nucleus accumbens and theentral tegmental area in which acute cocaine did not alterlutamate release, while repeated cocaine producedarked glutamate release (Rahman et al., 2005; Hotsen-iller and Wolf, 2003; McFarland et al., 2003). Cocaine at0 mg/kg increased glutamate levels over saline controls

n the nucleus accumbens (Smith et al., 1995), suggestinghat glutamate release is most likely due to the high dosef cocaine. However, we could not exclude the possibilityhat extracellular glutamate levels can be altered by acuteocaine because our data were determined only at 30 minime point.

Although the results from this study and others indicatestrong stimulation of glutamate release in the striatum in

esponse to cocaine stimulation, precise mechanism(s)nderlying this event are poorly understood. It has beenuggested that changes in the physiological activity ofeveral intracellular signaling proteins such as activator of

protein signaling 3 (AGS3) and cystine-glutamate ex-hanger may account for the facilitation of striatal gluta-ate release after cocaine administration (Kalivas et al.,003). In addition, amphetamine, another indirect dopa-
ine receptor agonist, increased glutamate release in the m
triatum (Nash and Yamamoto, 1993; Del Arco et al.,999) and this effect was thought to be mediated throughrans-synaptic circuits in the basal ganglia (Rawls andcGinty, 2000). Nevertheless, data from this study

howed a stimulatory effect of cocaine on glutamate re-ease. These data were obtained through a glutamate

icrobiosensor and are consistent with the results fromrevious reports using different methods. Thus, the gluta-ate microbiosensor could be an effective tool for moni-

oring the real-time changes in extracellular glutamate lev-ls in response to stimulant drug exposure.

egulation of the ER stress proteins by cocainen the dorsal striatum

he present data showed that both acute and repeatedocaine significantly increased induction of the ER stressroteins such as BiP, Ire1� and perk at several differentime points, suggesting that cocaine upregulates ER stressrotein expression in the dorsal striatum. The ER is an

mportant organelle that participates in cellular homeosta-is by regulating calcium signaling cascades and proteinolding (Ermak and Davies, 2002). The disruption of intra-ellular homeostasis or oxidative stress can cause ERtress in primary neuronal cell cultures and other cell linesMcCullough et al., 2001; Paschen and Frandsen, 2001).ecent studies demonstrated that the ER stress pathwaylays an important role in neuronal apoptosis (Sandersnd Wride, 1995; Hale et al., 1996; Vaux and Strasser,996). However, there has been almost no report to illu-inate the effect of cocaine uptake on the expression of

he ER stress proteins in the brain. Nassogne and cowork-rs (1997) reported that methamphetamine increased thexpression of BiP that is considered to be associated withPR and participates in the ER-induced apoptosis (Pas-hen and Frandsen, 2001). The increase in BiP levelsight serve a protective function because BiP overexpres-

ion protects cells against apoptotic insults (Morris et al.,997). Ire1� is also an ER-associated protein and is be-

ieved to initiate UPR that is responsible for protein foldingn the ER under stressed conditions (Miyoshi et al., 2000).nder the conditions of the ER stress UPR is downregu-

ated and thus apoptotic process in neurons is enhancedKatayama et al., 1999).

Our results also showed a significant increase in BiPnd Ire1� IR in the dorsal striatum, suggesting the ERtress can be induced by cocaine administration. A UPRan be also identified by activation of the ER-residentransmembrane protein kinase called perk, which leads tohe hyper-phosphorylation of eIF2 alpha, upregulation ofPR78, increased amounts of GADD153/CHOP, and

leavage of procaspase-12 in immortalized astrocytes, C1ells, and primary cultured astrocytes (Liu et al., 2004).t can be conceivable that cocaine-induced ER stress in-reases perk expression, which further induces neuronalpoptosis in the striatum. These findings suggest that co-aine is closely related to the induction of the ER stressroteins and contributes to neuronal cell apoptosis in thetriatum. Furthermore, our data also indicate that pretreat-
ent of the group I mGluR antagonist PHCCC, the

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GluR5 subtype antagonist MPEP or the NMDA antago-ist MK801 significantly attenuated induction of the ERtress proteins by repeated cocaine. It is likely that in-reased levels of glutamate by repeated cocaine activatetriatal neurons expressing glutamate receptors includingroup I mGluRs and NMDA. Thus, activation of glutamateeceptors in striatal neurons by repeated cocaine plays aritical role in the upregulation of the ER stress genexpression. Parallel with these data, infusion of the selec-ive D1 antagonist SCH2330 significantly decreased acuter repeated cocaine induction of the IR of the ER stressroteins in the dorsal striatum, suggesting that activation ofopamine receptors in striatal neurons by cocaine alsolays an important role in the upregulation of the ER stressrotein expression probably by interacting with NMDAChoe and McGinty, 2000). Therefore interaction of gluta-ate and dopamine receptors alters NMDA-mediated in-

racellular calcium homeostasis leading to the ER stressrotein expression in the striatum, although mechanisms

nvolving the precise intracellular routes to express the ERtress proteins from the receptors remain to be investi-ated.

CONCLUSION

n conclusion, this study investigated the involvement oflutamate and dopamine receptors in the regulation of ERtress proteins in response to cocaine administration in theat dorsal striatum. The results demonstrated that repeatedocaine, but not acute, augmented extracellular glutamateelease in the dorsal striatum. However, both acute andepeated cocaine significantly increased expression of theR stress proteins via activation of dopamine and gluta-ate receptors. These data suggest that stimulation oflutamate and dopamine receptors by cocaine possessesapability to express the ER stress proteins in the dorsaltriatum.

cknowledgments—This work was supported by the Korea Re-earch Foundation (KRF2004-002-H00023), the Ministry of Healthnd Welfare (02-PJ3-PG6-EV05-0001) and the Postdoctoral Sup-orting Grant from Pusan National University, Korea.

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(Accepted 5 December 2006)(Available online 14 February 2007)

cocaine increases endoplasmic reticulum stress …

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