The Effect of N-Acetylcysteine in the Nucleus Accumbens on Neurotransmission and Relapse to Cocaine Yonatan M

The Effect of N-Acetylcysteine in the Nucleus Accumbens on Neurotransmission and Relapse to Cocaine Yonatan M. Kupchik, Khaled Moussawi, Xing-Chun Tang, Xiusong Wang, Benjamin C. Kalivas, Rosalia Kolokithas, Katelyn B. Ogburn, and Peter W. Kalivas Background: Relapse to cocaine seeking has been linked with low glutamate in the nucleus accumbens core (NAcore) causing potentiation of synaptic glutamate transmission from prefrontal cortex (PFC) afferents. Systemic N-acetylcysteine (NAC) has been shown to restore glutamate homeostasis, reduce relapse to cocaine seeking, and depotentiate PFC-NAcore synapses. Here, we examine the effects of NAC applied directly to the NAcore on relapse and neurotransmission in PFC-NAcore synapses, as well as the involvement of the metabotropic glutamate receptors 2/3 (mGluR2/3) and 5 (mGluR5). Methods: Rats were trained to self-administer cocaine for 2 weeks and following extinction received either intra-accumbens NAC or systemic NAC 30 or 120 minutes, respectively, before inducing reinstatement with a conditioned cue or a combined cue and cocaine injection. We also recorded postsynaptic currents using in vitro whole cell recordings in acute slices and measured cystine and glutamate uptake in primary glial cultures. Results: NAC microinjection into the NAcore inhibited the reinstatement of cocaine seeking. In slices, a low concentration of NAC reduced the amplitude of evoked glutamatergic synaptic currents in the NAcore in an mGluR2/3-dependent manner, while high doses of NAC increased amplitude in an mGluR5-dependent manner. Both effects depended on NAC uptake through cysteine transporters and activity of the cysteine/glutamate exchanger. Finally, we showed that by blocking mGluR5 the inhibition of cocaine seeking by NAC was potentiated. Conclusions: The effect of NAC on relapse to cocaine seeking depends on the balance between stimulating mGluR2/3 and mGluR5 in the NAcore, and the efﬁcacy of NAC can be improved by simultaneously inhibiting mGluR5.

mate (6). The cocaine-induced decrease in extracellular glutamate Key Words: Addiction, cocaine, mGluR2/3, mGluR5, N-acetylcyste- Ϫ ine, nucleus accumbens is associated with reduced system Xc function (4,7) and reduced levels of its catalytic subunit, the catalytic light chain of the system Ϫ Xc heterodimer (8). Hence, restoring nonsynaptic glutamate by ulnerability to relapse is a cardinal feature of cocaine addic- Ϫ tion, and the glutamatergic projection from the prefrontal targeting system Xc has been evaluated as a pharmacotherapy in V cortex (PFC) to the nucleus accumbens core (NAcore) has drug addiction. N-acetylcysteine (NAC) is a cystine prodrug that restores extracellular glutamate in the NAcore by stimulating sys- been identified as a potentially important neural mediator of re- Ϫ lapse (1,2). Accordingly, many enduring changes are produced in tem Xc (4,7). NAC reduces desire for cocaine use (9) and the num- the physiology of this projection by the chronic administration of ber of cigarettes smoked (10) in humans and cocaine and heroin addictive drugs that have been used as a biological rationale in relapse in animal models (5,11,12). These effects are caused by NAC developing potential pharmacotherapies for treating addiction (3). normalizing extrasynaptic glutamate levels that activate presynap- A key observation along these lines is that withdrawal from chronic tic mGluR2/3 and thereby reduce synaptic glutamate release (13) cocaine is associated with reduced nonsynaptic extracellular gluta- and cocaine and heroin seeking in animal models (14–16). mate levels in the NAcore (2,4). Normally, extrasynaptic glutamate The use of NAC to inhibit drug seeking by increasing nonsynap- Ϫ of glial origin negatively regulates synaptic glutamate release at tic glutamate via activating system Xc will restore tone onto other PFC-NAcore synapses through activating presynaptic group II extrasynaptic glutamate receptors, in addition to mGluR2/3. This metabotropic glutamate receptors (mGluR2/3) (2). Reduced extra- raises a potential paradox because restoring tone to metabotropic synaptic glutamate after chronic cocaine is insufficient for normal glutamate receptor 5 (mGluR5) should promote reinstatement of mGluR2/3 activation, thereby increasing PFC-NAcore glutamate re- drug seeking. Thus, mGluR5 stimulation with a positive allosteric lease probability (5). modulator promotes reinstatement of drug seeking, while inhibit- A major contributor to the maintenance of extracellular gluta- ing mGluR5 reduces the reinstatement of drug seeking (17–21). mate in the NAcore is glial cystine/glutamate exchange (system Ϫ Here, we tested the hypotheses that NAC acts directly in the NAcore Xc ), which exchanges extracellular cystine for intracellular gluta- to reduce the reinstatement of cocaine seeking and glutamate synaptic transmission in PFC-NAcore synapses and that NAC acts via activating system X Ϫ. We then tested the hypothesis that NAC- From the Departments of Neurosciences (YMK, KM, XW, PWK) and Psychia- c mediated stimulation of mGluR2/3 and mGluR5 produces opposite try (X-CT, BCK), Medical University of South Carolina; and Honors College (RK, KBO), College of Charleston, Charleston, South Carolina. effects on glutamate transmission, akin to the opposite effects on Authors RK and KBO contributed equally to this work. the reinstatement of cocaine seeking. Finally, having validated op- Address correspondence to Yonatan M. Kupchik, Ph.D., Medical University posite effects of NAC-mediated stimulation of mGluR2/3 and of South Carolina, Department of Neuroscience, 173 Ashley Avenue, BSB mGluR5, we show that by blocking mGluR5 the behavioral efficacy 403, Charleston, SC 29425; E-mail: [email protected]. of NAC to inhibit the reinstatement of cocaine seeking is aug- Received Aug 26, 2011; revised Sep 28, 2011; accepted Oct 19, 2011. mented.

Methods and Materials Self-Administration and Extinction Procedures Rats were trained to self-administer cocaine (National Institutes Animal Housing and Surgery of Health, Bethesda, Maryland) in operant chambers with two re- All experiments were conducted in accordance with the Na- tractable levers. The self-administration regimen consisted of 2 tional Institute of Health Guidelines for the Care and Use of Labora- weeks (12 days) self-administration at Ͼ10 infusions per session. tory Animals, and the Institutional Animal Care and Use Committee Daily sessions lasted 2 hours, with an active lever press resulting in at the Medical University of South Carolina approved all proce- .2 mg in .05 mL cocaine infusion (dissolved in sterile .9% saline) over dures. Male Sprague-Dawley rats (250g) (Charles River Laboratories, 3 seconds, while inactive lever presses were of no consequence. A Indianapolis, Indiana) were single housed under controlled temper- 5-second cue tone (2900 Hz) and light followed each cocaine infu- ature and humidity with a 12-hour light/dark cycle. All behavioral sion. Cocaine infusions were also followed by a 20-second time-out, training occurred during the dark cycle. Rats were acclimated in the during which active lever presses had no consequence. vivarium for 1 week before surgeries and fed and watered ad libi- tum until 2 days before behavioral training, during which food was Reinstatement of Cocaine Seeking restricted to four pellets per day. Rats were anesthetized with ket- After extinction training, rats underwent a cocaine ϩ cue- amine hydrochloride (87.5 mg/kg Ketaset; Fort Dodge Animal primed (10 mg/kg, intraperitoneal [IP]) reinstatement trial. Follow- Health, Fort Dodge, Iowa) and xylazine (5 mg/kg; Lloyd Laborato- ing a counterbalanced crossover design, we microinjected .5 ␮L/ ries, Shenandoah, Iowa) and implanted with intravenous catheters. side of artificial cerebrospinal fluid (aCSF) or NAC (1 ␮gor10␮g) at For microinjection experiments, rats were stereotaxically im- a rate of .25 ␮L/minute 30 minutes before starting reinstatement planted with bilateral guide cannulas (26 gauge) aimed at NAcore trials (Figure 1B). In a second experiment, NAC (10 ␮g/.5 ␮L/side) according to Paxinos et al. (22)(ϩ1.8 mm anteroposterior, 1.5 mm was microinjected 30 minutes before cue-induced reinstatement mediolateral, and Ϫ5.5 mm dorsoventral from the skull surface). (i.e., presenting the light and tone previously associated with co- Intravenous catheters were flushed daily with cefazolin (.2 mL of .1 caine infusion), and a systemic injection (1 mg/kg, IP) of (2S)- g/mL) and heparin (.2 mL of 100 IU) to prevent infection and main- 2-Amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) pro- tain catheter patency, and rats recovered for a week before behav- panoic acid (LY341495) was administered 5 minutes before NAC ioral training. Yoked saline control animals received a noncontin- and 5 minutes before cue (Figure 1B). In a third experiment, cocaine gent infusion of saline in parallel with cocaine rats receiving a seeking was reinstated by a challenge injection of cocaine (10 mg/ self-administered infusion of cocaine. kg, IP). Two hours before cocaine, subjects were injected with NAC

Figure 1. Microinjection of N-acetylcysteine (NAC) in the nucleus accumbens core inhibits cocaine-seeking reinstatement. (A) The training procedure included 2 weeks of cocaine self-administration followed by 2 weeks of extinction training. (B) Experimental paradigm for reinstatement testing. (C) Cocaine ϩ cue-primed reinstatement was reduced by intra-accumbens NAC (1 or 10 ␮g/side). Data were evaluated using a one-way analysis of variance with repeated measures [F(2,29) ϭ 6.48, p ϭ .008]. (D) NAC (10 ␮g/side) inhibited cue-primed reinstatement and was reversed by metabotropic glutamate receptor 2/3 antagonist (LY341495, 1 mg/kg, intraperitoneal). One-way repeated measures analysis of variance, F(2,23) ϭ 3.74, p ϭ .050. (E) Illustration of cannula tips in the p Ͻ .05 compared with microinjection of artificialء .(accumbens. mm are rostral to Bregma (reprinted from [22], with permission from Elsevier, copyright 2006 cerebrospinal fluid, using a Dunnett’s post hoc comparison with vehicle. aCSF, artificial cerebrospinal fluid; SA, self-administration.

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(10 mg/kg, IP) or saline vehicle, and 15 minutes before cocaine- in triplicate in bicarbonate buffered Krebs-Ringer solution (pH ϭ primed drug seeking, rats were injected with saline or 3-[(2-Methyl- 7.4; Sigma-Aldrich, St. Louis, Missouri) (26). To isolate uptake via Ϫ 1,3-thiazol-4-yl)ethynyl]pyridine hydrochloride (MTEP) (.1 mg/kg, system Xc , aspartate (1 mmol/L) and acivicin (1 mmol/L) were ␥ IP). Each rat received a maximum of three reinstatement trials and added into each well to block system XAG and -glutamyl transpep- underwent 3 days of extinction training between each trial. The tidase, respectively (27). Cystine uptake experiments were initiated treatment order was randomized. Reinstatement testing lasted for by adding .5 ␮Ci/mL of L-35S-cystine and incubating the culture for 2 hours and active lever presses were counted but resulted in no 15 minutes at room temperature. The uptake was terminated by drug delivery. Although three different stimuli were used to initiate rapidly washing the cells three times with ice-cold media. Cells were the reinstatement of cocaine seeking (e.g., cue, cocaine, or cue ϩ solubilized in .5 mL of radioimmunoprecipitation assay buffer (Sig- cocaine), all have been shown to require activation of the projec- ma-Aldrich). Radioactivity and protein content in the cell lysate tion from the PFC to the NAcore (2). were measured. To control for possible effects of NAC to inhibit the reduction of 35S-cystine, some experiments were repeated using Slice Preparation sodium-free 3H-glutamate uptake (250 nmol/L, 51 Ci/mmol) to es- Ϫ Slices were prepared from saline-yoked or naïve animals. Rats timate the activity of system Xc (28). were anesthetized with ketamine (see above) and decapitated. The brain was removed and coronal accumbens brain slices (220 ␮m) Statistics and Histology (VT1200S Leica vibratome; Leica Microsystems, Wetzlar, Germany) To determine cannula placement in the NAcore, animals were were collected into a vial containing aCSF (in mmol/L: 126 sodium anesthetized with pentobarbital (100 mg/kg, IP) followed by ␮ chloride, 1.4 sodium phosphate, 25 sodium bicarbonate, 11 glu- intracardiac perfusion of 10% formalin. Coronal slices (100 m cose, 1.2 magnesium chloride, 2.4 calcium chloride, 2.5 potassium thick) were obtained and bilateral cannula location determined chloride, 2.0 sodium Pyruvate, .4 ascorbic acid, bubbled with 95% by an individual blind to the rats’ behavioral responses (22). oxygen and 5% carbon dioxide) and a mixture of 5 mmol/L Treatment groups and drug concentrations were compared us- kynurenic acid and 50 ␮mol/L D-(-)-2-Amino-5-phosphonopen- ing analyses of variance. For electrophysiological data and some tanoic acid. Slices were incubated at 35°C for 30 to 40 minutes then behavioral data, within-subject measurements were made using stored at 25°C. a repeated measures analysis of variance. A Bonferroni post hoc test was employed for multiple comparisons between groups or In Vitro Whole Cell Recording a Dunnett’s t test was employed for multiple comparisons with a All recordings were collected at 32°C (TC-344B; Warner Instru- single control group. ment Corporation, Hamden, Connecticut) in the dorsomedial NA- Results core, where the prefrontal inputs are most dense (23,24) and where microinjections were localized (Figure 1E). Neurons were visualized NAC in the NAcore Inhibits the Reinstatement of Cocaine with an Olympus BX51WI microscope (Olympus America, Center Seeking Valley, Pennsylvania). Inhibitory synaptic transmission was blocked To determine whether NAC acts in the NAcore to inhibit the ␮ with picrotoxin (50 mol/L). Multiclamp 700B (Axon Instruments, reinstatement of cocaine seeking, we microinjected NAC into the Union City, California) was used to record excitatory postsynaptic NAcore of rats extinguished from cocaine self-administration (Fig- Ϫ currents (EPSCs) under 80 mV voltage clamp whole cell configu- ure 1A). Using a counterbalanced crossover design, rats were micro- ⍀ ration. Glass microelectrodes (1–2 M ) were filled with cesium- injected with aCSF or NAC (1 or 10 ␮g/side) into the NAcore 30 based internal solution (in mmol/L: 124 cesium methanesulfonate, minutes before initiating reinstatement by a combination of condi- 10 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid potassium, tioned cue and cocaine injection (Figure 1B). Both doses of micro- 1 ethylene glycol tetraacetic acid, 1 magnesium chloride, 10 sodium injected NAC reduced the reinstatement of lever pressing com- chloride, 2.0 magnesium adenosine triphosphate, and .3 sodium pared with microinjected aCSF (Figure 1C). guanosine 5’-triphosphate, 1 QX-314, pH 7.2–7.3, 275 mOsm). Data It was previously shown that pretreatment with systemic were acquired at 10 kHz and filtered at 2 kHz using AxoGraph X LY341495, a mGluR2/3 antagonist, prevents systemic NAC-medi- software (AxoGraph Scientific, Sydney, Australia). To evoke EPSCs, a ated inhibition of cocaine reinstatement (29). Here, we extend this ϳ ␮ bipolar stimulating electrode was placed 100 to 200 m dorso- finding and show (Figure 1D) that pretreatment with systemic medial of the recorded cell to maximize chances of stimulating PFC LY341495 inhibited the capacity of NAC administered into the NA- ϳ afferents. The stimulation intensity chosen evoked a 50% of max- core to reduce cue-induced lever pressing. Figure 1E shows the imal EPSC. Each cell was superfused with a single concentration of location of the microinjection sites in the NAcore. NAC, cysteine, or cystine, since their effects were produced following uptake into cells and not washable. Recordings were collected NAC Has a Biphasic Effect on EPSCs in the NAcore every 30 seconds and averaged over 1.5 minutes. Series resistance To examine the effect of NAC on neurotransmission in the NA- (Rs) measured witha2mVdepolarizing step (10 msec) given with core, we used whole cell patch recordings of spiny cells in acute each stimulus and holding current were always monitored online. slices from yoked-saline or drug-naïve rats. The application of a low Recordings with unstable Rs or when Rs exceeded 20 M⍀ were dose of NAC (.5 ␮mol/L) resulted in a reduction in the EPSC ampli- aborted. tude (Figure 2A), consistent with previous data showing that cys- Ϫ tine-induced activation of system Xc increases nonsynaptic gluta- Primary Cell Culture mate (4), which inhibits EPSC amplitude (29). However, the Primary cultures of astrocytes were prepared from cerebral cor- inhibitory effect of NAC reversed at higher concentrations, with 50 tices of 1- to 2-day-old rat pups (25). The tissue was dissociated and and 500 ␮mol/L NAC significantly increasing EPSC amplitude. A plated in 150 cm2 flasks at a density of 2.5 ϫ 105 cells/mL. When the change in paired pulse ratio (PPR) was employed as a marker of a primary culture reached confluence, the monolayer of cells were presynaptic change in the efficacy of synaptic transmission (30,31). trypsinized and plated into 12-well plates. Cells cultured for 21 to 35 To reduce variability, the PPR after NAC was divided by the PPR days were used in the experiments. Uptake studies were performed during baseline. Paired pulse ratio increased with .5 ␮mol/L NAC,

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but no change was observed at other concentrations (Figure 2B), suggesting that the decrease in EPSC at .5 ␮mol/L NAC involved a presynaptic mechanism, while the increase in EPSC at higher concentrations was more likely of postsynaptic origin.

Two NAC Derivatives, Cysteine and Cystine, Have Opposite Effects on EPSCs NAC is known to decompose to cysteine and then to cystine (32). Thus, its effect could be generated by one or both amino acids. Figure 2C shows that, akin to high concentrations of NAC, application of .5 or 500 ␮mol/L cysteine increased the EPSC amplitude. In contrast, both .5 and 500 ␮mol/L cystine inhibited EPSC amplitude. Also, the PPR was insensitive to cysteine but was increased by both cystine concentrations. Thus, at .5 ␮mol/L, NAC acts more like cystine to inhibit the EPSC through a presynaptic mechanism, while at high doses, NAC resembles the effect of cysteine to increase EPSC amplitude potentially via a postsynaptic mechanism.

NAC Effect on EPSCs Is Blocked by (S)-4-carboxyphenylglycine or 2-Amino-2-norbornanecarboxylic Acid/L-Serine The effects of NAC on EPSC amplitude are assumed to be mediated by increasing extracellular glutamate via activating system Ϫ ␮ Xc . To test this, we applied .5 or 500 mol/L NAC while blocking Ϫ ␮ system Xc with 10 mol/L (S)-4-carboxyphenylglycine (CPG). CPG alone did not change EPSC amplitude. However, the capacity of .5 ␮mol/L NAC to reduce and of 500 ␮mol/L NAC to increase the EPSC was blocked by CPG (Figure 3). Since the effect of 500 ␮mol/L NAC on EPSC amplitude resem- bled that of 500 ␮mol/L cysteine, we determined whether the effect

Figure 2. Dose-dependent effect of N-acetylcysteine (NAC), cysteine, and cystine on neurotransmission in the nucleus accumbens core. (A) In- creasing NAC concentrations produced a biphasic effect on excitatory postsynaptic current (EPSC) amplitude (). Excitatory postsynaptic currents were normalized to baseline and are shown as mean percentage of baseline. Inset—representative traces illustrating the effect of NAC. Traces are from different cells, baseline EPSCs were normalized. One-way p Ͻ .05 using a Dunnett’sء .analysis of variance, F(4,39) ϭ 18.60, p Ͻ .001 test comparing with 0. (B) Only .5 ␮mol/L NAC changed the paired pulse Figure 3. The effect of N-acetylcysteine (NAC) on excitatory postsynaptic Ͻ Ϫ ء ϭ ϭ ratio (PPR). One-way analysis of variance, F(4,37) 4.95, p .003. p current (EPSC) amplitude was antagonized by blocking system Xc with 10 .05 using a Dunnett’s test comparing with 0. (C) The effect of .5 and 500 ␮mol/L CPG or by blocking cysteine uptake with a mixture of 100 ␮mol/L ␮mol/L NAC, cysteine, or cystine on EPSC size (upper bars, compared BCH ϩ 500 ␮mol/L L-serine (Ser). (A) Representative traces for each drug with baseline EPSC) and PPR (bottom bars). NAC data are the same as in applied to a single cell. (B) CPG or BCH/Ser alone did not affect EPSC ampli- (A) and (B). Both .5 and 500 ␮mol/L cysteine increased the EPSC (124 Ϯ tude. The inhibitory effect of .5 ␮mol/L NAC was blocked by both drugs 13% and 141 Ϯ 2%, respectively) but did not alter the PPR. Both .5 and [F(2,17) ϭ 14.15, p Ͻ .001]. The excitatory effects of 500 or 50 ␮mol/L NAC 500 ␮mol/L cystine inhibited the EPSC (88 Ϯ 3% and 80 Ϯ 5%, respec- were blocked by CPG [t(12) ϭ 4.20, p ϭ .001] or BCH/Ser [t(10) ϭ 2.21, p ϭ tively) and changed the PPR (111 Ϯ 3% and 109 Ϯ 2%, respectively). .051], respectively. For the BCH/Ser experiment, 50 ␮mol/L NAC instead of Inset—representative traces of a single cell for both concentrations of 500 ␮mol/L NAC was used (see Results). Data for NAC alone taken from p Ͻ .05, compared with No blockers using a Dunnett’sء .p Ͻ .05 using a one-sample t test compared with 100. Figure 2A and 2Bء .the same drug post hoc test or Student t test.

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because higher doses of NAC required higher doses of BCH/Ser, which alone reduced EPSC amplitude. The results show that similar to CPG, BCH/Ser blocked both inhibitory and excitatory effects of NAC on the EPSC (Figure 3). These data indicate that activation of Ϫ system Xc and uptake via cysteine transporters are necessary for NAC to alter EPSCs in NAcore slices.

؊ NAC Enhances System Xc Activity by Acting on Cysteine Transporters The involvement of cysteine transporters in the effects of NAC was surprising, and to further examine the role of cysteine transporters in NAC-induced changes, we used primary astrocyte cultures and measured uptake of 35S-cystine (25). The cultures were incubated in35S-cystine alone and in the presence of increasing concentrations of unlabeled cystine. As expected, with increasing Ϫ concentrations of cystine (a competitive substrate for system Xc ), the uptake of 35S-cystine declined in a concentration-dependent

Figure 4. N-acetylcysteine (NAC) and cysteine increased 35S-cystine or 3H- Ϫ glutamate uptake by system Xc in cultured astrocytes and this effect was Ϫ ␮ blocked by the Xc blocker CPG (10 mol/L) or a mixture of cysteine transporter blockers BCH and L-serine (Ser) (500 ␮mol/L each). All experiments were performed in the presence of aspartate and acivicin to block system ␥ 35 XAGϪ and -glutamyl transpeptidase (27). (A) Uptake of S-cystine in the presence of increasing concentrations of cystine (), NAC (●), cysteine (Œ), and CPG (□) presented as % change from baseline (horizontal line, gray denotes SEM). Cystine [F(8,26) ϭ 46.8, p Ͻ .001] and CPG [F(5,17) ϭ 36.9, p Ͻ .001] inhibited 35S-cystine uptake, while NAC [F(5,23) ϭ 69.8, p Ͻ .001] and cysteine [F(5,17) ϭ 57.3, p Ͻ .001] increased 35S-cystine uptake. Gray area around the line at 100 indicates SEM in the absence of drug. (B) CPG or BCH/Ser reversed NAC- and cysteine-induced increase in 35S-cystine uptake [F(8,32) ϭ 25.89, p Ͻ .001]. (C) Unlabeled glutamate [F(3,11) ϭ 83.5, p Ͻ .001] and CPG [F(3,11) ϭ 83.5, p Ͻ .001] inhibited whereas NAC [F(2,8) ϭ 7.2, p ϭ Figure 5. The effects of N-acetylcysteine (NAC) on neurotransmission are .025] enhanced 3H-glutamate uptake. N ϭ 3 to 4 for each group and each mediated through metabotropic glutamate receptors (mGluRs). (A) Repre- (p Ͻ .05 compared sentative excitatory postsynaptic currents (EPSCs). (B; upper panelء .determination is the average of three replicate wells ␮ ␮ with baseline; ϩp Ͻ .05 compared with NAC or cysteine alone. Glu, gluta- LY341495 (1 mol/L) increased EPSC amplitude, while MTEP (5 mol/L) was ϭ ϭ mate. without effect [F(2,26) 6.42, p .006]. LY341495 prevented the reduction in EPSC amplitude induced by .5 ␮mol/L NAC, while MTEP was without effect [F(2,23) ϭ 16.13, p Ͻ .001]. Conversely, LY341495 was without effect of NAC on the EPSC also involved activation of cysteine transport- on the increase in EPSC amplitude produced by 500 ␮mol/L NAC, while ers. We applied .5 or 50 ␮mol/L NAC while blocking sodium (Naϩ)- MTEP abolished the increase [F(2,16) ϭ 8.58, p ϭ .004]. (Lower panel) Neither ␮ ϩ LY341495 nor MTEP alone affected the paired pulse ratio (PPR), while dependent (by 500 mol/L L-serine [Ser]) and Na -independent ␮ ␮ LY341495, but not MTEP, prevented the increase in PPR after .5 mol/L NAC (by 100 mol/L 2-amino-2-norbornanecarboxylic acid [BCH]) cys- [F(2,20) ϭ 5.76, p ϭ .012]. The PPR after 500 ␮mol/L NAC alone was not p Ͻ .05 compared with NACء .teine transporters (mixture denoted BCH/Ser) (33). We used 50 signiﬁcantly changed by MTEP or LY341495 ␮mol/L instead of 500 ␮mol/L NAC in the BCH/Ser experiment (white bar) using a Dunnett’s post hoc. Antag, antagonist.

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and mGluR5 are abundant in the nucleus accumbens (35–38), play a role in the induction of long-term potentiation and long-term depression in the NAcore (21), and are implicated in drug addiction (18). In addition, NAC has been shown to block relapse to cocaine through activation of mGluR2/3 (29). Thus, we measured the effect of NAC on EPSCs in the presence of LY341495, a mGluR2/3 antagonist, or MTEP, a mGluR5 negative allosteric modulator (Figure 5). LY314195 alone increased EPSC amplitude, while MTEP had no effect. LY314195 also prevented the reduction in EPSC amplitude produced by .5 ␮mol/L NAC and prevented the NAC-induced change in PPR, while MTEP did not. In contrast, the capacity of 500 ␮mol/L NAC to augment EPSC amplitude was not blocked by LY341495 but was blocked by MTEP. The PPR ratio was not affected by the addition of either drug compared with 500 ␮mol/L NAC alone. Together, these results show that while both the inhibitory and excitatory effects of NAC are induced by an elevation in extracellular glutamate concentrations, the inhibitory effect was mediated by presynaptic mGluR2/3, while the excitatory effect was mediated by postsynaptic mGluR5.

NAC and MTEP Synergize to Inhibit Cocaine Seeking The effect of NAC on EPSCs seems to be determined by the balance between mGluR2/3 and mGluR5 activation. This balance is potentially important for the efficacy of NAC in inhibiting the reinstatement of cocaine seeking. It was previously found that either an antagonist of mGluR2/3 or a positive allosteric modulator of mGluR5 blocks NAC-induced inhibition of cocaine-seeking reinstatement (21,29). Since NAC-induced elevation in nonsynaptic glutamate activates both mGluR2/3 and mGluR5, the stimulation of Figure 6. Blocking metabotropic glutamate receptor 5 with MTEP enhances mGluR5 by higher concentrations of NAC may actually promote the N-acetylcysteine (NAC)-induced inhibition of cocaine reinstatement. (A) cocaine-seeking reinstatement. Therefore, we determined whether Experimental paradigm for reinstatement testing. (B) NAC (10 mg/kg, intra- combining MTEP with NAC might improve the capacity of NAC to peritoneal [IP]) reduced active lever presses following cocaine priming, inhibit cocaine seeking. The same cocaine-training procedure as in while MTEP (.1 mg/kg, IP) had no effect. However, MTEP enhanced NAC- /p Ͻ .05 Figure 1 was used, but rats were injected with either NAC (10 mgء .[induced reduction in lever presses [F(4,69) ϭ 9.23, p Ͻ .001 compared with extinction, ϩp Ͻ .05 compared with Saline ϩ Saline, using a kg, IP) or saline 2 hours before cocaine priming (10 mg/kg IP) in Bonferroni post hoc analysis. SA, self-administration. combination with MTEP (.1 mg/kg, IP) or saline administered together with NAC and 30 minutes before cocaine (Figure 6A). At the manner (Figure 4A). A similar reduction in uptake was seen when doses used, neither NAC nor MTEP alone affected cocaine-induced Ϫ reinstatement of lever pressing compared with the saline control. adding increasing concentrations of CPG (system Xc inhibitor). Since NAC is a cystine prodrug, it was expected to mimic the effect However, Figure 6B shows that the combination of NAC and MTEP of cystine on 35S-cystine uptake. However, while 1 or 3 ␮mol/L NAC significantly reduced lever pressing to extinction levels. did not produce a significant effect, higher concentrations enhanced 35S-cystine uptake. Enhanced 35S-cystine uptake was also Discussion seen when cysteine was added to the incubating solution, and akin Systemic administration of NAC decreases the reinstatement of to EPSC amplitude, the effects of NAC and cysteine were blocked cocaine seeking (4,5,7,29,39). This has been proposed to be an both by CPG and by the cysteine transporter blockers BCH/Ser outcome of NAC being deacetylated to cysteine and dimerized to (Figure 4B). To ensure that 35S-cystine had not been reduced to Ϫ cystine, which activates glial system Xc to exchange extracellular cysteine and thereby transported by cysteine transporters, we mea- cystine for intracellular glutamate, thereby elevating nonsynaptic ϩ 3 sured the Na -independent uptake of H-glutamate (Figure 4C). glutamate (2). This, in turn, counteracts the reduced extrasynaptic 3 Again, while addition of glutamate or CPG reduced H-glutamate levels of glutamate found after withdrawal from cocaine and re- uptake, NAC produced a concentration-dependent facilitation. The stores extrasynaptic glutamate levels, which activate the mGluR2/3, overall results in Figure 4 suggest that NAC does not activate sys- thereby reducing synaptic glutamate release probability and de- Ϫ tem Xc by acting as a substrate either directly or by being con- creasing cocaine seeking (5,29). Here, we show that NAC acts di- verted into cystine but rather acts similar to cysteine by entering the rectly in the NAcore to inhibit cocaine seeking and demonstrate a cell through cysteine transporters (34) and facilitating the activa- requirement for both system X Ϫ and cysteine transporters in NAC’s Ϫ c tion of system Xc via an undetermined intracellular mechanism. ability to indirectly stimulate extrasynaptic mGluRs. While lower concentrations of NAC inhibit glutamate transmission onto NAcore The Effects of NAC on EPSCs are Differentially Mediated by spiny cells via activation of presynaptic mGluR2/3, higher concen- mGluR2/3 and mGluR5 trations countermand this effect by stimulating mGluR5 and poten- The results, so far, indicated that NAC altered glutamate trans- tiating EPSC amplitude (see Figure 7 for a schematic illustration). Ϫ mission in the NAcore by activating system Xc (4). We next deter- Finally, these opposite effects of mGluR2/3 and mGluR5 on the mined whether the effects of NAC on EPSCs were mediated by EPSC were revealed by blocking the capacity of NAC to stimulate metabotropic glutamate receptor (mGluR) activation. mGluR2/3 mGluR5, thereby potentiating the ability of NAC to inhibit cocaine

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Figure 7. The effect of N-acetylcysteine (NAC) on evoked excitatory postsynaptic currents (EPSCs) in the nucleus accumbens core. (A) A low dose of NAC (.5 ␮mol/L) is taken up by cysteine transporters (presumably following deacetylation in the extracellular space) and via an unknown mechanism stimulates Ϫ system Xc . The increase in nonsynaptic extracellular glutamate is sufficient to activate presynaptic inhibitory metabotropic glutamate receptor 2/3 (mGluR2/3) but not to activate postsynaptic metabotropic glutamate receptor 5 (mGluR5). This results in presynaptic inhibition of the EPSC amplitude. (B) A Ͼ ␮ Ϫ high dose of NAC ( 5 mol/L) enters the cell via cysteine transport and further accelerates the activity of the Xc . The higher extracellular glutamate levels activate both mGluR2/3 and mGluR5, but the excitatory effect of mGluR5 countermands the inhibitory effect of mGluR2/3, resulting in increased EPSC amplitude. AMPA, ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid. seeking, presumably by unmasking the presynaptic effect of an mGluR2/3 blocker increases in vivo field EPSCs (5), extracellular mGluR2/3 stimulation by NAC. glutamate concentrations (44), and in vitro EPSCs (Figure 5). In addition, the inhibitory effect of low-dose NAC was accompa- ؊ NAC Actions Require Cysteine Transporters and System Xc nied by a change in PPR, implying a presynaptic mechanism NAC is deacetylated to cysteine, which is rapidly oxidized to consistent with the presynaptic localization of mGluR2/3 (45,46) Ϫ cystine (40) and then transported by system Xc into the cells, versus the postsynaptic location of mGluR5 (35,45,46). Finally, where it is reduced to cysteine for glutathione synthesis (41). Here, the inhibitory effect of .5 ␮mol/L NAC on the EPSC and PPR was we demonstrated that the effects of NAC on mGluR regulation of blocked by an mGluR2/3 selective dose of LY341495 (47,48) but Ϫ synaptic strength depend not only on activating system Xc but not by MTEP. also on cysteine transporters. This is consistent with a previous Compared with the mGluR2/3-dependent effect of nmol/L NAC, report showing that NAC elevates intracellular cysteine levels by the increase in EPSC amplitude following higher concentrations of being deacetylated to cysteine and transported into cells (34). The NAC (Ͼ5 ␮mol/L) was mGluR5-dependent. This is consistent with involvement of cysteine transporters was further confirmed in cul- previous studies showing positive modulation of EPSCs by mGluR5 tured astroglia where NAC-elevated 35S-cystine or 3H-glutamate Ϫ (45,49). Presumably, the synaptic potentiation by higher doses uptake through system Xc required cysteine transporters. A simi- 35 arises from further NAC-induced increases in the activity of system lar effect of NAC on S-cystine uptake was reported previously for X Ϫ and the corresponding increases in extracellular glutamate nonglial cells (32,33). Although these data indicate that NAC must c that would activate the relatively lower affinity mGluR5. Consistent be internalized via cysteine transporters to increase system X Ϫ c with anatomical localization studies (35,45,46), the lack of change in activity, the intracellular signaling mechanism by which this occurs PPR after high-dose NAC supports a postsynaptic mechanism. In remains unknown. Interestingly, the dose-dependent bidirectional addition, mGluR5 is expressed by glia and regulates calcium-de- effect of NAC on excitatory transmission in the NAcore mimicked the actions of both cystine and cysteine. Thus, in the nanomolar pendent release of glutamate from glia, which could further con- range, NAC mimicked the effect of cystine by inhibiting EPSCs, and tribute to the stimulation of neuronal mGluRs (50). Although the akin to cysteine, NAC increased EPSC amplitude in the 50 to 500 doses of NAC required in vitro to indirectly stimulate mGluR5 were ␮mol/L range. It was surprising that high-dose cystine continued micromolar, this may reflect, in part, reduced basal extracellular to reduce EPSC amplitude and may be indicative of what has glutamate imposed by constant perfusion of tissue slices (29). Im- been observed where concentrations of cystine above 1 ␮mol/L portantly, since the effect of even the relatively low dose of 10 did not induce further increases in extracellular glutamate in the mg/kg NAC could be improved by blocking mGluR5, in vivo the NAcore (29). stimulation of mGluR5 by NAC appears to contribute to the reinstatement of cocaine seeking. Although we focused on mGluR2/3 NAC Stimulates Both mGluR2/3 and mGluR5 and mGluR5, NAC-induced elevation of glutamate may also acti- The inhibitory effect of low-dose NAC on EPSC amplitude was vate metabotropic glutamate receptor 1 and group III mGluRs, both blocked by a mGluR2/3 antagonist, while the potentiation of EPSC of which are expressed in the rat nucleus accumbens (51–53). Acti- amplitude was blocked by a mGluR5 antagonist. It has been sug- vation of presynaptic group III mGluRs negatively regulates EPSC gested that endogenous levels of glutamate have higher potency amplitude (54,55), while metabotropic glutamate receptor 1 stim- at mGluR2/3 than other mGluR subtypes (42,43). This is supported ulation increases the presynaptic release of glutamate (56,57), ei- by the fact that mGluR2/3, but not mGluR5, are tonically activated ther of which could contribute to our measurements of EPSC am- by low endogenous glutamate levels, as indicated by the facts that plitude and PPR. www.sobp.org/journal Y.M. Kupchik et al. BIOL PSYCHIATRY 2012;71:978–986 985

Balancing the Stimulation of mGluR2/3 and mGluR5 in NAC 13. Kalivas PW, Volkow N, Seamans J (2005): Unmanageable motivation in Treatment of Cocaine Relapse addiction: A pathology in prefrontal-accumbens glutamate transmis- Elevating nonsynaptic glutamate levels by NAC activation of sion. Neuron 45:647–650. system X Ϫ is thought to re-establish tone on mGluR2/3 in the 14. Baptista MA, Martin-Fardon R, Weiss F (2004): Preferential effects of the c metabotropic glutamate 2/3 receptor agonist LY379268 on condi- NAcore of cocaine-withdrawn animals, thereby inhibiting synaptic tioned reinstatement versus primary reinforcement: Comparison be- glutamate transmission and preventing the reinstatement of co- tween cocaine and a potent conventional reinforcer. J Neurosci 24: caine seeking (4,5,7,39). Our results support this conclusion by 4723–4727. showing that NAC microinfusion directly into the NAcore inhibited 15. Bossert JM, Gray SM, Lu L, Shaham Y (2006): Activation of group II cocaine seeking in an mGluR2/3-dependent manner. However, the metabotropic glutamate receptors in the nucleus accumbens shell at- present study also complicates this view of NAC action by showing tenuates context-induced relapse to heroin seeking. Neuropsychophar- macology 31:2197–2209. that, at least at higher concentrations, NAC can stimulate mGluR5 Ϫ 16. Peters J, Kalivas PW (2006): The group II metabotropic glutamate recep- also via activating system Xc release of glutamate. Importantly, tor agonist, LY379268, inhibits both cocaine- and food-seeking behav- activating mGluR5 reduces the efficacy of NAC (21), and antagoniz- ior in rats. Psychopharmacology (Berl) 186:143–149. ing mGluR5 blocks cocaine seeking (19,20,58–60). Thus, NAC 17. Gass JT, Osborne MP, Watson NL, Brown JL, Olive MF (2009): mGluR5 activation of mGluR5 should countermand the capacity of NAC- antagonism attenuates methamphetamine reinforcement and pre- induced mGluR2/3 activation to inhibit the reinstatement of co- vents reinstatement of methamphetamine-seeking behavior in rats. caine-seeking. Our finding that blocking mGluR5 augments the Neuropsychopharmacology 34:820–833. 18. Kenny PJ, Markou A (2004): The ups and downs of addiction: Role of capacity of NAC to inhibit cocaine-seeking supports an mGluR- metabotropic glutamate receptors. Trends Pharmacol Sci 25:265–272. dependent bidirectional effect of NAC on cocaine seeking. Re- 19. Kenny PJ, Paterson NE, Boutrel B, Semenova S, Harrison AA, Gasparini F, cently, NAC has been used with only partial success in clinical trials et al. (2003): Metabotropic glutamate 5 receptor antagonist MPEP de- to reduce cocaine or tobacco craving and relapse (9,10,61–63). creased nicotine and cocaine self-administration but not nicotine and Taken together, the data in this report indicate that combining NAC cocaine-induced facilitation of brain reward function in rats. AnnNY with an mGluR5 antagonist might improve the efficacy of NAC in Acad Sci 1003:415–418. 20. Martin-Fardon R, Baptista MA, Dayas CV, Weiss F (2009): Dissociation of treating addiction. the effects of MTEP [3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]piperidine] on conditioned reinstatement and reinforcement: Comparison be- This research was supported by United States Public Health Associ- tween cocaine and a conventional reinforcer. J Pharmacol Exp Ther 329: ation Grants DA015369, DA012513, and DA003906. XW is currently 1084–1090. affiliated with the Program in Neuroscience, Washington State Univer- 21. 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