Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2021 Sep 1.
Published in final edited form as: Addict Biol. 2019 Jul 22;25(5):e12797. doi: 10.1111/adb.12797

Accumbens Neuroimmune Signaling and Dysregulation of Astrocytic Glutamate Transport Underlie Conditioned Nicotine Seeking Behavior

Mark D Namba 1, Yonatan M Kupchik 2, Sade M Spencer 3, Constanza Garcia-Keller 4, Julianna G Goenaga 1, Gregory L Powell 1,5, Ian A Vicino 5,6, Ian B Hogue 5,6, Cassandra D Gipson 1,*
PMCID: PMC7323912  NIHMSID: NIHMS1032587  PMID: 31330570

Abstract

Nicotine self-administration is associated with decreased expression of the glial glutamate transporter (GLT-1) and the cystine-glutamate exchange protein xCT within the nucleus accumbens core (NAcore). N-acetylcysteine (NAC) has been shown to restore these proteins in a rodent model of drug addiction and relapse. However, the specific molecular mechanisms driving its inhibitory effects on cue-induced nicotine reinstatement are unknown. Here, we confirm that extinction of nicotine-seeking behavior is associated with impaired NAcore GLT-1 function and expression and demonstrate that reinstatement of nicotine seeking rapidly enhances membrane fraction GLT-1 expression. Extinction and cue-induced reinstatement of nicotine seeking was also associated with increased tumor necrosis factor alpha (TNFα) and decreased glial fibrillary acidic protein (GFAP) expression in the NAcore. NAC treatment (100 mg/kg/day, i.p., for 5 days) inhibited cue-induced nicotine seeking and suppressed AMPA to NMDA current ratios, suggesting that NAC reduces NAcore post-synaptic excitability. In separate experiments, rats received NAC and an antisense vivo-morpholino to selectively suppress GLT-1 expression in the NAcore during extinction and were subsequently tested for cue-induced reinstatement of nicotine seeking. NAC treatment rescued NAcore GLT-1 expression and attenuated cue-induced nicotine seeking, which was blocked by GLT-1 antisense. NAC also reduced TNFα expression in the NAcore. Viral manipulation of the NF-κB pathway, which is downstream of TNFα, revealed that cue-induced nicotine seeking is regulated by NF-κB pathway signaling in the NAcore independent of GLT-1 expression. Ultimately, these results are the first to show that immunomodulatory mechanisms may regulate known nicotine-induced alterations in glutamatergic plasticity that mediate cue-induced nicotine-seeking behavior.

Keywords: GLT-1, GFAP, IKK, TNFα, NF-κB, reinstatement

Introduction

Tobacco use is a significant health concern and remains a leading cause of preventable death worldwide, accounting for nearly 7 million deaths annually that are attributable to smoking and second-hand smoke (Reitsma et al., 2017). Current smoking cessation treatments primarily replace stimulation of nicotinic acetylcholine receptors (nAChRs) with drugs that have a different pharmacokinetic profile compared to smoking (Le Houezec, 2003) to attenuate drug craving without producing rewarding effects (Nides, 2008). These treatments have shown some clinical efficacy in helping individuals achieve abstinence (Kasza et al., 2015; McClure et al., 2013). Yet, the risk of relapse persists even for individuals receiving replacement therapy (Etter & Stapleton, 2006; Medioni et al., 2005), highlighting the need for more effective pharmacotherapies that better promote long-term abstinence from tobacco use.

Maladaptive glutamatergic plasticity underlying cue-induced drug seeking has been implicated across several major drugs of abuse (Scofield et al., 2016a; van Huijstee & Mansvelder, 2014). For example, increased prefrontal glutamate release into the nucleus accumbens core (NAcore) mediates cue- and drug-induced reinstatement of drug-seeking (Gipson et al., 2013a; LaLumiere & Kalivas, 2008; McFarland et al., 2003; Smith et al., 2017). Specifically, glutamatergic projections from the prelimbic cortex to the NAcore facilitate rapid, transient synaptic potentiation of GABAergic medium spiny neurons (MSNs) within the first 15 minutes (i.e., T(time)=15) of cue-induced reinstatement of cocaine-(Gipson et al., 2013a) and nicotine-seeking (Gipson et al., 2013b) as measured by increases in dendritic spine head diameter and AMPA to NMDA current ratios (AMPA/NMDA). Chronic self-administration and extinction also downregulates both the glial cystine-glutamate exchanger, system xc− (Sxc−), and the glial glutamate transporter 1 (GLT-1; Alhaddad et al., 2014; Baker et al., 2003; Gipson et al., 2013b; Knackstedt et al., 2009; Knackstedt et al., 2010; Sari et al., 2009; Shen et al., 2014). Both Sxc− and GLT-1 are highly expressed in astrocytes and regulate the majority of basal extracellular glutamate (Scofield et al., 2016a), thus serving as critical regulators of glutamate neurotransmission and are putative targets for treating substance use disorders (SUDs) (Roberts-Wolfe & Kalivas, 2015).

The cysteine pro-drug N-acetylcysteine (NAC) is an antioxidant and anti-inflammatory compound that is commonly used to treat acetaminophen poisoning (Yarema et al., 2009). NAC has also been used to treat a multitude of psychiatric disorders, including cocaine-, methamphetamine-, cannabis-, and tobacco use disorders (Berk et al., 2013; Deepmala et al., 2015; Elbini Dhouib et al., 2016; Froeliger et al., 2015). Drugs such as NAC and the β-lactam antibiotic ceftriaxone upregulate GLT-1 and the catalytic subunit of Sxc−, xCT. Restoration of these two proteins is associated with decreases in drug- and cue-induced reinstatement of drug seeking (Alhaddad et al., 2014; Knackstedt et al., 2010; Sari et al., 2009; Sondheimer & Knackstedt, 2011). Interestingly, subchronic NAC treatment successfully inhibits cue-induced cocaine reinstatement when xCT expression is suppressed but is ineffective if GLT-1 is not restored (Reissner et al., 2015). Therefore, the studies described herein attempt to clarify whether NAC’s therapeutic mechanism of action likewise depends on restoration of GLT-1.

In addition to restoring glutamate transport following cocaine self-administration, NAC has also been shown to normalize the expression of pro- and anti-inflammatory cytokines in the brains of alcohol-withdrawn rats (Schneider et al., 2017) and inhibits tumor necrosis factor alpha (TNFα)-induced activation of the nuclear factor kappa B (NF-κB) pathway through inhibition of IκB kinases (IKKs) (Oka et al., 2000). Both TNFα and downstream NF-κB signaling are heavily implicated in drug-induced neuroinflammation (Cui et al., 2014) and are key regulators of learning, memory, and synaptic plasticity (Albensi & Mattson, 2000; Kaltschmidt & Kaltschmidt, 2015; Meffert et al., 2003). As well, accumbens NF-κB signaling mediates cocaine conditioned place preference and associated changes in dendritic spine density (Russo et al., 2009). Taken together, it is likely that neuroimmunomodulation of glutamatergic plasticity is a key mechanism underlying drug relapse and associated synaptic plasticity. Understanding the mechanistic properties of NAC’s therapeutic potential may provide useful insights into the molecular underpinnings of cue-induced relapse across drugs of abuse. Thus, we also examined in the present study whether NAcore neuroimmune signaling underlies the inhibitory effect of NAC on conditioned nicotine seeking.

Methods

Subjects

197 Male Sprague Dawley rats (Charles River, 250-300 g) were individually housed on a 12-hour reverse light-dark cycle and received 20 g of food/day and ad libitum water. Experimentation was conducted during the dark phase. All experiments conducted were approved by either the Arizona State University (ASU) or the Medical University of South Carolina (MUSC) Institutional Animal Care and Use Committees. Experiments in Figures 1 and 4 were conducted at MUSC and experiments in Figures 2, 3, 5, S1–4 were conducted at ASU.

Figure 1. Nicotine self-administration, extinction, and reinstatement alter glutamate uptake and GLT-1 expression.

Figure 1.

Open in a new tab

(A) Timeline of experimental procedures. (B) Lever presses and nicotine infusions (0.02 mg/kg/infusion) across self-administration and lever presses across extinction training. (C) Average active and inactive lever presses during the first 15-mins of the last two extinction sessions and during a 15-min cue-induced reinstatement test (T=15). Rats significantly reinstated active lever pressing in response to contingent, nicotine-conditioned cues within the first 15-mins of the session. *p<0.05. (D) NAcore membrane GLT-1 expression between yoked saline, T=0, and T=15 conditions, normalized to Saline. Extinction of nicotine seeking is associated with a significant reduction in membrane GLT-1 expression within the NAcore, which is rapidly restored within the first 15-mins of cue-induced reinstatement. *p<0.05. (E) NAcore Na+-dependent and Na+-independent glutamate uptake after 14 days of extinction (T=0) versus yoked saline. At T=0, some rats were sacrificed to examine glutamate uptake in the NAcore. Nicotine self-administration and extinction significantly reduced Na+-dependent glutamate uptake (*p<0.05), while Na+-independent uptake was unchanged. Numbers in bars represent number of animals per group. Error bars = SEM.

Figure 4. NAC inhibits AMPA/NMDA and associated cue-induced nicotine seeking.

Figure 4.

Open in a new tab

(A) Timeline of experimental procedures. (B) Following 2-weeks of extinction, vehicle-treated rats demonstrated a significant increase in active lever pressing in response to drug-paired cues at T=15, which was blocked by NAC treatment (100 mg/kg, i.p., 5-days). *p<0.05 relative to extinction active lever presses and NAC active lever presses. (C) NAcore AMPA/NMDA following T=15 reinstatement in NAC or vehicle-treated rats. Rats receiving NAC treatment displayed inhibited AMPA/NMDA in the NAcore relative to vehicle-treated controls, as measured immediately after T=15 reinstatement. *p<0.05. AMPA/NMDA normalized to vehicle. The recording electrode (R), outlined in black, was placed in the dorsomedial NAcore as depicted in the representative image. AC = anterior commissure, numbers in bars in (B) represents number of animals, numbers in bars in (C) represents number of cells. Error bars = SEM.

Figure 2. Nicotine self-administration and extinction alters NAcore TNFα and GFAP expression.

Figure 2.

Open in a new tab

(A) Timeline of experimental procedures. (B) NAcore TNFα expression following nicotine self-administration (Nicotine), extinction (T=0), and 15-min cue-induced reinstatement (T=15), normalized to yoked saline (Saline). Extinction of nicotine seeking was associated with elevated TNFα, which persisted into cue reinstatement. *p<0.05 relative to Saline. (C) NAcore IL-6 expression between Nicotine, T=0, T=15, and Saline conditions, normalized to Saline. IL-6 expression was not altered under any of the test conditions. (D) NAcore GFAP expression between Nicotine, T=0, T=15, and Saline conditions, normalized to Saline. Extinction of nicotine seeking was associated with downregulated GFAP, which persisted into cue reinstatement. *p<0.05 relative to Saline. Numbers in bars represent number of animals per group. Error bars = SEM.

Figure 3. NAC rescues GLT-1, inhibits TNFα, and GLT-1 knockdown impairs NAC inhibition of cue-induced reinstatement.

Figure 3.

Open in a new tab

(A) Timeline of experimental procedures. (B) Lever presses and nicotine infusions across self-administration and lever presses across extinction training. (C) Average active lever presses during the last two extinction sessions and during a 2-hour cue-induced reinstatement session (26-hours following last NAC or vehicle injection). NAC significantly attenuated active lever pressing relative to Vehicle (Veh). This effect was blocked by administration of GLT-1 antisense morpholino. *p<0.05 and #p<0.10 relative to CTRL-NAC. (D) Crude membrane fraction GLT-1 expression across treatment groups, normalized to Sal-Veh. Membrane GLT-1 was significantly reduced in Nic-CTRL-Veh rats measured after a 2-hour cue reinstatement test. NAC restored membrane GLT-1 to yoked saline levels and GLT-1 antisense treatment blocked this effect. *p<0.05 relative to Sal-Veh. (E) NAcore TNFα expression (collapsed across morpholino conditions) following NAC treatment, normalized to vehicle. NAC significantly inhibited TNFα expression measured after a 2-hour cue reinstatement test. *p<0.05 relative to Vehicle. Numbers in bars represent number of animals per group. Sal = yoked saline. Nic = nicotine. CTRL = control morpholino. AS = antisense morpholino. Lever pressing behavior for yoked saline animals treated with NAC or vehicle is presented in Figure S2. Error bars = SEM.

Figure 5. Constitutive Activation of IKK Blocks the Attenuating Effect of NAC on Cue-Induced Nicotine Seeking.

Figure 5.

Open in a new tab

(A) Timeline of experimental procedures. (B) Schematic of TNFα and NF-κB Signaling and HSV manipulations. In the canonical TNFα→NF-κB signaling pathway, (1) TNFα binds to TNFR1, which promotes the association of adaptor proteins at the intracellular domain of TNFR1 (adaptor proteins not depicted). This leads to (2) activation of IKK complexes that (3) trigger the phosphorylation, ubiquitination, and proteasomal degradation of the inhibitory protein IκBα, which normally keeps NF-κB heterodimers in an inhibited state within the cytoplasm. Following IκBα phosphorylation, (4) NF-κB translocates into the nucleus and alters gene transcription, which includes genes such as GLT-1 (i.e., EAAT-2), TNFα, and many others. (C) Average active lever presses during the last two extinction sessions and during a 2-hour cue-induced nicotine reinstatement session (26-hours following last NAC or vehicle injection). Rats receiving GFP/Veh, IKKca/NAC, or IKKca/Veh significantly reinstated nicotine seeking in response to contingent cues as compared to the other three treatment conditions. #p<0.05 relative to GFP/NAC, IKKdn/NAC, and IKKdn/Vehicle. *p<0.05 relative to respective extinction. (D) Total NAcore GLT-1 expression across virus treatment. Regardless of NAC or vehicle treatment, both IKKca and IKKdn inhibited GLT-1 expression compared to GFP controls. *p<0.001. Data collapsed across treatment and normalized to GFP. Numbers in bars represent number of animals per group. Error bars = SEM.

Drugs

Nicotine tartrate (MP Biomedicals, LLC, Solon, OH, USA) was dissolved in 0.9% saline and the pH adjusted to 7.4. Nicotine doses were calculated based on free base weight. NAC (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 27 mg/mL sodium hydroxide in saline to physiological pH immediately before injection. Vivo-morpholinos (Gene Tools, LLC, Philomath, OR, USA) were dissolved in sterile phosphate buffered saline (PBS, 10 mM Na2HPO4, 2 mM KH2PO4, 137 mM NaCl, and 2.7 mM KCl) and stored at room temperature. The GLT-1 antisense (i.e., AS) vivo-morpholino sequence is 5’-TGTTGGCACCCTCGGTTGATGCCAT-3’ and the reverse sequence was used as a control (i.e., CTRL; Reissner et al., 2015, 2012).

Surgery

Rats were anesthetized with ketamine HCL (100 mg/kg, i.m.) and xylazine (8 mg/kg, i.m.) and underwent surgical implantation of intravenous catheters as previously described (Powell et al., 2019). For experiments in Figures 3 and 5, rats were fixed onto a stereotaxic frame and intracerebral guide cannulae (26G, 15 mm, PlasticsOne, Roanoke, VA, USA) were implanted bilaterally into the NAcore (+1.5 A/P, +1.8 M/L, −5.5 D/V, from Paxinos & Watson, 2007). Obturators were inserted into guide cannulae to prevent clogging. Cefazolin (100 mg/kg, i.v.) and meloxicam (1 mg/kg, s.c.) were administered at the end of surgery. Meloxicam was administered for three days following surgery to provide analgesia. Cefazolin and heparin (10 usp/day, i.v.) were infused through the catheter for seven days following surgery and heparin alone was administered daily throughout self-administration to maintain catheter patency.

Nicotine Self-Administration and Extinction Training

Behavioral testing took place in operant chambers containing two-levers, a stimulus light above each lever, and a food receptacle (Med Associates, Inc., Fairfax, VT, USA). Prior to surgery and self-administration procedures, rats received a 15-hour food training session where active lever presses dispensed a food pellet (45 mg, Bio-Serv, Flemington, NJ, USA) into the receptacle. Food delivery was not paired with conditioned cues. Following food training, rats underwent nicotine self-administration for 2-hours/day on a fixed-ratio 1 schedule of reinforcement where each active lever press resulted in a single infusion of nicotine (0.02 mg/kg/infusion across 5.9 secs) that was paired with a compound stimulus light and auditory tone followed by a 20-sec time-out period. The cue-light remained illuminated until the 20-sec time-out period expired and the auditory tone persisted only for the duration of the infusion. Inactive lever presses had no consequence but responses were still recorded. For experiments in Figures 1–3, some rats received non-contingent yoked saline infusions that were paired with the aforementioned cues. Self-administration criteria were set at a minimum of 10 infusions per session for 10 sessions and a 2:1 active-to-inactive lever press ratio. Upon completion of self-administration, some rats entered extinction training, where active lever presses no longer resulted in nicotine infusions or associated cues. Extinction training persisted for 14-16 days prior to sacrifice (Figures 1–2), a 15-min cue-induced reinstatement test (Figures 1, 2, 4), or a 2-hour (Figures 3, 5) cue-induced reinstatement test. Active lever presses during reinstatement testing resulted in presentation of nicotine-paired cues but not nicotine. Only animals that extinguished to below 65 active lever presses prior to the onset of virus or vivo-morpholino and NAC treatment were included in the study as described previously (Reissner et al., 2015).

Herpes Simplex Virus (HSV) Vector Verification

See Supplemental Methods for virus verification procedures.

Morpholino, Virus, and NAC Treatment

Beginning on day 10 of extinction training, rats received 3-days of bilateral microinjections (0.5 μl/min for 2 min, 30 pmol/injection) of either GLT-1 antisense or control sequence vivo-morpholino, 2-hours following each extinction session. Microinjectors were placed 2 mm below the guide cannulae into the NAcore and were left in place for 1-min following the injection to allow for the morpholino to fully diffuse through the tissue. Rats were sham injected 1-day prior to their first microinjection so the animals could habituate to the procedure. This morpholino treatment schedule is based on previous studies showing effective knockdown of NAcore GLT-1 protein expression and inhibition of NAC-induced restoration of GLT-1 (Reissner et al., 2015, 2012). During the last 5-days of extinction, rats received NAC (100 mg/kg, i.p.) or saline 2-hours prior to each extinction session. This treatment regimen was based on previous studies that demonstrated an increase in cystine-glutamate exchange and GLT-1 expression following NAC treatment and a decrease in cue-induced cocaine reinstatement (Knackstedt et al., 2010; Moussawi et al., 2011). To examine the role of NF-κB in conditioned nicotine seeking, HSV vectors that express a dominant negative (IKKdn) or a constitutively active (IKKca) mutant of IKK were utilized. Upon activation by TNFα or other upstream signals, IKK facilitates the activation and nuclear translocation of NF-κB heterodimers (Figure 5B, Gilmore, 2006). Rats received an intra-NAcore microinjection of either HSV-IKKdn, HSV-IKKca, or HSV-GFP (1.0–1.5 μl/hemisphere, 0.5 μl/min) on day 10 of extinction. Rats were sham microinjected the day before receiving HSV. NAC was administered for 5-days beginning on day 10 of extinction exactly as described above. Cannula placement (denoted by clear cannula track marks and tissue damage surrounding the injection site) was visually inspected prior to tissue collection and only animals with cannula placement in the dorsomedial NAcore were included in the study.

Tissue Preparation and Western Blotting

Following extinction or reinstatement testing, 1 mm × 1 mm × 2 mm tissue samples were collected from the NAcore over ice. To examine GLT-1 expression, crude membrane fractions of the NAcore were prepared. Briefly, tissue was homogenized in 200 μl of ice-cold sucrose buffer containing HEPES (pH=7.4). Samples were centrifuged at 1,000 × g for 10-mins at 4°C, resulting in a pellet that was re-suspended in another 200 μl of ice-cold sucrose buffer. The pellet was centrifuged again at 1,000 × g for 10-mins to remove nuclei and larger debris. Supernatants were pooled and concentrated at 12,000 × g for 20-mins. The resulting pellet was re-suspended in 30 μl of RIPA lysis buffer and centrifuged at 10,000 × g for 5-mins to remove insoluble matter. All buffers contained 1:100 protease and phosphatase inhibitors. Preparation of NAcore whole cell lysates and all western blotting procedures were conducted as previously described (Powell et al., 2019). Table 1 provides a list of antibodies and concentrations used. Band density was analyzed using NIH ImageJ software and protein expression was normalized to calnexin or GAPDH.

Table 1.

Antibodies for Western Blot and Immunohistochemistry Experiments

Target Catalog # RRID 10 Concentration 20 Concentration
GLT-1 Abcam ab41621 AB_941782 1:1,000 1:40,0001
CD40 Abcam ab13545 AB_1951619 1:1,000 1:5,0001
TNFα Abcam ab6671 AB_305641 1:1,000 1:2,0001
IL-6 Santa Cruz sc-57315 AB_2127596 1:1,000 1:5,0002
GFAP Abcam ab53554 AB_880202 1:1,000 1:5,0003
GFP Abcam ab13970 AB_300798 1:500 1:2,0004
Calnexin Enzo ADI-SPA-860 AB_10616095 1:1,000 1:25,0001
GAPDH CST D16H11 AB_10622025 1:1,000 1:25,0001
Open in a new tab

Note.

1

All secondary antibodies were Goat pAb to Rb IgG (HRP) (Abcam, ab97080)

2

Horse pAb to Mouse IgG (HRP) (Cell Signaling Technology, #7076)

3

Donkey pAb to Goat IgG (HRP) (Abcam, ab97110)

4

or Goat pAb to Chk IgY (Alexa Fluor® 488) (Abcam, ab150173).

Glutamate Uptake

See Supplemental Materials for glutamate uptake procedures.

Slice Preparation and Whole-Cell Electrophysiology

NAcore slice preparations and whole-cell electrophysiology were conducted as previously described (Gipson et al., 2013a; 2013b). These Methods are also provided in the Supplemental Materials.

Statistics

Active lever pressing during T=15 reinstatement (Figure 1C) was analyzed with a two-way ANOVA with “session” (extinction vs reinstatement) and “lever” (active vs inactive) as fixed factors. Membrane GLT-1 expression comparing yoked saline (Saline), 14-days extinction of nicotine seeking (T=0), and 15-min cue-induced reinstatement of nicotine seeking (T=15) conditions was analyzed with a one-way ANOVA (Figure 1D). A two-way ANOVA was used to assess differences in Na+-dependent and Na+-independent glutamate uptake between Saline and T=0 conditions (Figure 1E), with “drug” (T=0 vs Saline) and “condition” (Na+-dependent vs Na+-independent uptake) as fixed factors. One-way ANOVAs were used to analyze NAcore TNFα (Figure 2B), interleukin-6 (IL-6) (Figure 2C), and glial fibrillary acid protein (GFAP) expression (Figure 2D) between Saline, Nicotine, T=0, and T=15 conditions. Active lever pressing during a 2-hour cue-induced reinstatement test (Figure 3C) in morpholino-treated rats that received NAC or vehicle injections was analyzed using a two-way ANOVA, with “treatment” (NAC vs Vehicle) and “morpholino” (antisense vs control) as fixed factors. Membrane GLT-1 expression was analyzed using a one-way ANOVA comparing yoked saline rats treated with NAC or vehicle to nicotine rats treated with morpholinos and NAC or vehicle (Figure 3D). A two-way ANOVA was used to examine NAcore TNFα expression in rats treated with morpholinos and NAC or vehicle, with “morpholino” and “treatment” as fixed factors (Figure 3E). Active lever pressing during T=15 reinstatement compared between NAC- and vehicle-treated rats was analyzed with a three-way ANOVA, with “session”, “lever”, and “treatment” (NAC vs vehicle) as fixed factors (Figure 4B). AMPA/NMDA compared between NAC- and vehicle-treated rats (Figure 4C) was analyzed using a two-tailed unpaired t-test. Active lever pressing during a 2-hour cue-induced reinstatement test (Figure 5C) in HSV-treated rats that received NAC or vehicle injections was analyzed using a two-way ANOVA, with “treatment” and “virus” (GFP vs IKKdn vs IKKca) as fixed factors. GLT-1 expression was similarly analyzed using a two-way ANOVA (Figure 5D). For statistical analysis of viral verification experiments, see Supplemental Materials. Post hoc comparisons were made using a Bonferroni, Tukey HSD, or Dunnett’s multiple comparisons tests using SPSS statistical software. Significance level was set at α = 0.05 for all analyses. Timelines of experimental procedures are provided in each figure (Figures 1–5A).

Results

Nicotine Self-Administration, Extinction, and Reinstatement Decrease Glutamate Uptake and Alter GLT-1 Expression in the NAcore.

Here we examined whether (1) chronic nicotine self-administration and extinction (Figure 1B) impairs glutamate uptake and (2) if GLT-1 is susceptible to reinstatement-dependent alterations in membrane expression. First, we examined nicotine seeking behavior during a 15-min reinstatement session (T=15). A two-way ANOVA revealed significant main effects of session (F(1,36)=37.98, p<0.001) and lever (F(1,36)=23.16, p<0.001), as well as a significant session X lever interaction (F(1,36)=18.54, p<0.001). Post hoc comparisons revealed a significant increase in active lever presses during T=15 reinstatement relative to extinction (Figure 1C, *p<0.05). Next, we examined NAcore GLT-1 expression between yoked saline, T=0, and T=15 conditions. A one-way ANOVA revealed a significant effect of test condition (F(2,37)=4.58, p<0.05). Post hoc comparisons revealed a significant decrease in GLT-1 expression at T=0 and a return to saline levels at T=15 (Figure 1D, *p<0.05). Lastly, we examined NAcore glutamate uptake after extinction training (T=0) compared to yoked saline. A two-way ANOVA revealed significant main effects of drug (F(1,18)=9.73, p<0.05) and condition (F(1,18)=156.95, p<0.001), as well as a significant drug X condition interaction (F(1,18)=8.27, p=<0.05). Post hoc comparisons indicated significantly lower Na+-dependent uptake at T=0 compared to saline, and significantly higher Na+-dependent glutamate uptake overall compared to Na+-independent uptake (Figure 1E, *p<0.05). Na+-independent glutamate uptake was not altered by extinction of nicotine seeking. These data suggest that chronic nicotine self-administration and extinction dysregulates glial glutamate transport and that dynamic, activity-dependent changes in GLT-1 trafficking may underlie the reinstatement of nicotine-seeking.

Extinction of Nicotine Seeking is Associated with Upregulated TNFα Expression and Downregulated GFAP in the NAcore.

Dysregulated glutamate transport and elevated extracellular glutamate levels are potent stimulators of pro-inflammatory TNFα signaling (Olmos & Lladó, 2014). Thus, we hypothesized that nicotine self-administration and extinction may alter neuroimmune signaling in the NAcore. First, we examined NAcore TNFα expression between yoked saline, nicotine, T=0, and T=15 conditions. A one-way ANOVA revealed a significant effect of condition on TNFα expression (F(3,32)=7.208, p=0.0008). Post hoc comparisons revealed that TNFα was unchanged immediately after chronic nicotine self-administration, but significantly elevated at T=0 and T=15 relative to saline (Figure 2B, *p<0.05). Next, we assessed NAcore GFAP expression between these conditions. A one-way ANOVA revealed a significant effect of condition on GFAP expression (F(3,33)=3.992, p=0.016). Post hoc comparisons showed significant reductions in GFAP expression at T=0 and T=15 relative to saline (Figure 2D, *p<0.05). No significant differences in IL-6 expression were detected between test conditions (Figure 2C). Altogether, these results suggest that extinction of nicotine seeking and withdrawal may dysregulate specific immunomodulatory signaling pathways within the NAcore, which may underlie cue-induced nicotine-seeking behavior.

NAC Inhibits Cue-induced Nicotine Seeking Through a GLT-1-dependent Mechanism and Inhibits NAcore TNFα Expression.

Considering nicotine self-administration and extinction dysregulates GLT-1 function and expression and increases TNFα expression, it is possible that NAC’s mechanism is GLT-1-dependent as in cocaine (Reissner et al., 2015) and may inhibit TNFα. Figure 3B depicts lever presses and infusions during nicotine self-administration and lever presses during extinction training. First, we examined active lever pressing during a 2-hour cue-induced reinstatement test between morpholino and drug treatment conditions. A two-way ANOVA revealed a significant treatment X morpholino interaction (but no main effects of treatment or morpholino alone) (F(1,27)=5.978, p=0.02). Post hoc comparisons revealed a significant decrease in cue-induced nicotine reinstatement during a 2-hour session due to NAC treatment, which was blocked by GLT-1 antisense (Figure 3C, *p<0.05). Lever press behavior for yoked saline controls receiving NAC or vehicle is presented in Figure S2. Next, we assessed crude membrane fraction GLT-1 expression in the NAcore across morpholino and drug treatment conditions compared to yoked saline controls. A one-way ANOVA revealed a significant difference among treatment groups (F(5,29)=12.16, p<0.0001). Post hoc comparisons revealed that GLT-1 was rescued in Nic-CTRL-NAC rats, and that Nic-CTRL-Veh, Nic-Antisense-NAC, Nic-Antisense-Veh conditions produced significantly lower membrane fraction GLT-1 expression relative to yoked saline-Veh controls (Figure 3D, *p<0.05). Lastly, we assessed whether NAC and morpholino treatment altered NAcore TNFα expression. A two-way ANOVA revealed a significant main effect of NAC treatment on TNFα expression (F(1,27)=8.771, p=0.006). Post hoc comparisons indicated that regardless of morpholino treatment, NAC inhibited TNFα (Figure 3E, *p<0.05). We also examined CD40 expression, which is a marker of pro-inflammatory activation of immune cells (Kawahara et al., 2009). Results indicate that antisense morpholino treatment, regardless of NAC, upregulated CD40 (see Figure S3). These results show that NAC inhibits cue-induced nicotine seeking through a GLT-1-dependent mechanism and inhibits TNFα expression, suggesting that immunomodulation may underlie NAC’s therapeutic mechanism.

NAC Inhibits Rapid Synaptic Potentiation and Associated Cue-Induced Nicotine Seeking.

Since NAC depends on NAcore GLT-1 restoration to inhibit cue-induced reinstatement of nicotine seeking, we hypothesized that NAC would also inhibit AMPA/NMDA in the NAcore compared to vehicle in nicotine-extinguished rats. A three-way ANOVA was performed to assess the effect of NAC treatment on T=15 reinstatement. This test revealed significant main effects of session (F(1,76)=10.13, p<0.05) and lever (F(1,76)=34.94, p<0.001), as well as significant session X lever (F(1,76)=16.32, p<0.001), session X treatment (F(1,76)=5.31, p<0.05), and session X lever X treatment (F(1,76)=4.039, p<0.05) interactions. Post hoc comparisons revealed a significant reduction in active lever presses in NAC-treated rats compared to vehicle (Figure 4B, *p<0.05). In addition, a two-tailed t-test revealed a significant decrease in AMPA/NMDA within the NAcore of NAC-treated rats (Figure 4C, t(15)=3.015, p=0.01). These findings suggest that NAC treatment may inhibit cue-induced nicotine seeking by inhibiting post-synaptic excitability in the NAcore, consistent with previous reports (Kupchik et al., 2012; Moussawi et al., 2009, 2011).

NF-κB pathway signaling in the NAcore mediates conditioned nicotine seeking independent of GLT-1 expression.

NAC inhibits pro-inflammatory NF-κB signaling in vitro, and NF-κB is known to regulate learning, memory, and synaptic plasticity (Oka et al., 2000; Meffert et al., 2003). Therefore, we utilized viral vectors expressing a constitutively active or dominant negative mutant of IKK (IKKca or IKKdn, respectively) to assess whether NAcore NF-κB pathway signaling underlies cue-induced reinstatement of nicotine seeking and NAC’s mechanism of action. Figure 5B provides a schematic of the TNFα→NF-κB pathway. First, we examined active lever presses during a 2-hour cue reinstatement test among virus-treated rats that received NAC or vehicle (self-administration and extinction curves are provided in Figure S4). A two-way ANOVA revealed significant main effects of treatment (F(1,38)=18.74, p<0.001) and virus (F(2,38)=13.341, p<0.001), as well as a significant treatment X virus interaction (F(2,38)=9.938, p<0.001) (Figure 5C). Post hoc analyses revealed that GFP/Veh, IKKca/Veh, and IKKca/NAC conditions all significantly reinstated relative to extinction (*p<0.05) and displayed significantly higher reinstatement compared to the other treatment conditions (#p<0.05). Next, we assessed the effect of virus and NAC or vehicle treatment on total NAcore GLT-1 expression. A two-way ANOVA revealed a significant main effect of virus (F(2,34)=12.103, p<0.001), with no significant main effect of treatment or treatment X virus interaction (Figure 5D). Post hoc analysis of virus revealed significantly higher GLT-1 expression in GFP-treated rats compared to both IKKdn and IKKca (*p<0.001). Taken together, these results indicate that NAC itself did not alter total levels of GLT-1 expression (consistent with our previous findings (Powell et al., 2019)), but that constitutive activation or inhibition of IKK may suppress total GLT-1 expression regardless of NAC treatment. This suggests that the NF-κB pathway modulates conditioned nicotine seeking through a GLT-independent mechanism and that the therapeutic efficacy of NAC may depend on its immunomodulatory activity described in Figure 3.

Discussion

The present findings indicate that nicotine self-administration and extinction impairs glial glutamate transport, decreases GFAP expression, and that decreased membrane GLT-1 expression is rapidly reversed by cue-induced reinstatement. In addition, these results demonstrate that NAC inhibits nicotine seeking and AMPA/NMDA in the NAcore and that NAC inhibition of nicotine seeking depends on GLT-1 expression. Lastly, this study provides the first evidence to suggest that (1) nicotine self-administration and subsequent changes in glutamate transport may alter neuroimmune function, (2) NAC treatment may ameliorate these nicotine-induced dysregulations, and (3) the NF-κB pathway mediates conditioned nicotine seeking as well as the inhibitory effects of NAC on this behavior independent of GLT-1 expression.

Downregulation of GLT-1 is a consistent neuroadaptation that has been observed across drug classes, and restoration of GLT-1 expression is associated with a decrease in cue-induced reinstatement of cocaine seeking (Scofield et al., 2016a). Here, we confirm nicotine self-administration and extinction downregulates the function and expression of GLT-1 in the NAcore. Notably, GLT-1, which is a Na+-dependent transporter expressed primarily in astrocytes, is responsible for >90% of glutamate uptake in the CNS (Haugeto et al., 1996). Whether this downregulation is contingent on extinction training is not entirely clear, although a previous study examining GLT-1 after withdrawal from non-contingent exposure to the synthetic cathinone MDPV showed that accumbens GLT-1 is downregulated after at least two days of withdrawal (Gregg et al., 2016). However, another study found that GLT-1a mRNA was only downregulated in the accumbens following long-access cocaine self-administration and 45 days of withdrawal (Kim et al., 2018). Despite somewhat mixed results, these studies seem to suggest that GLT-1 downregulation is not necessarily contingent on extinction training but rather may develop after some period of withdrawal. Beyond downregulated GLT-1, we also demonstrate here that extinction of nicotine-seeking is associated with reduced expression of the astrocytic filament protein GFAP, which corroborates previous findings by Scofield et al. (2016b). This study reported decreased NAcore GFAP expression and concomitant decreases in astrocyte surface area, volume, and synaptic co-localization. Here, we extend these previous findings by demonstrating that reduced GFAP expression persists through T=15 reinstatement. Reduced GFAP expression has been demonstrated at both the clinical and preclinical levels with neuropsychiatric disorders such as depression and schizophrenia, both of which are highly comorbid with smoking (Cotter et al., 2001; Lasser et al., 2000). Downregulation of GFAP may indicate that astrocytes remain in a hyporeactive state following extinction from drug seeking, as previously hypothesized by Scofield et al. (2016b). In support of this hypothesis, chemogenetic activation of astrocytes prior to cue-induced reinstatement of cocaine seeking increased glial glutamate release and inhibit cocaine seeking through a mGlu2/3-dependent mechanism (Scofield et al., 2015). Taken together, reductions in GLT-1 and GFAP expression following extinction of nicotine seeking likely represent astrocytic maladaptations that contribute to nicotine relapse vulnerability.

While many studies suggest that GLT-1 expression is downregulated following chronic drug self-administration and withdrawal, this study is the first to show that GLT-1 expression may undergo activity-dependent regulation in its membrane expression. Although this rapid increase in GLT-1 could theoretically contribute to increased glutamate uptake during reinstatement, this process could account for the decay of transient synaptic potentiation (t-SP) that we have observed during cue-induced nicotine- (Gipson et al., 2013b) and cocaine-seeking (Gipson et al., 2013a). In support, we previously found that throughout a 2-hour session of cue-induced reinstatement of nicotine seeking, both active lever press behavior and extracellular glutamate concentration decline across the session, likely due to the within-session extinction curve seen when examining time-course behavior (Gipson et al., 2013a; Gipson et al., 2013b). Moreover, another study showed that inhibiting NAcore (but not NAshell) GLT-1 glutamate uptake with dihydrokainic acid prevented ceftriaxone from inhibiting cue-induced cocaine seeking during a 60-min session (Fischer et al., 2013). Interestingly, exogenous glutamate has been shown to rapidly increase glutamate uptake within 15 minutes of exposure in vitro (Duan et al., 1999). This supports our hypothesis that the rapid elevation in membrane GLT-1 observed here, likely due to increased membrane trafficking, may represent an activity-dependent compensatory mechanism that is sensitive to rapid changes in extracellular glutamate. Taken together, it is possible that rapidly increased membrane GLT-1 expression is transient and mediates the within-session changes in dendritic spine head diameter and AMPA/NMDA that are characteristic of t-SP (Gipson et al., 2013b). While not directly assessed in this study, we show that membrane GLT-1 is reduced after a 2-hour cue-induced reinstatement test relative to yoked saline controls (Figure 3D), suggesting that the rapid increase at T=15 is transient. Alternatively, if the rapid increase in membrane GLT-1 increases glutamate uptake efficiency, this could potentially reduce tone on presynaptic mGlu2/3 autoreceptors, thus contributing to the net enhanced glutamate efflux observed during conditioned cocaine and nicotine seeking (Gipson 2013a; Gipson et al., 2013b). While not directly quantified in the present study, these hypotheses warrant further investigation.

Although NAC has shown checkered success both clinically and pre-clinically (Knackstedt et al., 2009; Powell et al., 2019; Schmaal et al., 2011), several previous studies have shown that NAC inhibits both cue-induced reinstatement of cocaine (Reissner et al., 2015) and nicotine seeking (Moro et al., 2018; Ramirez-Niño et al., 2013; Goenaga et al., 2019). Importantly, impairing Sxc− (a Na+-independent glutamate transporter) has no effect on NAC inhibition of cue-induced cocaine seeking, while impairing the rescue of GLT-1 expression potentiates cue-induced cocaine seeking in NAC-treated rats through a mGlu5-dependent mechanism (Reissner et al., 2015). We did not observe potentiation of nicotine seeking in NAC-antisense treated rats, which suggests that the mechanism by which NAC inhibits cue-induced nicotine seeking may not be entirely analogous to that of cue-induced cocaine seeking. While nicotine self-administration shares some common neurobiological consequences with other drugs of abuse, nicotine differentially alters NAcore proteins associated with glutamatergic signaling as compared to cocaine and heroin (e.g., AMPA and NMDA receptor subunit composition; Conrad et al., 2008; Gipson et al., 2013b; Shen et al., 2011). Nevertheless, we demonstrate here that NAC inhibits NAcore AMPA/NMDA during T=15 reinstatement, which is when AMPA/NMDA and dendritic spine head diameter are transiently increased (Gipson et al., 2013a; 2013b). This is consistent with previous reports demonstrating a reduction in EPSC amplitude in the NAcore following NAC treatment (Kupchik et al., 2012) and a rescue of AMPA/NMDA to yoked saline levels following withdrawal from cocaine self-administration (Moussawi et al., 2011). Taken together, the present findings highlight the need to examine nicotine-specific mechanisms underlying cue-triggered nicotine relapse, which might guide the development of new and effective pharmacotherapies that improve treatment outcomes.

Nicotine uniquely modulates neuroimmune signaling in the CNS through its full-agonist activity at α7 nAChRs. Specifically, cholinergic activity at α7 nAChRs on microglia plays a significant role in modulating immune responses to inflammatory stimuli (Shytle et al., 2004). For example, nicotine exposure can disrupt the blood brain barrier (Hawkins et al., 2004) and induce oxidative stress and NF-κB activation (Barr et al., 2007). Here, we demonstrate that extinction of nicotine seeking is associated with marked increases in NAcore TNFα expression, which was attenuated by NAC. We did not observe an increase in IL-6 expression following nicotine self-administration and extinction, contrary to some previous observations (Lau et al., 2012; Lee et al., 2012). However, the effects of nicotine (versus smoking) on immune signaling may vary between brain regions considering the heterogeneity of brain nAChR expression (Gotti et al., 2009). Nevertheless, our results are consistent with recent findings demonstrating increased TNFα, but not IL-6, gene expression in the frontal cortex of nicotine- and cigarette smoke-exposed rats (Royal et al., 2018). TNFα, which is a known modulator of learning, memory, and glutamatergic plasticity (Albensi & Mattson, 2000; Stellwagen et al., 2005; Stellwagen & Malenka, 2006), is primarily derived from microglia under pathological conditions (Welser-Alves & Milner, 2013). Indeed, our results corroborate previous findings indicating an inhibitory effect of NAC on brain TNFα expression (Saleh, 2015). TNFα both increases surface expression of calcium permeable AMPA receptors and decreases surface expression of GABAA receptors within hippocampal slices, resulting in significant increases in excitatory synaptic strength (Stellwagen et al., 2005). This effect was shown to occur within 15 minutes of TNFα treatment (Stellwagen et al., 2005), which is the same time point at which we have previously observed t-SP of MSNs during cue-induced nicotine reinstatement (Gipson et al., 2013b). Recently, it has been suggested that TNFα might decrease excitatory activity at striatal synapses and inhibit cocaine-induced synaptic plasticity (Lewitus et al., 2016). However, this study did not utilize a range of physiologically-relevant concentrations of TNFα (compare to Habbas et al., 2015) and utilized five days of experimenter-delivered cocaine as opposed to self-administration, which are both models of drug delivery that produce profoundly different neurobehavioral consequences (Namba et al., 2018). Altogether, it is possible that chronic nicotine could uniquely alter neuroimmune activity that functions as a modulator of glutamatergic plasticity and subsequent drug-seeking behavior.

This study is the first to describe a role for accumbens NF-κB pathway signaling in mediating conditioned nicotine-seeking behavior and provides a novel mechanism of action for NAC-induced suppression of nicotine-seeking behavior. As expected, NAC sufficiently blocked cue-induced reinstatement of nicotine seeking in GFP controls, which was blocked by IKKca. Strikingly, IKKdn alone sufficiently prevented reinstatement of nicotine seeking despite downregulated GLT-1 expression. While seemingly counterintuitive, it is likely that both constitutive activation and inhibition of the NF-κB pathway lead to downregulated GLT-1 considering NF-κB is a bi-directional regulator of GLT-1 (Sitcheran et al., 2005). Indeed, neuronal activation of astrocytic NF-κB is required for activity-dependent induction of GLT-1 expression (Ghosh et al., 2011). These findings support our present results demonstrating rapid elevations in GLT-1 expression during reinstatement, when prelimbic inputs into the NAcore are activated. Given that GLT-1 expression was impaired in both IKKdn and IKKca conditions, it is probable that neuronal NF-κB (likely located post-synaptically in MSNs) drove these observed effects here on cue-induced nicotine seeking. This corroborates a previous study using these same vectors, where IKKca facilitated cocaine conditioned place preference and associated increases in MSN dendritic spine density, which was inhibited by IKKdn (Russo et al., 2009). Additionally, chronic social defeat stress increases accumbens IKK signaling, and IKKca was found to facilitate synaptic alternations in MSNs induced by this stress (Christoffel et al., 2011). The present findings are also supported by a recent study suggesting that specific rescuing of cocaine-induced impairments of NAcore GLT-1a expression alone is not sufficient to attenuate cue-induced cocaine seeking (Logan et al., 2018). Taken together, these findings support the growing hypothesis that GLT-1-independent mechanisms may underlie the therapeutic efficacy of drugs such as NAC.

Clinical studies investigating NAC as a treatment for SUDs have shown inconsistent results. A placebo-controlled study showed that NAC had no effect on nicotine craving or withdrawal but showed a decrease in self-reported cigarette use per day when two outliers were removed due to excessive alcohol consumption (Knackstedt et al., 2009). Similarly, another study showed that NAC demonstrated a trend towards reducing nicotine craving (Schmaal et al., 2011). NAC exhibits antioxidant and anti-inflammatory properties, as described here and elsewhere, that may underlie its observed therapeutic effects on cue-induced nicotine seeking (Zafarullah et al., 2003). One major limitation of NAC is its poor oral bioavailability, which could be improved by using novel delivery vectors such as nanoparticles. One recent study demonstrated that the delivery of NAC via nanoparticles significantly enhanced its anti-inflammatory potential (Markoutsa & Xu, 2017). Thus, it is possible that NAC’s checkered clinical success could be due at least in part to its poor bioavailability, which would attenuate NAC’s anti-inflammatory potential. Similarly, ceftriaxone has been shown to increase GLT-1 expression through a NF-κB-dependent mechanism (Lee et al., 2008), suggesting that ceftriaxone may also exert its therapeutic efficacy through immunomodulatory mechanisms. Ultimately, these findings are the first to describe potential immunomodulatory mechanisms of NAC within a nicotine self-administration paradigm that may be relevant to its capacity to attenuate nicotine craving and relapse in humans. Thus, these data provide support for the potential use of immunomodulatory pharmacotherapeutics as adjunctive relapse-prevention tools and establish a precedent for further investigation into the role of neuroimmunomodulation in regulating nicotine-seeking behavior and associated synaptic plasticity.

Supplementary Material

Supp Mat
Supp info

Acknowledgements

The authors thank Dr. Peter Kalivas for use of equipment and morpholino experimental design. The authors thank Armani Del Franco, Joseph McCallum, Yeyoung Jun, Sandy Phan, Matt Miller, Jose Piña, Hanaa Ulangkaya, Vincent Carfagno, and Neringa Stankeviciute for their technical assistance. We also thank the laboratories of Dr. M. Foster Olive and Dr. Heather A. Bimonte-Nelson for providing equipment necessary for western blot experiments, as well as Dr. Scott J. Russo for kindly gifting us the HSV-IKK vectors. We also thank Dr. Jonna M. Leyrer-Jackson for providing helpful feedback on this manuscript. This work was supported by the Arizona Alzheimer’s Consortium, DA 036569 and -S1, DA044479, and DA045881 (to CDG).

References

  1. Albensi BC, & Mattson MP (2000). Evidence for the Involvement of TNF and NF-κB in Hippocampal Synaptic Plasticity. Synapse, 35(2), 151–159. [DOI] [PubMed] [Google Scholar]
  2. Alhaddad H, Das SC, & Sari Y (2014). Effects of ceftriaxone on ethanol intake: a possible role for xCT and GLT-1 isoforms modulation of glutamate levels in P rats. Psychopharmacology, 231(20), 4049–4057. 10.1007/s00213-014-3545-y [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baker DA, McFarland K, Lake RW, Shen H, Tang X-C, Toda S, & Kalivas PW (2003). Neuroadaptations in cystine-glutamate exchange underlie cocaine relapse. Nature Neuroscience, 6(7), 743–749. 10.1038/nn1069 [DOI] [PubMed] [Google Scholar]
  4. Beattie EC, Stellwagen D, Morishita W, Bresnahan JC, Ha BK, Von Zastrow M, … Malenka RC (2002). Control of Synaptic Strength by Glial TNFalpha. Science, 295(5563), 2282–2285. 10.1126/science.1067859 [DOI] [PubMed] [Google Scholar]
  5. Christoffel DJ, Golden SA, Dumitriu D, Robison AJ, Janssen WG, Ahn HF, … Russo SJ (2011). IκB Kinase Regulates Social Defeat Stress-Induced Synaptic and Behavioral Plasticity. Journal of Neuroscience, 31(1), 314–321. 10.1523/JNEUROSCI.4763-10.2011 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cui C, Shurtleff D, & Harris RA (2014). Neuroimmune mechanisms of alcohol and drug addiction. International Review of Neurobiology, 118, 1–12. 10.1016/B978-0-12-801284-0.00001-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fischer KD, Houston ACW, & Rebec GV (2013). Role of the Major Glutamate Transporter GLT1 in Nucleus Accumbens Core Versus Shell in Cue-Induced Cocaine-Seeking Behavior. Journal of Neuroscience, 33(22), 9319–9327. 10.1523/JNEUROSCI.3278-12.2013 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gipson CD, Kupchik YM, Shen H, Reissner KJ, Thomas CA, & Kalivas PW (2013a). Relapse induced by cues predicting cocaine depends on rapid, transient synaptic potentiation. Neuron, 77(5), 867–872. 10.1016/j.neuron.2013.01.005 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gipson CD, Reissner KJ, Kupchik YM, Smith ACW, Stankeviciute N, Hensley-Simon ME, & Kalivas PW (2013b. Reinstatement of nicotine seeking is mediated by glutamatergic plasticity. Proceedings of the National Academy of Sciences of the United States of America, 110(22), 9124–9129. 10.1073/pnas.1220591110 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Haugeto O, Ullensvang K, Levy LM, Chaudhry FA, Honoré T, Nielsen M, … Danbolt NC (1996). Brain glutamate transporter proteins form homomultimers. Journal of Biological Chemistry, 271(44), 27715–27722. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/8910364 [DOI] [PubMed] [Google Scholar]
  11. Kalivas PW, LaLumiere RT, Knackstedt L, & Shen H (2009). Glutamate transmission in addiction. Neuropharmacology, 56, 169–173. 10.1016/j.neuropharm.2008.07.011 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kaltschmidt B, & Kaltschmidt C (2015). NF-KappaB in Long-Term Memory and Structural Plasticity in the Adult Mammalian Brain. Frontiers in Molecular Neuroscience, 8, 69 10.3389/fnmol.2015.00069 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kim R, Sepulveda-Orengo MT, Healey KL, Williams EA, & Reissner KJ (2018). Regulation of glutamate transporter 1 (GLT-1) gene expression by cocaine self-administration and withdrawal. Neuropharmacology, 128, 1–10. 10.1016/j.neuropharm.2017.09.019 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Knackstedt LA, LaRowe S, Mardikian P, Malcolm R, Upadhyaya H, Hedden S, … Kalivas PW (2009). The Role of Cystine-Glutamate Exchange in Nicotine Dependence in Rats and Humans. Biological Psychiatry, 65(10), 841–845. 10.1016/j.biopsych.2008.10.040 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Knackstedt LA, Melendez RI, & Kalivas PW (2010). Ceftriaxone Restores Glutamate Homeostasis and Prevents Relapse to Cocaine Seeking. Biological Psychiatry, 67(1), 81–84. 10.1016/j.biopsych.2009.07.018 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kupchik YM, Moussawi K, Tang X-C, Wang X, Kalivas BC, Kolokithas R, … Kalivas PW (2012). The effect of N-acetylcysteine in the nucleus accumbens on neurotransmission and relapse to cocaine. Biological Psychiatry, 71(11), 978–986. 10.1016/j.biopsych.2011.10.024 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. LaLumiere RT, & Kalivas PW (2008). Glutamate release in the nucleus accumbens core is necessary for heroin seeking. Journal of Neuroscience, 28(12), 3170–3177. 10.1523/JNEUROSCI.5129-07.2008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lee J, Taneja V, & Vassallo R (2012). Cigarette smoking and inflammation: cellular and molecular mechanisms. Journal of Dental Research, 91(2), 142–149. 10.1177/0022034511421200 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lee S-G, Su Z-Z, Emdad L, Gupta P, Sarkar D, Borjabad A, … Fisher PB (2008). Mechanism of Ceftriaxone Induction of Excitatory Amino Acid Transporter-2 Expression and Glutamate Uptake in Primary Human Astrocytes. Journal of Biological Chemistry, 283(19), 13116–13123. 10.1074/jbc.M707697200 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Logan CN, LaCrosse AL, & Knackstedt LA (2018). Nucleus accumbens GLT-1a overexpression reduces glutamate efflux during reinstatement of cocaine-seeking but is not sufficient to attenuate reinstatement. Neuropharmacology, 135, 297–307. 10.1016/j.neuropharm.2018.03.022 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Markoutsa E, & Xu P (2017). Redox Potential-Sensitive N -Acetyl Cysteine-Prodrug Nanoparticles Inhibit the Activation of Microglia and Improve Neuronal Survival. Molecular Pharmaceutics, 14(5), 1591–1600. 10.1021/acs.molpharmaceut.6b01028 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. McClure EA, Vandrey RG, Johnson MW, & Stitzer ML (2013). Effects of varenicline on abstinence and smoking reward following a programmed lapse. Nicotine & Tobacco Research, 15(1), 139–148. 10.1093/ntr/nts101 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. McFarland K, Lapish CC, & Kalivas PW (2003). Prefrontal glutamate release into the core of the nucleus accumbens mediates cocaine-induced reinstatement of drug-seeking behavior. Journal of Neuroscience, 23(8), 3531–3537. https://doi.org/23/8/3531[pii] [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Meffert MK, Chang JM, Wiltgen BJ, Fanselow MS, & Baltimore D (2003). NF-κB functions in synaptic signaling and behavior. Nature Neuroscience, 6(10), 1072–1078. 10.1038/nn1110 [DOI] [PubMed] [Google Scholar]
  25. Moro F, Orrù A, Marzo CM, Di Clemente A, & Cervo L (2018). mGluR2/3 mediates short-term control of nicotine-seeking by acute systemic N-acetylcysteine. Addiction Biology, 23(1), 28–40. 10.1111/adb.12443 [DOI] [PubMed] [Google Scholar]
  26. Moussawi K, Pacchioni A, Moran M, Olive MF, Gass JT, Lavin A, & Kalivas PW (2009). N-Acetylcysteine reverses cocaine-induced metaplasticity. Nature Neuroscience, 12(2), 182–189. 10.1038/nn.2250 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Moussawi K, Zhou W, Shen H, Reichel CM, See RE, Carr DB, & Kalivas PW (2011). Reversing cocaine-induced synaptic potentiation provides enduring protection from relapse. Proceedings of the National Academy of Sciences of the United States of America, 108(1), 385–390. 10.1073/pnas.1011265108 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Namba MD, Tomek SE, Olive MF, Beckmann JS, & Gipson CD (2018). The Winding Road to Relapse: Forging a New Understanding of Cue-Induced Reinstatement Models and Their Associated Neural Mechanisms. Frontiers in Behavioral Neuroscience, 12(February), 1–22. 10.3389/fnbeh.2018.00017 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. O’Neill LA ., & Kaltschmidt C (1997). NF-kB: a crucial transcription factor for glial and neuronal cell function. Trends in Neurosciences, 20(6), 252–258. 10.1016/S0166-2236(96)01035-1 [DOI] [PubMed] [Google Scholar]
  30. Oka S, Kamata H, Kamata K, Yagisawa H, & Hirata H (2000). N-acetylcysteine suppresses TNF-induced NF-kappaB activation through inhibition of IkappaB kinases. FEBS Letters, 472(2–3), 196–202. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/10788610 [DOI] [PubMed] [Google Scholar]
  31. Olmos G, & Lladó J (2014). Tumor necrosis factor alpha: a link between neuroinflammation and excitotoxicity. Mediators of Inflammation, 2014, 861231 10.1155/2014/861231 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Powell GL, Leyrer-Jackson JM, Goenaga J, Namba MD, Piña J, Spencer S, … Gipson CD (2019). Chronic treatment with N -acetylcysteine decreases extinction responding and reduces cue-induced nicotine-seeking. Physiological Reports, 7(1), e13958 10.14814/phy2.13958 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ramirez-Niño AM, D’Souza MS, & Markou A (2013). N-acetylcysteine decreased nicotine self-administration and cue-induced reinstatement of nicotine seeking in rats: Comparison with the effects of N-acetylcysteine on food responding and food seeking. Psychopharmacology, 225(2), 473–482. 10.1007/s00213-012-2837-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Reissner KJ, Gipson CD, Tran PK, Knackstedt LA, Scofield MD, & Kalivas PW (2015). Glutamate transporter GLT-1 mediates N-acetylcysteine inhibition of cocaine reinstatement. Addiction Biology, 20(2), 316–323. 10.1111/adb.12127 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Reissner KJ, Sartor GC, Vazey EM, Dunn TE, Aston-Jones G, & Kalivas PW (2012). Use of vivo-morpholinos for control of protein expression in the adult rat brain. Journal of Neuroscience Methods, 203(2), 354–360. 10.1016/j.jneumeth.2011.10.009 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Roberts-Wolfe DJ, & Kalivas PW (2015). Glutamate transporter GLT-1 as a therapeutic target for substance use disorders. CNS & Neurological Disorders Drug Targets, 14(6), 745–756. 10.1016/bs.mcb.2015.01.016.Observing [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Royal W, Can A, Gould TD, Guo M, Huse J, Jackson M, … Bryant J (2018). Cigarette smoke and nicotine effects on brain proinflammatory responses and behavioral and motor function in HIV-1 transgenic rats. Journal of Neurovirology, 24(2), 246–253. 10.1007/s13365-018-0623-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Russo SJ, Wilkinson MB, Mazei-Robison MS, Dietz DM, Maze I, Krishnan V, … Nestler EJ (2009). Nuclear factor kappa B signaling regulates neuronal morphology and cocaine reward. Journal of Neuroscience, 29(11), 3529–3537. 10.1523/JNEUROSCI.6173-08.2009 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Saleh AAS (2015). Anti-neuroinflammatory and antioxidant effects of N-acetyl cysteine in long-term consumption of artificial sweetener aspartame in the rat cerebral cortex. Journal of Basic & Applied Zoology, 72, 73–80. 10.1016/J.JOBAZ.2015.05.001 [DOI] [Google Scholar]
  40. Sari Y, Smith KD, Ali PK, & Rebec GV (2009). Upregulation of Glt1 Attenuates Cue-Induced Reinstatement of Cocaine-Seeking Behavior in Rats. Journal of Neuroscience, 29(29), 9239–9243. 10.1523/JNEUROSCI.1746-09.2009 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Scofield MD, Boger HA, Smith RJ, Li H, Haydon PG, & Kalivas PW (2015). Gq-DREADD Selectively Initiates Glial Glutamate Release and Inhibits Cue-induced Cocaine Seeking. Biological Psychiatry, 78(7), 441–451. 10.1016/j.biopsych.2015.02.016 [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Scofield MD, Heinsbroek JA, Gipson CD, Kupchik YM, Spencer S, Smith ACW, … Kalivas PW (2016). The Nucleus Accumbens: Mechanisms of Addiction across Drug Classes Reflect the Importance of Glutamate Homeostasis. Pharmacological Reviews, 68(3), 816–871. 10.1124/pr.116.012484 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Scofield MD, Li H, Siemsen BM, Healey KL, Tran PK, Woronoff N, … Reissner KJ (2016). Cocaine Self-Administration and Extinction Leads to Reduced Glial Fibrillary Acidic Protein Expression and Morphometric Features of Astrocytes in the Nucleus Accumbens Core. Biological Psychiatry, 80(3), 207–215. 10.1016/j.biopsych.2015.12.022 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Shen H, Moussawi K, Zhou W, Toda S, & Kalivas PW (2011). Heroin relapse requires long-term potentiation-like plasticity mediated by NMDA2b-containing receptors. Proceedings of the National Academy of Sciences of the United States of America, 108(48), 19407–19412. 10.1073/pnas.1112052108 [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Shen H, Scofield MD, Boger H, Hensley M, & Kalivas PW (2014). Synaptic Glutamate Spillover Due to Impaired Glutamate Uptake Mediates Heroin Relapse. Journal of Neuroscience, 34(16), 5649–5657. 10.1523/JNEUROSCI.4564-13.2014 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sitcheran R, Gupta P, Fisher PB, & Baldwin AS (2005). Positive and negative regulation of EAAT2 by NF-kappaB: a role for N-myc in TNFalpha-controlled repression. The EMBO Journal, 24(3), 510–520. 10.1038/sj.emboj.7600555 [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Smith ACW, Scofield MD, Heinsbroek JA, Gipson CD, Neuhofer D, Roberts-Wolfe DJ, … Kalivas PW (2016). Accumbens nNOS interneurons regulate cocaine relapse. Journal of Neuroscience. 10.1523/JNEUROSCI.2673-16.2016 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sondheimer I, & Knackstedt LA (2011). Ceftriaxone prevents the induction of cocaine sensitization and produces enduring attenuation of cue- and cocaine-primed reinstatement of cocaine-seeking. Behavioural Brain Research, 225(1), 252–258. 10.1016/j.bbr.2011.07.041 [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Stellwagen D, Beattie EC, Seo JY, & Malenka RC (2005). Differential Regulation of AMPA Receptor and GABA Receptor Trafficking by Tumor Necrosis Factor-α. Journal of Neuroscience, 25(12), 3219–3228. 10.1523/JNEUROSCI.4486-04.2005 [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Stellwagen D, & Malenka RC (2006). Synaptic scaling mediated by glial TNF-α. Nature, 440(7087), 1054–1059. 10.1038/nature04671 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp Mat
NIHMS1032587-supplement-Supp_Mat.docx (548.1KB, docx)
Supp info
NIHMS1032587-supplement-Supp_info.docx (17.1KB, docx)

RESOURCES