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Department of Pharmacology and Toxicology, State University of New York at Buffalo, Buffalo, New YorkResearch Institute on Addictions, Program in Neuroscience, Department of Psychology, State University of New York at Buffalo, Buffalo, New York
Department of Pharmacology and Toxicology, State University of New York at Buffalo, Buffalo, New YorkResearch Institute on Addictions, Program in Neuroscience, Department of Psychology, State University of New York at Buffalo, Buffalo, New York
Department of Pharmacology and Toxicology, State University of New York at Buffalo, Buffalo, New YorkResearch Institute on Addictions, Program in Neuroscience, Department of Psychology, State University of New York at Buffalo, Buffalo, New York
Department of Pharmacology and Toxicology, State University of New York at Buffalo, Buffalo, New YorkResearch Institute on Addictions, Program in Neuroscience, Department of Psychology, State University of New York at Buffalo, Buffalo, New York
Department of Pharmacology and Toxicology, State University of New York at Buffalo, Buffalo, New YorkResearch Institute on Addictions, Program in Neuroscience, Department of Psychology, State University of New York at Buffalo, Buffalo, New York
Department of Pharmacology and Toxicology, State University of New York at Buffalo, Buffalo, New YorkResearch Institute on Addictions, Program in Neuroscience, Department of Psychology, State University of New York at Buffalo, Buffalo, New York
Address correspondence to David M. Dietz, University at Buffalo (SUNY), Department of Pharmacology and Toxicology, 3435 Main St, 613 Biomedical Research Building, Buffalo, NY 14214.
Department of Pharmacology and Toxicology, State University of New York at Buffalo, Buffalo, New YorkResearch Institute on Addictions, Program in Neuroscience, Department of Psychology, State University of New York at Buffalo, Buffalo, New YorkDepartment of Psychology, California State University Bakersfield, Bakersfield, California
Drug addiction is defined as a chronic disease characterized by compulsive drug seeking and episodes of relapse despite prolonged periods of drug abstinence. Neurobiological adaptations, including transcriptional and epigenetic alterations in the nucleus accumbens, are thought to contribute to this life-long disease state. We previously demonstrated that the transcription factor SMAD3 is increased after 7 days of withdrawal from cocaine self-administration. However, it is still unknown which additional factors participate in the process of chromatin remodeling and facilitate the binding of SMAD3 to promoter regions of target genes. Here, we examined the possible interaction of BRG1—also known as SMARCA4, an adenosine triphosphatase–containing chromatin remodeler—and SMAD3 in response to cocaine exposure.
Methods
The expression of BRG1, as well as its binding to SMAD3 and target gene promoter regions, was evaluated in the nucleus accumbens and dorsal striatum of rats using western blotting, co-immunoprecipitation, and chromatin immunoprecipitation following abstinence from cocaine self-administration. Rats were assessed for cocaine-seeking behaviors after either intra-accumbal injections of the BRG1 inhibitor PFI3 or viral-mediated overexpression of BRG1.
Results
After withdrawal from cocaine self-administration, BRG1 expression and complex formation with SMAD3 are increased in the nucleus accumbens, resulting in increased binding of BRG1 to the promoter regions of Ctnnb1, Mef2d, and Dbn1. Intra-accumbal infusion of PFI3 attenuated, whereas viral overexpression of Brg1 enhanced, cocaine-reinstatement behavior.
Conclusions
BRG1 is a key mediator of the SMAD3-dependent regulation of cellular and behavioral plasticity that mediates cocaine seeking after a period of withdrawal.
Drug addiction is one of the most debilitating psychiatric disorders and is characterized by compulsive drug seeking and episodes of relapse despite prolonged periods of abstinence. In drug addicts, relapse and craving during abstinence are often triggered by environmental cues that were previously associated with drug use (
). Such studies have shown that neurobiological adaptations leading to changes in neuronal plasticity in key brain areas, such as the nucleus accumbens (NAc), contribute to increased drug seeking and craving (
Accumulating evidence suggests that the long-term changes in cellular and behavioral function after drug exposure are mediated by alterations in gene transcription (
). We previously identified SMAD3, a transcription factor within the transforming growth factor-β signaling pathway, as an essential mediator of cocaine-induced plasticity and craving behavior that regulates the expression of β-catenin (Ctnnb1), adenylyl cyclase-associated protein 2 (Cap2), and drebrin (Dbn1) (
). Binding of SMAD3 to DNA and the subsequent regulation of gene expression are thought to require interactions with adenosine triphosphate (ATP)-dependent swItch/sucrose non-fermentable (SWI/SNF) nucleosome repositioning complexes (
). Although a role of these transcriptional complexes has been documented in regulation of cognitive processes, such as memory-related synaptic plasticity (
), the role of the BRG1-SMAD3 complex in drug addiction remains unknown. The data presented here provide evidence that this complex is involved in drug seeking after a period of abstinence. Withdrawal from cocaine self-administration in rats resulted in increased expression and functional interaction of BRG1 and SMAD3 in the NAc. Furthermore, pharmacologic inhibition of BRG1 attenuated its interaction with SMAD3 and suppressed cue-induced reinstatement of cocaine seeking, whereas overexpression of BRG1 exacerbated cocaine-seeking behavior. These results implicate the SMAD3 and BRG1 transcriptional complex in the regulation of gene expression and behavioral changes after cocaine exposure, providing a novel mechanism for potential therapeutic targets in the treatment of cocaine addiction.
Methods and Materials
Subjects
Male Sprague Dawley rats (300–375 g; Harlan, Indianapolis, IN) were allowed to habituate to the colony room for 7 days on arrival. Rats had ad libitum access to food and water and were singly housed after surgery and for the duration of the self-administration phase of the experiments to protect the catheter/harness assembly. Behavioral testing took place during the dark phase of the 12-hour light/dark cycle. This study was conducted in accordance with the guidelines set up by the Institutional Animal Care and Use Committee of the State University of New York at Buffalo.
Self-administration Test Chambers
Twenty-four standard experimental test chambers (MED Associates, Inc., St. Albans, VT) were used for self-administration and reinstatement experiments. The test chambers contain two snout-poke holes located on one wall, with a stimulus light mounted above each, and a house light mounted in the middle of the back wall. Snout pokes were monitored with infrared detectors. Test chambers were computer controlled by a MED Associates interface with MED-PC.
Drugs
(–)-Cocaine hydrochloride, generously supplied by the National Institute on Drug Abuse drug supply program, was dissolved in sterile 0.9% saline. Cocaine solutions (4.5 mg/mL) were prepared on a weekly basis and were delivered by a syringe pump. Pump durations/injection volumes were adjusted on a daily basis according to body weight to deliver 1.0 mg/kg per infusion. The BRG1 inhibitor PFI3 (Tocris, Minneapolis, MN) was dissolved in a mixture of 1 part absolute ethanol, 1 part Emulphor-620 (Rhodia of Solvay; S.A., Brussels, Belgium), and 18 parts saline (
The SMARCA2/4 ATPase domain surpasses the bromodomain as a drug target in SWI/SNF-mutant cancers: Insights from cDNA rescue and PFI-3 inhibitor studies.
). Rats were allowed 7 days to recover after surgery. The catheters were flushed daily with 0.2 mL of a solution of enrofloxacin (4 mg/mL) in heparinized saline (50 IU/mL in 0.9% sterile saline) to preserve catheter patency. At the end of behavioral testing, each animal received an intravenous infusion of ketamine hydrochloride (0.5 mg/kg in 0.05 mL), and the behavioral response was observed to verify catheter patency. Loss of muscle tone and righting reflexes served as behavioral indicators of patency.
Self-administration and Cue-Induced Reinstatement
Self-administration procedures were conducted as previously described (
). After the 7-day recovery from jugular catheter surgery, rats were assigned to self-administer either saline or 1.0 mg/kg cocaine per infusion. Rats were subjected to 10 test sessions (2 hours per session), during which time responses to the active alternative snout-poke hole resulted in intravenous injections of cocaine or saline according to a fixed-ratio 1 (FR1) schedule of reinforcement, followed by a 30-second time-out period. Infusions were accompanied by a 5-second illumination of the stimulus light above the active snout-poke hole, and the house light was extinguished for the duration of the time-out period. Responses to the inactive hole resulted in no programmed consequences. The criterion for acquisition of cocaine self-administration was an average of 10 cocaine infusions per day during the 10-session test phase.
Extinction tests were initiated 24 hours after the last self-administration session. Rats were exposed to multiple within-session extinction sessions, during which time the chambers were dark and responses were recorded but resulted in no programmed consequences. Extinction sessions were 1 hour in duration, separated by 5 minutes, and were continued until responding levels fell to fewer than 20 responses per session (8–10 sessions) (
). Animals were then allowed an additional 6 days of abstinence, followed by 1-hour test of cue-induced reinstatement, during which time active responses produced cues previously paired with drug delivery.
Tissue Collection
After a 7-day withdrawal (or after cue-induced reinstatement for animals receiving PFI3 microinfusions), animals were euthanized by rapid decapitation, brains were removed and sliced into 1-mm-thick sections using a brain matrix (Braintree Scientific, Inc., Braintree, MA), and 2-mm-diameter tissue punches from the NAc and dorsal striatum/caudoputamen (CPU) were collected and rapidly frozen on dry ice.
Western Blotting and Immunoprecipitation
NAc or CPU tissue punches from each rat that self-administered cocaine or saline (n = 6 per group) were homogenized in 25 mmol/L Tris (pH 8.0) and 0.25 mol/L sucrose buffer. Total protein was extracted, and 30-μg samples were loaded onto 10% Tris-sodium dodecyl sulfate (SDS) polyacrylamide gels for electrophoresis separation, then transferred to nitrocellulose membranes and blocked with 5% nonfat milk in phosphate-buffered saline. Membranes were incubated overnight at 4°C with primary antibodies diluted in blocking buffer (Rockland Immunochemicals, Inc., Limerick, PA), including rabbit anti-BRG1 (1:2,000; Abcam, Cambridge, MA), rabbit anti–phospho(p)-SMAD3 (1:500; Calbiochem of Millipore Corp., Billerica, MA), and mouse anti–β-actin (1:10,000; Cell Signaling Technologies, Inc., Danvers, MA). Membranes were incubated with IRDye secondary antibodies (1:5,000; LI-COR, Inc., Lincoln, NE) for 1 hour at room temperature. The blots were imaged using the Odyssey Infrared Imaging system (LI-COR, Inc.) and quantified by densitometry using ImageJ (National Institutes of Health, Bethesda, MD). β-Actin was used as a loading control.
For immunoprecipitation, NAc or CPU tissue punches from rats (n = 8 for the saline group, n = 7 for the cocaine group) were homogenized in 500 μL of homogenization buffer that contained 50 mmol/L β-glycerol phosphate, 1 mmol/L dithiothreitol (DTT), 2 mmol/L ethylene glycol tetraacetic acid (EGTA), 10 mmol/L sodium fluoride (NaF), 1 mmol/L sodium orthovanadate, 20 mmol/L N-(2-hydroxyethyl)-1-piperazine N′-(2-ethanesulfonic acid) (HEPES) (pH 7.4), 1% Triton X-100, 10% glycerol, 0.5% SDS, phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich, St. Louis, MO), and protease inhibitors (Roche, Basel, Switzerland). A total of 600 μg of protein was used for immunoprecipitation with protein G beads (GE Healthcare, Little Chalfont, United Kingdom) as previously described with some modification (
). Briefly, samples were brought to a final volume of 500 μL with lysis buffer, and 5 μL of anti-BRG1 antibody (Abcam) was added, followed by gentle tumbling overnight at 4°C. Next, 50 μL of protein G agarose slurry was added to each sample, followed by gentle tumbling at 4°C overnight. Beads were pelleted by centrifugation and washed three times with 1 mL of wash buffer. After the final wash, reducing sample buffer that contained SDS and dithiothreitol was added to the pelleted beads, and samples were heated to 95°C for 5 minutes and subjected to SDS-polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose and blotted for mouse anti-SMAD3 (1:1,000; Abcam) and anti-BRG1 (for normalization).
Quantitative Polymerase Chain Reaction and Chromatic Immunoprecipitation--Quantitative Polymerase Chain Reaction
Bilateral NAc punches were obtained after 7 days of withdrawal from cocaine self-administration. Chromatin immunoprecipitation (ChIP) was performed for BRG1 as described previously (
), with minor modifications. Briefly, four punches each from two rats were pooled for each sample (n = 7 samples from 14 rats for the saline group; n = 6 from 12 rats for the cocaine group) and fixed for 12 minutes in 1% formaldehyde, then quenched with 2 mol/L glycine for 5 minutes. The chromatin was solubilized and extracted by cell and nuclear lysis. The chromatin was sheared using a Bioruptor 300 (Diagenode Diagnostics, Seraing, Belgium) at 4°C at high sonication intensity for 30 seconds on and 30 seconds off for 10 minutes, followed by a 10-minute rest, which was repeated three times. Fragment sizes of 250–1000 bp were verified by agarose gel electrophoresis. Magnetic sheep anti-rabbit beads (Invitrogen of Thermo Fisher Scientific, Waltham, MA) were incubated with anti-BRG1 antibody at 4°C overnight on a rotator. After the prescribed wash steps, 70 μL of the magnetic bead/antibody complex slurry was incubated with the sheared chromatin sample for 16 hours at 4°C. Five percent of sheared chromatin from each sample was used as an input control. Samples were washed with lithium chloride (LiCl) and Tris-ethylenediaminetetraacetic acid (EDTA) buffers. Reverse cross-linking was performed at 65°C overnight, and proteins and RNA were removed using proteinase K (Invitrogen) and RNase (Roche), respectively. DNA was purified using a DNA purification kit (Qiagen, Hilden, Germany). In addition, an immunoglobulin G control was used to test for nonspecific binding. Quantitative polymerase chain reaction (qPCR) was performed (iQ5 system; Bio-Rad Laboratories, Inc., Hercules, CA) to identify binding of BRG1 to proximal promoter regions of target genes that contain a SMAD3 binding site (
) (primers listed in Supplemental Table S1). Amplification reactions were run in triplicate with iQ SYBR Green (Bio-Rad Laboratories, Inc.), and each sample was normalized to the immunoglobulin G control. Fold changes were calculated as cocaine relative to saline control.
For qPCR after PFI3 microinfusions, NAc tissue punches were collected after cue-induced reinstatement, and total RNA was extracted using Trizol (Invitrogen) and the RNeasy Micro Kit (Qiagen), as previously described (
) [primers used for housekeeping (glyceraldehyde 3-phosphate dehydrogenase [GAPDH]) and all other genes are listed in Supplemental Table S2].
Pharmacologic Inhibition of BRG1
Rats received jugular catheters and implantation of bilateral guide cannulas (C235G-2.4; Plastics One, Inc., Roanoke, VA) that were aimed at the NAc [coordinates from bregma according to Paxinos and Watson (
): anteroposterior, +1.7; mediolateral, +1.2; dorsoventral, −6.5]. Animals were assigned to receive microinjections (1 μL/hemisphere) of PFI3 (n = 7) or vehicle (n = 8, counterbalanced based on their performance during cocaine self-administration and extinction) infused at a rate of 0.5 μL/min; injectors were left in place for an additional 3 minutes to allow for diffusion. Rats received microinjections once per day for 4 days during the withdrawal period, beginning on the fourth day after the last cocaine exposure. Thirty minutes after the last microinjection, animals were tested for cue-induced reinstatement for 1 hour, and then immediately they were sacrificed for tissue collection.
Food Reinforcement
Commercially available two-lever operant chambers located within sound-attenuating, ventilated enclosures (Coulbourn Instruments, LLC, Whitehall, PA) were used for all food reinforcement experiments. Rats were trained to lever press for a food reward (45-mg pellet; Bio-Serv, Flemington, NJ). During the daily 1-hour training sessions, rats could press either lever (both active) under an FR1 schedule and earn up to 50 food pellets. The response requirement was gradually increased to FR10 over a period of 10 days. After food training, rats were implanted with bilateral guide cannulas aimed at the NAc for PFI3 or vehicle (n = 7 per group) infusions as described above. Rats were tested for the response rate 30 minutes after the last microinjection.
Locomotor Activity
Locomotor activity was recorded by an infrared motion-sensor system (AccuScan Instruments, Inc., Columbus, OH) fitted outside transparent plastic cages (40 × 40 × 30 cm). The Versa Max animal activity software (Omnitech Electronics, Inc., Columbus, OH) monitors the distance the animal travelled in 1 hour. Locomotor testing was performed 30 minutes after the final microinfusion for animals receiving PFI3 or vehicle (n = 8 per group) or after complete expression of virus after viral microinjection (n = 8 per group).
Viral Overexpression of BRG1 and dnSmad3 in the NAc
The generation of the dominant negative Smad3 (dnSmad3) construct has been described in detail previously (
). Briefly, serines in the SSXS motif at the C-terminus of SMAD3 (which confer activation when phosphorylated and allow for translocation) were mutated to alanines (SAXA) (
) and dnSmad3 were cloned into a p1005 herpes simplex virus (HSV) transcription cassette. Such HSV vectors exhibit maximal expression 3–5 days after infection (
). Viruses were validated both in vitro and in vivo before use in behavioral experiments.
Intra-accumbal viral injections were performed after the 7-day withdrawal period after within-session extinction from self-administration. Rats were counterbalanced according to self-administration and extinction performances and were assigned to receive bilateral injections of HSV-dnSmad3 (n = 9), HSV-Brg1 (n = 8), or HSV-green fluorescent protein (GFP) (as a control: for HSV-dnSmad3 control, n = 8; for HSV-Brg1 control, n = 10) aimed at the NAc; injectors were set at a 10° angle; Paxinos and Watson (
Behavioral and structural responses to chronic cocaine require a feedforward loop involving DeltaFosB and calcium/calmodulin-dependent protein kinase II in the nucleus accumbens shell.
). Viruses (1 μL/hemisphere) were infused at a rate of 0.2 μL/min, and injectors were left in place for an additional 10 minutes to allow for diffusion. Three days later, when the transgene overexpression is maximal, rats were tested for cue-induced reinstatement for 1 hour.
Statistical Analyses
Statistical analyses were conducted using Prism software (GraphPad Software, Inc., La Jolla, CA). Performance during self-administration and extinction was analyzed using repeated measures two-way within-subject analyses of variance (ANOVAs), followed by Bonferroni post hoc tests. Student t tests were conducted for analysis of cue-induced reinstatement, food reinforcement, locomotor activity, and immunoblotting. One-way ANOVAs were conducted on ChIP data, followed by Fisher’s least significant difference post hoc tests. Significance was set at p < .05, and data are presented as the mean ± SEM.
Results
Upregulation of p-SMAD3 and BRG1 After Abstinence From Cocaine Self-administration
Rats self-administered significantly more infusions of cocaine than saline during the 10-session training paradigm (Figure 1A, B; two-way repeated-measures ANOVA, interaction effect F9,90 = 2.559, p = .011; drug effect F1,10 = 95.21, p < .001; session effect F9,90 = 1.237, p = .282). Tissue punches from NAc and CPU (Figure 1C) were taken 7 days after the last cocaine exposure. After this withdrawal period, levels of p-SMAD3 (t test, t10 = 2.241, p = .049) and BRG1 (t test, t10 = 2.470, p = .033) were significantly increased in the NAc (Figure 1D, E), whereas no change was found in total SMAD3 protein (saline: 1.000 ± 0.040; cocaine: 0.949 ± 0.082; t test, t10 = 0.552, p = .592). In contrast, no significant changes were found in the CPU (Figure 1F, G).
Figure 1Withdrawal from cocaine self-administration increases phospho-(p)-SMAD3 and BRG1 protein levels and interaction. (A) Timeline of behavioral testing and tissue collection. (B) Mean number of saline (Sal) and cocaine (Coc) (1 mg/kg per infusion) infusions per session during self-administration training (n = 6 per group). (C) Representative anatomic location of tissue punches taken from rat nucleus accumbens (NAc) and caudoputamen (CPU). (D−G) Relative p-SMAD3 and BRG1 expression in the NAc (D, E; n = 6 per group) and CPU (F, G;n = 5–6 per group). Data are expressed as mean ± SEM; *p < .05 vs. Sal.
Increased BRG1-SMAD3 Interaction After Abstinence From Cocaine Self-administration
To evaluate protein interactions, another cohort of rats was trained to self-administer cocaine (Figure 2A, B; two-way repeated-measures ANOVA: interaction effect F9,351 = 3.389, p < .001; drug effect F1,39 = 168.1, p < .001; session effect F9,351 = 1.340, p = .214). A significantly greater amount of SMAD3 co-immunoprecipitated with BRG1 in the NAc (t test, t13 = 2.399, p = .032) but not the CPU (t test, t8 = 0.4731, p = .649) after 7 days of withdrawal (Figure 2C). Using ChIP assay, there was increased BRG1 at the promoter regions of several SMAD3-dependent target genes that have previously been implicated in actin dynamics and cocaine-induced synaptic plasticity (
), including Ctnnb1 (p = .026), Mef2d (p = .016), and Dbn1 (p = .012), with a small increase in binding to Cap2 (p = .080) but not Grin2a (p = .610) or Pdyn (p = .477) (Figure 2D; one-way ANOVA, F11,62 = 2.573, p = .009).
Figure 2Withdrawal from cocaine (Coc) self-administration increases functional BRG1-SMAD3 interaction. (A) Timeline of behavioral testing and tissue collection. (B) Mean number of saline (Sal) and Coc (1 mg/kg per infusion) infusions per session during self-administration training (n = 19–22 per group). (C) Protein lysates from rat nucleus accumbens (NAc) and caudoputamen (CPU) collected after withdrawal from Coc or Sal self-administration were immunoprecipitated (IP) with anti-BRG1 antibody and probed for SMAD3 binding (n = 5–8 per group). (D) Occupancy of BRG1 on the promoter regions of β-catenin (Ctnnb1), NMDA receptor 2A (Grin2a), myocyte enhancer factor 2d (Mef2d), adenylyl cyclase-associated protein 2 (Cap2), drebrin (Dbn1), and prodynorphin (Pdyn) in the NAc as measured by quantitative chromatin immunoprecipitation after 7-day withdrawal from Sal or Coc self-administration (n = 5–7 samples per group; two animals count as one sample). Data are expressed as mean ± SEM; *p < .05, #p < .10 vs. Sal. IB, immunoblot.
BRG1-SMAD3 Interaction Regulates Cue-Induced Reinstatement of Cocaine Seeking
To test whether the BRG1 activity is required for cocaine-seeking behavior, rats received intra-accumbal microinfusions of PFI3 or vehicle during the withdrawal period after self-administration (Figure 3A). Rats were assigned to vehicle or PFI3 groups such that there was no difference in cocaine self-administration training (Figure 3B; two-way repeated-measures ANOVA, treatment effect F1,13 = 0.388, p > .05) or extinction (Figure 3C; two-way repeated-measures ANOVA, treatment effect F1,13 = 0.342, p > .05) before BRG1 inhibition. Rats receiving microinfusions of PFI3 had significantly fewer total active responses during cue-induced reinstatement (Figure 3D; t test, t13 = 2.374, p = .033). Tissue samples from the NAc were collected. Co-immunoprecipitation experiments revealed reduced binding of SMAD3 to BRG1 after PFI3 infusion (Figure 3E; t test, t10 = 2.519, p = .030). Repeated intra-accumbal infusions resulted in decreased expression of several BRG1-SMAD3 target genes, including Mef2d, Cap2, and Dbn1 (Supplemental Figure S1). The decreased responses during cue-induced reinstatement was not due to reduced overall activity because there was no difference between groups in total distance traveled during a locomotor test (Figure 3F; t test, t14 = 0.082, p = .935). Importantly, PFI3 treatment did not alter the response rate for a food reinforcer (Figure 3G; t test, t12 = 0.139, p = .891), demonstrating that the effect of PFI3 was specific to cue-induced reinstatement.
Figure 3Pharmacologic inhibition of BRG1 in the nucleus accumbens (NAc) decreases cue-induced reinstatement of cocaine seeking. (A) Timeline of behavioral testing and intra-accumbal microinjections. (B) Mean number of infusions per session during cocaine self-administration (1 mg/kg per infusion) and (C) total responses during extinction did not differ between groups assigned to receive BRG1 inhibitor PFI3 or vehicle. (D) Number of active responses during cue-induced reinstatement after microinjection (1 μL/hemisphere) of vehicle or PFI3 (30 mmol/L) (n = 7–8 per group). (E) SMAD3 and BRG1 co-immunoprecipitation (IP) after microinjection of vehicle or PFI3 (n = 6 per group). (F) Total distance traveled in a locomotor test and (G) responding for a food reward did not differ between groups (n = 7–8 per group). Data are expressed as mean ± SEM; *p < .05 vs. vehicle. IB, immunoblot; IgG, immunoglobulin G.
To confirm the importance of the SMAD3 transcriptional complex in cue-mediated cocaine-seeking behaviors, HSV constructs overexpressing dnSmad3 or a GFP control were microinjected into the NAc. Consistent with our previous work (
), blockade of SMAD3 by viral overexpression of dnSmad3 significantly reduced cue-induced reinstatement as measured by the attenuation of the total active responses (HSV-GFP group, 47.50 ± 7.693; HSV-dnSmad3 group, 28.56 ± 4.197; t test, t5 = 2.231, p = .041). Taken together, these data demonstrate that SMAD3 and BRG1 activity are essential for cocaine seeking after a period of withdrawal, because inhibition of either suppresses reinstatement behaviors.
Overexpression of BRG1 Potentiates Cue-Induced Reinstatement of Cocaine Seeking
To determine whether increased expression of BRG1 could further enhance cocaine-seeking behavior, rats received intra-accumbal injections of HSV-Brg1 during the period of withdrawal from cocaine self-administration (Figure 4A, B). As in the above experiments with PFI3 infusions, groups receiving either HSV-Brg1 or HSV-GFP did not differ with respect to the amount of cocaine taken during self-administration (Figure 4C; two-way repeated-measures ANOVA, treatment effect F1,16 = 0.013, p > .05) or extinction responses (Figure 4 D; two-way repeated-measures ANOVA, treatment effect F1,16 = 0.024, p > .05) before viral injection. Viral-mediated overexpression of BRG1 in the NAc significantly increased the number of active responses in the cue-induced reinstatement test (Figure 4E; t test, t16 = 2.312, p = .034) compared with HSV-GFP controls. This increase in responding was not due to differences in general locomotor activity, because overexpression of BRG1 in the NAc had no effect on total distance traveled during a locomotor test (Figure 4F; t test, t14 = 0.279, p = .783).
Figure 4Overexpression of Brg1 in the nucleus accumbens enhances cue-induced reinstatement of cocaine seeking. (A) Timeline of behavioral testing and intra-accumbal virus injection. (B) Representative image of a coronal section of the rat brain (1.7 mm from bregma), depicting green fluorescent protein (GFP)-infected cells in the nucleus accumbens. Representative western blots showing increased BRG1 protein levels in the nucleus accumbens after herpes simplex virus (HSV)-Brg1 overexpression. (C) Mean number of infusions per session during cocaine self-administration (1 mg/kg per infusion) and (D) total responses during extinction procedure did not differ between groups assigned to receive HSV-Brg1 or HSV-GFP. (E) Number of active responses during cue-induced reinstatement after overexpression of HSV-GFP or HSV-Brg1 (n = 8–10 per group). (F) Total distance traveled in a locomotor test (n = 8 per group). Data are expressed as mean ± SEM, *p < .05 vs. HSV-GFP. AC, anterior commissure.
Accumulating evidence suggests that alterations in gene regulation contribute substantially to the long-term changes in brain structure and function after drug exposure [reviewed in (
). In addition, our previous work identified SMAD3 as another important regulator in the expression of genes that are heavily implicated in neuronal plasticity (
). The results presented here confirm the importance of SMAD3 and demonstrate an important and novel role for BRG1 and its interaction with SMAD3 in cocaine-mediated behavioral adaptations. After a 7-day withdrawal from cocaine self-administration, the interaction between SMAD3 and BRG1 in the NAc was significantly increased and was associated with increased binding of BRG1 to the promoter regions of SMAD3-regulated genes. Moreover, pharmacologic or viral-mediated modification of this interaction bidirectionally altered cue-induced reinstatement in a model of cocaine seeking.
It is well accepted that transcription factors control gene expression by interacting with other chromatin-remodeling factors (
). Abnormalities in components of chromatin remodeling complexes have been implicated in many psychiatric diseases. For example, mutations of SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin proteins are involved in psychiatric disorders such as schizophrenia (
). In addition, the ATP-utilizing chromatin assembly and remodeling factor is a critical component of a chromatin-remodeling complex involved in the development of susceptibility to depression and in regulating stress-related behaviors (
). Recent data also suggest an underlying role of chromatin remodeling factors in the neurobiology of drug abuse. For example, chronic cocaine exposure results in increased expression of the bromodomain and extraterminal protein BRD4, an epigenetic reader protein (
). We recently demonstrated that an accessory ATPase subunit of the ISWI family of chromatin remodeling complexes, bromodomain adjacent to zinc finger domain 1B, is also upregulated in the NAc following cocaine exposure (
In line with the emerging evidence, our data implicate BRG1, a key component of SWI/SNF chromatin remodeling complex, in addiction and drug abuse. In addition to its role in regulating differentiation and development in the central nervous system (
), the data implicate BRG1 in long-term neuroplasticity. BRG1 was found to interact with SMAD3 in vivo and to modulate behaviors in response to withdrawal from cocaine treatment. Importantly, inhibition of BRG1 decreased, whereas overexpression increased behavioral responses in a cocaine-seeking test. We propose that the inhibitor PFI3 disrupts the BRG1-SMAD3 interaction because the same effect was observed by interfering with SMAD3 activity by dnSmad3 overexpression. Thus, the expression levels of SMAD3 and BRG1, as well as the integrity of the BRG1-SMAD3 transcriptional complex, play important roles in regulating cocaine-seeking behavior.
Withdrawal from cocaine exposure increased the binding of BRG1 to DNA targets, which is consistent with previous in vivo work demonstrating that BRG1 predominantly interacts with SMAD3 on target promoters to facilitate transforming growth factor-β–mediated gene transcription (
). These data suggest that BRG1 may be an essential component of multiple transcriptional complexes that mediate gene expression after cocaine exposure. In vitro studies demonstrate that BRG1 is involved in the organization of actin filaments (
). The binding of BRG1 to the promoter regions of these genes may in turn alter the synaptic plasticity in NAc medium spiny neurons and thus regulate the behavioral response to cocaine (
Identifying effective treatments for addiction has been a subject of intensive investigation. Our data establish that the SWI/SNF chromatin-remodeling component BRG1 in the NAc is involved in SMAD3-dependent gene regulation in withdrawal after cocaine self-administration. Furthermore, pharmacologic disruption of the BRG1-SMAD3 interaction by the BRG1 inhibitor PFI3 in the NAc was sufficient to decrease cocaine-seeking behavior. Here, we demonstrate the essential role of BRG1 and SMAD3 interactions in the NAc after cocaine self-administration. The ability of the BRG1 inhibitor to alter drug-seeking behavior offers potential therapeutic value; however, future studies will be necessary to determine the ability of PFI3 to readily cross the blood-brain barrier in addition to fully elucidating BRG1-SMAD3 regulation in other mesolimbic dopamine brain regions. Together, these findings provide new insight into epigenetic mechanisms underlying cocaine addiction and provide a new direction for the design of improved treatments for drug addiction.
Acknowledgments and Disclosures
This work was supported by National Institutes of Health; National Institute on Drug Abuse Grant No. R01DA037257 (to DMD).
All authors report no biomedical financial interests or potential conflicts of interest.
The SMARCA2/4 ATPase domain surpasses the bromodomain as a drug target in SWI/SNF-mutant cancers: Insights from cDNA rescue and PFI-3 inhibitor studies.
Behavioral and structural responses to chronic cocaine require a feedforward loop involving DeltaFosB and calcium/calmodulin-dependent protein kinase II in the nucleus accumbens shell.
Chronic cocaine use results in neuroplasticity in the nucleus accumbens that underlies vulnerability to relapse, even after protracted abstinence. It is well accepted that epigenetic changes in gene transcription contribute to the addicted phenotype, and three transcription factors in particular have received a great deal of attention: cyclic adenosine monophosphate response element binding protein, nuclear factor-κB, and ΔFosB. Targeting transcription factors is seen as a promising point of pharmacological intervention, in part because inhibition of a single transcription factor may pleiotropically alter multiple genes that contribute to the complex disorder.
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