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. Author manuscript; available in PMC: 2023 Jan 23.
Published in final edited form as: Eur J Neurosci. 2021 Sep 14;54(7):6382–6396. doi: 10.1111/ejn.15441

Isolation housing elevates amphetamine seeking independent of nucleus accumbens glutamate receptor adaptations

Erik J Garcia 1, Mary E Cain 1
PMCID: PMC9869284  NIHMSID: NIHMS1863408  PMID: 34481424

Abstract

Overdose death rates caused by psychostimulants have increased by 22.3% annually from 2008 to 2017. Cue-evoked drug craving progressively increases and contributes to perpetual relapse. Preclinical models have determined that glutamate receptor plasticity within the nucleus accumbens (NAc) drives amplified cue-evoked drug seeking after prolonged abstinence (>40 days). Isolated condition (IC) rearing increases cocaine and amphetamine (AMP) self-administration and cue-induced reinstatement. We tested the hypothesis that housing in the IC will augment AMP seeking after short and prolonged abstinence from AMP self-administration when compared with rats reared in the enrichment condition (EC). EC and IC male rats acquired stable AMP or SAL self-administration and were tested in a cue-induced AMP-seeking test after 1 and 40 days of abstinence. After the seeking test, the whole NAc was extracted and prepared for western blot analysis. Results indicate that IC rats had more active lever presses during a brief extinction interval and during the cue-induced seeking test. After 40 days of abstinence, IC rats had more active lever presses than EC rats during the cue-induced seeking test. Western blots indicated that the expression ratio between GluA1:mGlur5 was reduced only in IC-AMP-trained rats and the ratio between GluA1:mGlur1 was positively correlated with AMP seeking after prolonged abstinence in IC-AMP rats. These results indicate that IC housing engenders a vulnerable phenotype prone to persistent AMP seeking. The behavioural momentum of this vulnerable phenotype is further revealed when AMP-associated cues are presented following prolonged abstinence.

Keywords: AMPA receptors, cue-induced drug seeking, environmental enrichment, incubation of craving, metabotropic glutamate receptors

1 |. INTRODUCTION

The rates of overdose deaths caused by psychostimulants (methamphetamine, 3,4-methylenedioxy-methamphetamine, methylphenidate and amphetamine/dextroamphetamine [AMP]) have increased by 22.3% annually from 2008 to 2017 (Kariisa et al., 2019). During drug abstinence, drug craving progressively increases a phenomenon termed the incubation of drug craving. The incubation of drug craving occurs for many illicit drugs, suggesting that the progressive increase in drug craving is related to the abstinence period and not specific to the pharmacology profile of a ligand (Bossert et al., 2005; Grimm et al., 2001; Parvaz et al., 2017). In rodent models of drug self-administration and cue-induced drug seeking, rodents display a time-dependent increase in drug seeking suggesting that aspects of the incubation phenomenon can be modelled in rodents (Pickens et al., 2011). Determining the behavioural, environmental, and neurobiological underpinnings of the time-dependent increase in drug-seeking is critical to treating substance use disorders (SUDs).

Preclinical rodent experiments have determined that amplified cue-evoked drug seeking after prolonged abstinence (>40 days) from cocaine is accompanied by dysregulated glutamate receptor expression within the nucleus accumbens (NAc) (Ma et al., 2014; Wolf, 2016; Wolf & Ferrario, 2010). High-conductance calcium (Ca2+) permeable amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (CP-AMPA) mediate the amplified response for drug cues after prolonged abstinence (Conrad et al., 2008). Additionally, once the functional tone of metabotropic glutamate receptor 1 (mGlur1) tone is lost, CP-AMPA accumulates rampantly increasing the synaptic expression onto medium spiny neurons in the NAc that drive cue-evoked cocaine seeking. The mGlur1 modulates synaptic depression in the NAc after cocaine self-administration and the membrane expression of mGlur1 decreases during cocaine abstinence (Loweth, Scheyer, et al., 2014; Loweth, Tseng, & Wolf, 2014; McCutcheon et al., 2011). Importantly, decreased mGlur1 membrane expression precedes the accumulation of CP-AMPA membrane expression, suggesting that mGlur1 governs CP-AMPA surface expression (Loweth, Scheyer, et al., 2014).

While the effects of cocaine self-administration and prolonged abstinence on glutamate receptor expression are robustly characterized (Wolf, 2010a, 2010b; Wolf, 2016), other psychomotor stimulants that carry abuse potential are less understood. However, it appears that the time-dependent increase in methamphetamine seeking peaks more rapidly when compared with cocaine (Murray et al., 2019; Scheyer et al., 2016). Despite the different time courses, amplified methamphetamine seeking during abstinence is driven in part, by CP-AMPA receptor accumulation in the NAc, an effect suppressed by the mGlur1 positive allosteric modulator, SYN119 (Scheyer et al., 2016, but see Murray et al., 2019). These results suggest augmented CP-AMPA expression underlies the progressive increase in drug seeking. However, the incubation of craving has not been characterized after amphetamine (AMP) self-administration and prolonged abstinence.

Enriched condition (EC) rearing or environmental enrichment is an effective anti-addiction/SUD strategy. EC reduces the responding for conditioned cues associated with cocaine (Chauvet et al., 2009), AMP (Stairs et al., 2006) and nondrug reinforcers (Grimm et al., 2008) when compared with rodents housed in an isolated condition (IC) (Bardo et al., 2013). However, EC housing has proven less robust to suppress the incubation of cocaine craving; some experiments conclude that EC housing protects against cocaine and sucrose incubation (Chauvet et al., 2012; Grimm et al., 2008; Ma et al., 2016), whereas others determined that EC is not effective at suppressing the incubation effect (Thiel et al., 2012) or requires sustained EC housing (Ma et al., 2016). These seemingly discrepant results may be attributed to an EC ‘intervention’ type model (EC housing after drug exposure). Less research has utilized the EC ‘protection’ model (differential housing before drug exposure) in an incubation of drug craving model. Previous experiments suggest that maintaining an EC is necessary to suppress behaviours related to drug vulnerability (Chauvet et al., 2012; Garcia et al., 2017; Solinas et al., 2010; Yates et al., 2017). Although a definitive mechanism has not been identified that explains the differences in differentially housed rodents, housing manipulations impact NAc AMPA subunit profiles. Optogenetically induced long-term depression (LTD) effectively removes CP-AMPA in the NAc and blocks augmented cocaine seeking. However, the removal of CP-AMPA was not long lasting unless the rats were maintained in an EC environment (Ma et al., 2016). Alternatively, IC rats may be more vulnerable to develop CP-AMPA and augmented drug seeking and following abstinence from AMP self-administration.

We tested the hypothesis that sustained EC and IC housing manipulations will alter AMP seeking after short and prolonged abstinence from AMP self-administration. We hypothesized that sustained IC housing would amplify AMP seeking, reducing mGlur1 expression and increase GluA1 expression in the whole NAc. The results suggest that the IC engineers a vulnerable phenotype prone to highly motivated AMP seeking following 1 and 40 days of abstinence when compared with EC rats.

2 |. MATERIALS AND METHODS

2.1 |. Animals and environmental housing conditions

Naïve male Sprague–Dawley rats (n = 81, Charles River, Portage, MI, USA) arrived at Kansas State University at PND 21. The colony room was maintained on a 12:12-h reverse light–dark cycle at 22°C and 30% to 45% humidity. Rats were reared in the EC or IC for 30 days (PND 22–51) (Arndt, Johns, et al., 2015; Garcia et al., 2019). EC rats were housed with 8–16 other male rats in a large metal cage (60 × 120 × 45 cm) that was lined with pine wood chip bedding. During the rearing period, EC rats were handled every day for approximately 1 min. The environmental novelty was maintained by arranging 14 (seven new + seven old) plastic toys and PVC pipe sections in unique arrangements daily. IC rats were housed individually in hanging metal wire cages (17 × 24 × 20 cm). During the 30-day rearing period, IC rats were not handled and were not be exposed to novel objects or pine chip bedding. After the rearing period, all rats remained housed in their respective rearing environments during all subsequent experiments and manipulations. All experimental procedures were conducted in accordance with the Institutional Animal Care and Use Committee at Kansas State University and the NIH guidelines (National Research Council, 1996).

2.2 |. Drugs and solutions

Dextroamphetamine (AMP; Sigma Aldrich, TX, USA) was dissolved in sterile SAL (0.9%) to a concentration of 0.1 mg/kg/infusion. Cefazolin was dissolved in sterile water and administered in a volume of 0.1 ml at (50 mg/ml) intravenously (iv) daily to minimize infection.

2.3 |. Apparatus

Lever press training, AMP self-administration and AMP-seeking tests were conducted in standard operant conditioning chambers (ENV-001, Med Associates, St. Albans, VT, USA) enclosed in a sound-attenuating and ventilated cubicle. AMP or SAL infusions were administered via a syringe pump (PHM-100, Med Associates) connected to a 10-ml syringe (Arndt, Garcia, et al., 2015;Arndt, Johns, et al., 2015; Garcia et al., 2019; Garcia & Cain, 2020).

2.4 |. Lever press training

At PND 52 rats were food restricted to 85% to facilitate lever press training (~3 days) (Arndt et al., 2019; Garcia et al., 2019). During a 30-min lever press training session, a single active lever press (FR-1) delivered a 20% sucrose solution in a recessed food receptacle. Inactive lever presses had no consequence. During lever training, the house light was always turned on, and the 3-cm white cue lights centred above each lever remained off during the entire session and were never explicitly paired with sucrose reinforcement.

2.5 |. Indwelling jugular catheter surgical procedures

Rats were anesthetized with ketamine (80 mg/kg, ip) and diazepam (5 mg/kg, ip) (Arndt, Johns, et al., 2015; Garcia et al., 2019; Garcia & Cain, 2020). Polyurethane catheters (12-cm length, 0.2-mm internal diameter; SAI Infusion Technologies) were implanted into the left jugular vein. Catheter tubing was connected subcutaneously to a 22-g back-mounted cannula (Plastics One, Roanoke, VA, USA; Biomedical Structures, Warwick, RI, USA). To maintain catheter patency, catheters were flushed before and after self-administration sessions with heparinized SAL (0.1 ml, 10–30 IU/ml) and once per day with cefazolin (0.1 ml, 50 mg/ml) to minimize infection. Rats were allowed 7 days to recover before AMP self-administration sessions.

2.6 |. AMP (0.1 mg/kg/infusion) self-administration

Rats were trained to self-administered AMP (0.1 mg/kg/infusion) or SAL (equal volume to AMP) on an FR-1 schedule of reinforcement in daily 1-h sessions. Active lever presses infused AMP or SAL over ~5.9 s. Infusions of AMP and SAL were paired with the illumination of the white cue light above the active lever for 5.9 s and pump infusion sound (Arndt et al., 2019; Arndt, Johns, et al., 2015; Garcia et al., 2019; Garcia & Cain, 2020). After infusions, the cue light and infusion pump then turned off, and the house light remained on to signal a 20-s timeout. Active lever presses during the timeout period were recorded but had no programmed consequence. The inactive lever always had no programmed consequence. Acquisition criteria were defined as (1) an average of at least 10 infusions per hour and (2) an active:inactive lever press ratio greater than 2:1. Catheter patency was checked with methohexital (Brevital, 0.1 ml, 10 mg/ml) before the first self-administration and after the last self-administration session. Rats (n = 81) meeting all criteria were included in the AMP-seeking tests, biochemical analysis and data analyses (Figure 1a).

FIGURE 1.

FIGURE 1

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Male EC and IC rats acquire stable amphetamine (AMP) 0.1 mg/kg/infusion or saline (SAL) self-administration. (a) Experimental timeline and (b) EC (black circle) and IC (black triangle) rats self-administer equal amounts of AMP across the last 15 training sessions. IC rats (white triangle) self-administer more saline than EC rats (white circle). (c) Inactive lever presses during corresponding AMP or SAL self-administration sessions. The average across all training sessions revealed EC-AMP rats had more inactive lever presses when compared with IC-AMP rats. IC-SAL rats had more inactive lever presses when compared with EC-SAL rats (not denoted on the figure). Sample sizes: EC-AMP: n = 16; EC-SAL: n = 20, IC-AMP: n = 25; IC-SAL: n = 20. *p < .05 EC-AMP versus IC-AMP; ^p < EC-SAL versus IC-SAL

2.7 |. Context- and discrete cue-induced AMP-seeking tests

After 24 h, all rats were tested in a context and discrete cue-induced seeking procedure. Rats were tethered to the intravenous pump leash as if it were a normal self-administration session, but AMP or SAL was not infused. During the initial 10-min interval, all lever pressing had no programmed consequence but was recorded. This manipulation was used to determine the effects of the contextual cues on AMP and SAL seeking (Anastasio et al., 2014). After the initial interval elapsed, a single non-contingent 5.9-s presentation of the conditioned discrete cue complex (cue light + infusion pump sound) was presented. Subsequent active lever presses resulted in a 5.9-s presentation of the discrete cue complex and the illumination of the house light for the 20-s timeout (Anastasio et al., 2013; Anastasio et al., 2014). Responses on the inactive lever had no programmed consequence. The discrete cue-induced seeking interval was 60 min.

2.8 |. NAc western blot

Immediately following the context and cue-induced seeking tests, half of the rats were anesthetized with 4% isoflurane gas and rapidly decapitated. Whole brains were harvested and immediately frozen in powdered dry ice and stored at −80°C until dissected. The remaining rats were housed in their respective EC or IC home cage environments for 40 days of forced abstinence. After 40 days of forced abstinence, the rats were tested in the context and cue-induced seeking test, sacrificed and brains harvested using the same procedures mentioned above.

The frozen brain was sliced in 1-mm coronal sections (Brain Matrix Kent Scientific), and the whole NAc (core and shell) was dissected on ice. The NAc was homogenized in 1 ml of sucrose harvest buffer (0.32 M sucrose, 2 mM EDTA, 50 mM Tris–HCl pH 7.4) + 10 μl of protease inhibitors (ThermoScientific Protease Cocktail). Homogenized NAc tissue incubated on ice for 15 min. NAc samples were centrifuged at 1000 g for 5 min at 4°C (P1). The supernatant was transferred into a new tube (S1) and then centrifuged at 10,000 g for 20 min at 4°C. The resulting supernatant (S2) was discarded, and the resulting cellular membrane pellet (P2) was resuspended in 200 μl of NP-40 lysis buffer (150 mM NaCl, 1% NP-40, 50 mM Tris-Base pH 8.0) + protease inhibitors. The P2 fraction was used for all western blot analysis analyses (LaCrosse et al., 2016).

Total protein of each sample was determined using the Pierce BCA assay and Nanodrop 8000 (ThermoScientific). NAc samples (10 μg) were run on precast acrylamide gels (4% to 20% bis-acrylamide; SDS-PAGE, Bio-Rad) under reducing conditions (βME). The samples were separated at 100 V for 90 min and wet transferred to a PVDF membrane at 75–90 V for 120 min. The PVDF membrane was blocked using a non-fat milk solution (3% non-fat dry milk + Tris-buffered SAL [TBST + 0.1% Tween 20]). The primary antibody (GluA1: rabbit polyclonal 1:2000 AB31232 Abcam; GluA2: mouse monoclonal 1:1000 AB106515 Abcam; mGlur1: rabbit polyclonal 1:1000 AB82211 Abcam; mGlur5: rabbit monoclonal 1:5000 AB76316 Abcam) was applied overnight at 4°C. Then, the PVDF membrane was washed in TBST for 1 h, exchanging the used TBST with fresh TBST every 10 min. Horseradish peroxidase-conjugated secondary antibodies (anti-rabbit AB97051 or anti-mouse AB205719) were mixed in 5% non-fat dry milk + TBST and applied at room temperature for 1 h. The PVDF membrane was again washed with TBST for 1 h exchanging with fresh TBST every 10 min. Chemiluminescent signal was visualized with Clarity ECL (Bio-Rad). Primary and secondary antibodies were removed using a 0.015% glycine, 0.001 SDS, 0.01 Tween 20 and pH 2.2 solution. The PVDF membrane was reblocked and probed for additional proteins of interest or the loading control. Each protein of interest was normalized to calnexin (rabbit polyclonal AB22595, 1:10,000–1:20,000). IC-SAL rats were normalized to 100% to make comparisons across rearing groups and drug groups. All image analyses were performed in ImageJ.

2.9 |. Statistical and data analyses

AMP self-administration sessions were analysed with a repeated measures factorial ANOVA, with rearing group and drug as between-subjects variables and training session as a within-subjects factor. The context and cue-induced seeking tests were analysed using repeated measures factorial ANOVAs with rearing group, drug and incubation groups as between-subjects factors. For the animals that were tested after 1 and 40 days of abstinence, we analysed their data with a separate within-subjects ANOVA (Adhikary et al., 2017). Significant interactions were probed with simple effects or planned t-test comparisons. Western blot data were analysed using a factorial ANOVA, with rearing group, drug and incubation group treated as between-subjects factors. A significance statistical difference was set at p < .05, and the family-wise error rate for multiple comparisons was Sidak corrected. All data are stored in on the publically available Open Science Framework repository. The repository is available at https://osf.io/r4bzp/. Additionally, the data are available for download in a GitHub repository, erikgarcia22/EC_IC_AMP_incubation. All other files or analyses are attainable by contacting the corresponding author.

3 |. RESULTS

3.1 |. Environmental housing does not affect AMP (0.1 mg/kg/infusion) self-administration

A repeated measures ANOVA revealed main effects of group (F1,75 = 36.93, p = 4.70E−8), drug (F1,75 = 126.80, p = 8.73E−18) and session (F15,1125 = 56.03, p = 4.44E−16) on the number of infusions earned and significant two-way interactions of session × drug (F15,1125 = 2.30, p = .003) and group × drug (F1,75 = 33.37, p = 1.63E−7). The session × group interaction was not significant (F15,1125 = 1.28, p = .20). There was a three-way interaction between group, drug and session (F15,1125 = 8.92, p = 1.33E−15), indicating that EC and IC rats self-administered AMP or SAL differently across the 16 training sessions. This interaction was probed with simple effects analyses and revealed that in the AMP-trained rats, EC rats earned more infusions of AMP when compared with IC rats (F1,1125 = 13.83, p = .0021) during the first session (Figure 1b). No other differences were observed between EC and IC rats across the subsequent AMP training sessions. In the SAL-trained rats, IC rats earned more SAL infusions when compared with EC rats in Sessions 1–15 (F1,1125 ≥ 10.01, p < .0016; Figure 1b). When corrected for type I error, EC and IC rats in the SAL group were not different in the number of infusions earned in the last self-administration session (F1,1125 = 9.17, p = .005).

Total active lever presses (actives + timeout actives) were analysed to determine if EC and IC rats were different in active lever presses during the timeout intervals. The ANOVA revealed main effects of drug (F1,75 = 49.70, p = 4.40E−10) and session (F1,75 = 39.27, p = 4.25E−8) but no main effect of group (F1,75 = 0.96, p = .315). There were significant interactions between group × drug (F1,75 = 19.04, p = .00003), session × group (F15,1125 = 2.45, p = .0017) and session × drug (F15,1125 = 2.32, p = .0014). There was a significant three-way interaction of group × drug × session (F15,1125 = 2.22, p < 1.42E−16) on total active lever presses. For AMP-trained rats, EC rats had significantly more total active lever presses during the first (F1,1125 = 119.33, p = 1.99E−15) and second AMP self-administration sessions (F1,1125 = 36.48, p = 4.19E−9) when compared with IC rats. No other differences were observed across the subsequent training sessions. For SAL-trained rats, IC rats had more total active lever presses compared with the EC rats during session one (F1,1125 = 21.13, p = 9.55E−6) and for session two (F1,1125 = 13.15, p = .0006). No other differences were observed between EC and IC rats in the SAL group (Figure S1).

Analysis of inactive lever presses revealed no effects of group (F1,75 = 2.55, p = .11), or drug (F1,75 = 0.38, p = .54), but there was a significant main effect of session (F1,75 = 14.76, p = 1.11E−15). There were interactions between group × drug (F1,75 = 23.93, p = 6.00E−6) and drug × session (F15,1125 = 2.32, p = .003). The average number of inactive lever presses was greater in EC rats when they self-administered AMP (F1,75 = 5.50, p = .022) when compared with IC rats, and alternatively, in SAL-trained rats, IC rats had greater inactive lever presses (F1,75 = 20.78, p = 1.96E−5) (Figure 1c).

3.2 |. Isolation housing augments context-induced seeking after 1 day of abstinence

All lever presses during context-induced seeking had no programmed consequence. The ANOVA revealed main effects of group (F1,77 = 42.11, p = 1.51E−8), drug (F1,77 = 50.37, p = 1.09E−9) and an interaction between group × drug (F1,77 = 7.11, p = .019) on active lever presses. Simple effects revealed that EC (F1,77 = 8.83, p = .008) and IC (F1,77 = 53.61, p = 4.06E−10) rats in the AMP group had more active lever presses compared with SAL controls (Figure 2a). IC-AMP-trained rats had more active lever presses when compared with EC-AMP-trained rats (F1,77 = 41.40, p = 1.92E−8) and IC-SAL-trained rats had more active lever presses than EC-SAL-trained rats (F1,77 = 7.40, p = .016). Analysis of inactive lever presses revealed main effects of group (F1,77 = 14.73, p = .0005) and drug (F1,77 = 11.08, p = .003), but there was no interaction of group × drug (F1,77 = 0.8, p = .37) (Figure 2a, bottom).

FIGURE 2.

FIGURE 2

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IC housing augments AMP seeking during a brief extinction interval (Cue OFF) and reinstatement (Cue ON). (a) Previously active lever presses during the extinction interval (10 min). IC-AMP have elevated previously active lever presses compared with EC-AMP rats (#p < .05). EC-AMP- and IC-AMP-trained rats have greater previously active lever presses compared with SAL-trained counterparts (*p = .05). After 40 days of abstinence, EC-AMP and EC-SAL increase previously active lever presses during extinction (^p = .05), whereas IC-AMP rats decrease extinction responses (^p = .05). (b) Inactive lever presses during the extinction interval. (c) Previously active lever presses after 1 and 40 days of abstinence during the cue-induced reinstatement interval (60 min). IC-AMP rats have greater previously active lever presses during cue-induced reinstatement (Cue ON) compared with EC-AMP rats (#p < .05). (d) Inactive lever presses during the cue-induced reinstatement. Generally, IC rats had greater inactive lever presses compared with EC rats. Day 1 sample sizes: EC-AMP: n = 16; EC-SAL: n = 20, IC-AMP: n = 25; IC-SAL: n = 20. Day 40 sample sizes: EC-AMP: n = 9; EC-SAL: n = 10, IC-AMP: n = 12; IC-SAL: n = 10

3.3 |. Isolation housing augments discrete cue-induced AMP seeking after 1 day of abstinence

To assess cue-induced seeking, a single non-contingent discrete cue was presented, and subsequent active lever presses were reinforced by the discrete cue. The ANOVA determined main effects of group (F1,77 = 10.19, p = .002) and drug (F1,77 = 53.45, p = 2.13E−10) but no interaction of group × drug (F1,77 = 2.15, p = .15). AMP-trained rats had more active lever presses compared with SAL-trained rats (IC-AMP vs. IC-SAL, t43 = 5.78, p = 3.18E−7 and EC-AMP vs. EC-SAL, t34 = 4.97, p = 9.39E−6). IC rats had more active lever presses compared with EC rats (IC-AMP vs. EC-AMP, t39 = 2.46, p = .018 and IC-SAL vs. EC-SAL, t38 = 2.69, p = .011; Figure 2c). Analysis of inactive lever presses revealed main effects of group (F1,77 = 13.71, p = 7.98E−4) and drug (F1,77 = 13.36, p = 9.35E−4), but there was no group × drug interaction for inactive lever presses (F1,77 = 1.26, p = .27). The average number of inactive lever presses was greater in AMP-trained rats (EC-AMP vs. EC-SAL, t34 = 2.54, p = .016 and IC-AMP vs. IC-SAL, t43 = 2.99, p = .005). IC rats had more inactive lever presses compared with EC rats (t79 = 2.75, p = .009; Figure 2d).

3.4 |. Prolonged abstinence increases extinction responding independent of AMP

A three-way repeated measures ANOVA was used to determine the effects of prolonged abstinence on AMP seeking. For these analyses, group and treatment were between-subjects factors and abstinence period was entered as a within-subjects factor because these rats were tested after 1 and 40 days of abstinence.

Analysis of active lever presses during the context-induced seeking test determined that there were main effects of group (F1,37 = 19.54, p = 8.31E−5), drug (F1,37 = 10.36, p = .003) and time (F1,37 = 12.76, p = .001). The analysis also revealed interactions between group × time (F1,37 = 18.29, p = 1.28E−4) and drug × time (F1,37 = 8.78, p = .005) but no group × drug (F1,37 = 2.97, p = .093) or group × drug × time interactions (F1,37 = 3.05, p = .09). Simple effects analysis determined that active lever presses increased following 40 days of abstinence for EC-AMP (F1,37 = 9.76, p = .003) and EC-SAL (F1,37 = 20.26, p = 6.50E−5) rats. Interestingly, IC-AMP rats decreased active lever presses (F1,37 = 8.67, p = .006) whereas IC-SAL rats had similar active lever presses following 40 days of abstinence (F1,37 = 3.95, p = .054; Figure 2a). The changes in active lever presses after prolonged abstinence are not attributable to AMP because the number of active lever presses in the AMP group did not exceed the SAL group for EC (F1,37 = 0.10, p = .77) or IC rats (F1,37 = 0.72, p = .40). Analysis of inactive lever presses revealed no effects of group (F1,37 = 2.37, p = .13) or drug (F1,37 = 0.10, p = .76) but a main effect of time (F1,37 = 14.21, p = .0006). The interaction between group × time was trending (F1,37 = 3.77, p = .06), and there was an interaction between drug × time (F1,37 = 10.07, p = .003). There was no group × drug interaction (F1,37 = 1.12, p = .30) and no group × drug × time interaction (F1,37 = 0.16, p = .69). Simple effects determined that inactive lever presses increased in EC-SAL (F1,37 = 17.75, p = 1.54E−4) and IC-SAL rats (F1,37 = 7.19, p = .01) but not EC-AMP (F1,37 = 1.92, p = .17) or IC-AMP rats (F1,37 = 0.90, p = .35; Figure 2b).

3.5 |. Isolation housing augments cue-induced drug seeking after 40 days of abstinence

After the single cue light presentation, active lever presses delivered the discrete cue complex. Analysis of active lever presses revealed main effects of group (F1,37 = 9.50, p = .004) and drug (F1,37 = 15.46, p = 3.56E−4) but no effect of time (F1,37 = 1.19, p = .28). There were no interactions between group × time (F1,37 = 0.60, p = .43), drug × time (F1,37 = 3.71, p = .06), group × drug (F1,37 = 1.43, p = .24) or group × drug × time (F1,37 = 0.06, p = .82). Planned comparisons determined that IC-AMP rats had greater active lever presses when compared with EC-AMP rats (F1,37 = 6.30, p = .016). There was no difference in active lever presses between IC-SAL and EC-SAL rats (F1,37 = 1.27, p = .26). The difference between IC-AMP and IC-SAL was trending toward significant (F1,37 = 3.81, p = .058), and there was no difference between EC-AMP and EC-SAL rats (F1,37 = 0.26, p = .62) on active lever presses (Figure 2c). Analysis of inactive lever presses revealed a main effect of group (F1,37 = 8.33, p = .006), but no effects of drug (F1,37 = 2.96, p = .09) or time (F1,37 = 1.16, p = .29). There were no interactions between group × time (F1,37 = 0.76, p = .39), drug × time (F1,37 = 2.92, p = .10), group × drug (F1,37 = 2.95, p = .09) or group × drug × time (F1,37 = 1.56, p = .22). Planned comparisons determined that IC-AMP rats had greater inactive lever presses when compared with EC-AMP rats (F1,37 = 5.24, p = .03). Inactive lever presses were not different between IC-SAL and EC-SAL rats (F1,37 = 0.20, p = .66). There was no difference in inactive lever presses between IC-AMP and IC-SAL (F1,37 = 1.97, p = .19) or between EC-AMP and EC-SAL rats (F1,37 = 1.75, p = .19; Figure 2d). Finally, the complete time course of AMP-seeking test demonstrates the persistent AMP seeking in IC rats when compared with EC rats (Figure S2A–D).

3.6 |. Environmental housing and AMP fail to alter the expression glutamate receptors in the NAc

A factorial ANOVA analysis of normalized GluA1 expression revealed no effects of group (F1,71 = 0.09, p = .77), drug (F1,71 = 0.77, p = .38) or time (F1,71 = 0.86, p = .36). There were no two-way interactions, and there was no evidence of an interaction between group × drug × time (F1,71 = 0.22, p = .64; Figure S3A). Analysis of normalized GluA2 expression determined no effects of group (F1,71 = 0.005, p = .94), drug (F1,71 = 0.008, p = .93) or time (F1,71 = 0.001, p = .97). There were no two-way interactions, and there was no evidence of an interaction between group × drug × time (F1,71 = 0.28, p = .60; Figure S3B). Analysis of normalized mGlur1 expression determined no main effects of group (F1,71 = 0.003, p = .96), drug (F1,71 = 0.88, p = .35) or time (F1,71 = 0.62, p = .44). The analysis determined that there were no two-way interactions and there was no evidence of an interaction between group × drug × time (F1,71 = 0.45, p = .50; Figure S3C). Analysis of normalized mGlur5 expression determined no main effects of group (F1,71 = 1.03, p = .32), drug (F1,71 = 1.10, p = .30) or time (F1,71 = 0.01, p = .91). The analysis determined that there were no two-way interactions and there was no evidence of an interaction between group × drug × time (F1,71 = 0.48, p = .49; Figure S3D).

Cocaine abstinence results in a time-dependent decrease in mGlur1 expression while concurrently, GluA1 expression increases. We hypothesized that the ratio of AMPA subunit to mGlur expression may be altered during this crucial interval and could illuminate our behavioural results. In EC-SAL rats, the expression ratios did not change after 40 days of abstinence (Figure 3), GluA1:mGlur1 (t17 = 1.21, p = .24), GluA2: mGlur1 (t17 = 1.37, p = .19), GluA1:mGlur5 (t17 = 1.45, p = .16) and GluA2:mGlur5 (t17 = 0.95, p = .36); for IC-SAL rats, GluA1:mGlur1 (t16 = 0.45, p = .96), GluA2: mGlur1 (t16 = 0.30, p = .77), GluA1:mGlur5 (t16 = 0.38, p = .71) and GluA2:mGlur5 (t16 = 1.60, p = .13); or EC-AMP rats, GluA1:mGlur1 (t13 = 0.83, p = .42), GluA2: mGlur1 (t13 = 0.45, p = .97), GluA2:mGlur5 (t13 = 1.56, p = .14) and GluA2:mGlur5 (t13 = 0.58, p = .57). However, in IC-AMP rats, 40 days of abstinence decreased GluA1:mGlur5 (t22 = 2.17, p = .04; Figure 3c). The other ratios were not changed following prolonged abstinence, GluA1:mGlur1 (t22 = 0.35, p = .73) or GluA2:mGlur1 (t22 = 0.40, p = .69). The ratio of GluA2:mGlur5 trended toward a decrease (t22 = 1.96, p = .06) (Figure 3d).

FIGURE 3.

FIGURE 3

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The normalized AMPA receptor subunit to mGlur expression ratios within the NAc following 1 and 40 days of AMP or SAL abstinence in EC and IC rats. Neither the (a) GluA1:mGlur1 nor (b) GluA2:mGlur1 expression ratios were altered following 40 days of abstinence from AMP or SAL. (c) The GluA1:mGlur5 expression ratio was significantly decreased following 40 days of abstinence from AMP in IC rats only (^p < .05). (d) The GluA2:mGlur5 trended toward a decrease following 40 days of abstinence from AMP in IC rats only (p = .06). Day 1 sample sizes: EC-AMP: n = 7; EC-SAL: n = 9, IC-AMP: n = 12; IC-SAL: n = 10. Day 40 sample sizes: EC-AMP: n = 7; EC-SAL: n = 10, IC-AMP: n = 12; IC-SAL: n = 10

3.7 |. NAc GluA1:mGlur1 expression ratio is correlated with AMP seeking in IC rats after prolonged abstinence

Finally, we conducted exploratory correlation analyses to determine the relationship between glutamate receptor ratios with total active and inactive lever presses during the seeking test. In EC-AMP rats, there was no correlation between GluA1:mGlur1 after 1 (r7 = −.32, p = .49) and 40 days of abstinence (r8 = −.24, p = .57). Interestingly, in IC-AMP rats, there was no correlation between active lever presses and GluA1:mGlur1 expression after 1 day of abstinence, (r12 = −.25, p = .44); however, after 40 days, the ratio of GluA1:mGlur1 was correlated with active lever presses (r11 = .69, p = .02). Table 1 summarizes the complete list of Pearson correlation coefficients for active lever presses and the calculated glutamate receptor ratios; inactive lever presses are reported in Table 2.

TABLE 1.

Correlation coefficients for relations between groups total (cue off + cue on) previously active lever presses and glutamate receptor expression ratios measured from NAc in male Sprague–Dawley rats

Previous active lever × NAc expression ratio
Group GluA1:mGlur1 GluA2:mGlur1 GluA1:mGlur5 GluA2:mGlur5
EC-AMP D1(7) −.3161 −.3582 .5878 .3216
IC-AMP D1(12) −.2487 −.2618 .1321 .02115
EC-SAL D1(9) .8305** .6839* .06368 −.4055
IC-SAL D1(8) .1315 .198 .0841 .2244
EC-AMP D40(8) −.2354 −.2539 .08795 −.08781
IC-AMP D40(11) .688* .5435 .4834 .2519
EC-SAL D40(10) .4693 .5889 .007667 .1597
IC-SAL D40(10) −.131 −.05624 −.1235 .002801
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Note: Subscript in parentheses denotes the number of rats in each correlation.

*

p < .05.

**

p < .01.

TABLE 2.

Correlation coefficients for relations between groups total (cue off + cue on) inactive lever presses and glutamate receptor expression ratios measured from NAc in male Sprague–Dawley rats

Inactive lever × NAc expression ratio
Group GluA1:mGlur1 GluA2:mGlur1 GluA1:mGlur5 GluA2:mGlur5
EC-AMP D1(7) −.4273 − .4806 .6345 .2927
IC-AMP D1(12) .01886 −.06486 .0947 −.09563
EC-SAL D1(9) .8667** .4313 .4567 −.4194
IC-SAL D1(8) −.1907 −.4164 −.09569 −.8098*
EC-AMP D40(8) .2695 .4034 −.1785 .1359
IC-AMP D40(11) .5863 .4317 .3159 .07186
EC-SAL D40(10) −.1919 −.1339 .02824 .1437
IC-SAL D40(10) .6352* .6493* .05398 .06859
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*

p < .05.

**

p < .01.

4 |. DISCUSSION

The current experiments manipulated the housing condition to determine the extent to which sustained environmental housing manipulations influence AMP seeking after short (1 day) and prolonged (40 days) abstinence. The experiments demonstrate that sustained IC housing engineers a vulnerable phenotype prone to persistent AMP seeking. We discovered that AMP seeking is elevated in IC rats after short and prolonged abstinence when compared with EC rats. The AMP-seeking response is modulated by abstinence interval, availability of the discrete cue complex and housing condition (Figure 2). Within the NAc, differential housing condition, AMP and abstinence interval did not alter the total expression of the AMPA receptor subunits: GluA1 or GluA2 or metabotropic mGlur1 or mGlur5. However, the expression ratio of GluA1:mGlur5 was significantly decreased in IC rats that self-administered AMP after prolonged abstinence. Therefore, our results suggest that early life isolation engineers a persistent AMP-seeking phenotype that is independent of abstinence interval and that prolonged abstinence from AMP alters the proteomic topography in the NAc.

4.1 |. Isolation housing augments AMP seeking

Rats reared in the IC self-administer more cocaine (Howes et al., 2000), amphetamine (Bardo et al., 2001; Green et al., 2002) and opiates (Hofford et al., 2017) when compared with EC rats. In the current experiments, IC rats earned more SAL infusions suggesting that alternative mechanisms may drive reinforcement learning in this phenotype and further experiments are required to fully determine the unique contributions of behavioural arousal and persistence in nondrug and/or sensory reinforcement (Cain et al., 2006; Wang et al., 2018). However, we hypothesize that IC rearing creates a vulnerable phenotype and successfully models distinct aspects of human SUDs. For example, IC rats escalate to higher intake (Gipson et al., 2011) and demonstrate less demand elasticity (more drug taking at lower doses/higher prices) compared with EC rats, suggesting that drug reinforcers have more value in IC rats (Yates et al., 2017). An augmented drug value would enhance the associative relationship between the drug and the discrete conditioned cue and facilitate a strong drug-seeking response and delay extinction (Chauvet et al., 2009; Stairs et al., 2006; Thiel et al., 2009; Thiel et al., 2012). In the current experiments, IC rats had augmented AMP seeking during the brief contextual cue-seeking interval and demonstrated an elevated cue-induced relapse response after a single day of abstinence. Interestingly, the dynamics of the AMP-seeking response were altered after prolonged abstinence in EC and IC rats.

After prolonged abstinence, EC-SAL and EC-AMP rats increased the number of active lever presses during the context cue test. The general increase in both SAL and AMP-trained rats suggests a general arousal or spontaneous recovery of behaviour and one not necessarily specific to AMP seeking. IC-SAL rats also increased active levers during the brief extinction interval, but this was not significant. To our surprise, the IC-AMP rats decreased active lever presses during the brief context cue interval, suggesting that the AMP-seeking response driven by the context may be weakened indicating an intact extinction memory. Nonetheless, regardless of housing group, context-induced drug seeking after prolonged abstinence does not exceed the behavioural arousal mechanisms driven by spontaneous recovery. Therefore, prolonged abstinence from AMP does not significantly elevate relapse-like behaviour when the conditioned cue is not presented.

Once the discrete cue complex was presented, IC-AMP rats had a robust cue-evoked AMP-seeking response that exceeded the response from the EC-AMP rats. The augmented cue-induced AMP-seeking response observed in IC rats was observed after brief and prolonged abstinence and indicates that IC rats are highly susceptible to the motivational components of drug-associated cues. In accordance with other literature, environmental enrichment suppressed cue-induced relapse-like behaviour (Garcia et al., 2019) and protected against any increase in drug seeking after prolonged abstinence (Chauvet et al., 2009; Chauvet et al., 2012; Solinas et al., 2008; Thiel et al., 2009). Generally, the most consistent effects of environmental enrichment on mitigating relapse and SUD-like behaviours are observed when the enriched environment is maintained (Chauvet et al., 2009; Galaj et al., 2020; Garcia et al., 2017).

Our current results suggest that prolonged abstinence from AMP does not result in a time-dependent increase in AMP seeking. This result was surprising and reveals several critical considerations that may define augmented AMP seeking after prolonged abstinence. The time-dependent increase in drug seeking has been observed with cocaine, methamphetamine and other drug reinforcers (Pickens et al., 2011), suggesting that neuroadaptations mediating the amplified drug seeking response are independent of specific drug kinetics or receptor/transporter dynamics. Furthermore, the development of maximal drug-seeking responses varies considerably across drugs and nondrug reinforcers (Abdolahi et al., 2010; Grimm et al., 2001; Murray et al., 2019; Scheyer et al., 2016; Shalev et al., 2001). Although many prolonged abstinence/incubation of craving models employ daily long access (6 h) self-administration sessions because long access self-administration consumption is associated with long-lasting neuroadaptations in critical nodes that modulate reward and motivated behaviour (Ahmed, 2011; Koob et al., 2004; Roth & Carroll, 2004; Vanderschuren & Everitt, 2004), long access self-administration is not required. Short access (1–3 h) (Li & Frantz, 2009; Swinford-Jackson et al., 2016) or even a single long access cocaine self-administration (Halbout et al., 2014) sufficiently produced an incubated cocaine-seeking response. Critically, it is still not clear if long access self-administration procedures increase responding for cues in late withdrawal or if it suppresses responding for cues during early withdrawal. In this latter instance, ‘incubation’ would occur once the inhibitory mechanisms dissipated across time. Indeed, after 1 day of abstinence, rats trained in a short access self-administration model had more cocaine-seeking lever presses when compared with rats trained in a long access model (Pacchioni et al., 2011). Comparatively, the current AMP-seeking response after 1 day of abstinence exceeds the ‘incubated’ methamphetamine-seeking response in animals trained in long access model (Murray et al., 2019; Scheyer et al., 2016). To fully characterize the extent to which AMP incubates and elucidate how short versus long access moderates the development of AMP seeking, future experiments are required, especially when the question remains about whether the incubation of drug craving is driven by a suppression of Day 1 drug seeking or a true time-dependent increase in cue-induced drug-seeking behaviour.

4.2 |. Amplified AMP seeking without NAc alterations

Under drug naïve conditions, the NAc primarily expresses CI-AMPA on medium spiny neurons (Conrad et al., 2008). However, after long access cocaine self-administration and 40 days of abstinence, homomeric GluA1 CP-AMPA is expressed more robustly throughout the whole NAc. Additionally, mGlur1 expression levels are inversely related to GluA1 CP-AMPA receptor expression, and loss of mGlur1-dependent LTD enables the rampant increase of CP-AMPA in the NAc core (Loweth et al., 2019; Loweth, Scheyer, et al., 2014). Together, the dynamic interaction between AMPA subunits and mGlur1 receptors are the putative mechanism that drives the time-dependent increase in cocaine seeking. Here, we used a similar number of self-administration sessions and incorporated short and prolonged abstinence periods to characterize the expression GluA1 and GluA2 AMPA receptor subunits and mGlur1 and mGlur5 following AMP or SAL self-administration. Our results indicate that neither AMP nor differential housing changed the absolute expression of GluA1, GluA2, mGlur1 or mGlur5 within the membrane fraction of the NAc after short or prolonged abstinence.

Given the inverse relationships that mGlur1 and mGlur5 have on synaptic plasticity and protein translation (Stefanik et al., 2018), we hypothesized that subtle changes could be revealed if the subunit/receptors were analysed as a ratio. When analysed as a ratio, IC-AMP rats had a significant decrease in the expression of GluA1:mGlur5 and a trend toward a decrease in the GluA2:mGlur5 in the NAc when compared with IC-AMP rats after 1 day of abstinence. These results indicate that the relative expression of mGlur5 to CP-AMPA and CI-AMPA receptors within the NAc increases after prolonged abstinence from AMP self-administration specifically in IC rats. This provides insight into possible LTD mechanisms that underlie AMP seeking after prolonged abstinence. In drug naïve conditions (SAL self-administration), mGlur5 negatively regulates protein translation in the NAc, such that reducing mGlur5 transmission with a negative allosteric modulator increases protein translation whereas reducing mGlur1 signalling is without effect. However, after prolonged abstinence from cocaine, the synaptic depression mechanism switches to a mGlur1-dependent mechanism (McCutcheon et al., 2011). This provides the hypothesis that prolonged abstinence from AMP does not robustly downregulate mGlur5 expression. Instead, our results reveal a dynamic interaction between environmental housing condition, AMP and NAc protein expression. It remains to be determined if the functional status of either the mGlur1 or mGlur5 in the NAc following IC housing is left intact, but our other results determined that IC rats require higher doses of a mGlur5 negative allosteric modulator (MTEP) to suppress AMP intake on FR and PR schedules of reinforcement when compared with EC rats (Arndt, Johns, et al., 2015; Gill et al., 2012). Therefore, despite minimal differences in the expression profiles across groups, the functional status of these receptors requires further investigation to understand how they relate to persistent drug seeking in early and late abstinence.

Recent evidence indicates that prolonged abstinence from methamphetamine does not alter CP-AMPA or mGlur1 membrane expression in the NAc core (Murray et al., 2019). Interestingly, prolonged abstinence from methamphetamine and cocaine yield comparable magnitudes of incubated cue-induced drug-seeking responses, an effect suppressed by blocking NAc core CP-AMPA receptors (Conrad et al., 2008; Scheyer et al., 2016). Importantly, repeated administration of a PAM mGlur1 ligand (SYN119) during early abstinence mitigates incubated cocaine (Loweth, Scheyer, et al., 2014) but not methamphetamine seeking (Murray et al., 2019), suggesting independent mechanisms that govern LTD. Our results are in accordance with previous literature (Murray et al., 2019) and indicate that mGlur1 has a limited role in the development or expression of an incubated AMP-seeking response. Similarly, AMP may not directly alter GluA1 membrane expression in the NAc. GluA1 is not upregulated following repeated AMP injections but is following cocaine injections when measured 2–3 weeks after exposure. Instead, AMP increases GluA1 phosphorylation in the postsynaptic density fraction (Wang et al., 2017). When vulnerable individuals are exposed to AMP the GluA1:mGlur1 expression ratio may predict intensified AMP seeking, because in IC-AMP rats, the GluA1:mGlur1 expression ratio was positively correlated with AMP seeking following prolonged abstinence. Pharmacological and biochemical experiments aimed at fully characterizing these receptors’ environmental interactions are still needed but suggest that the NAc is a critical brain region affected by early life environmental manipulations (Kelly & Hannan, 2019).

These experiments clearly demonstrated that early life development and social isolation augment cue-induced AMP seeking when compared with rats reared and housed in the EC. Interestingly, females exhibit high cue-induced relapse behaviour and self-administer more psychomotor stimulants when compared with males (Becker & Koob, 2016; Lynch, 2018). These outcomes provide another avenue of research and opportunity to reveal the neurobiological substrates that govern relapse behaviour. While we did not test the effects of differential housing or incubation duration in female rats, others have begun to understand the role of sex in SUDs and preclinical models of relapse. Interestingly, there seems to be a stimulant drug by sex interaction because cue-induced drug seeking is different for cocaine and methamphetamine (Fuchs et al., 2005; Kippin et al., 2005; Venniro et al., 2017) in females.

5 |. CONCLUSION

To conclude, EC and IC rats equally self-administered a high dose of AMP. EC and IC rats did not show the typical time-dependent increase in cue-induced AMP seeking. IC-AMP rats had greater AMP seeking after brief and prolonged abstinence when compared with EC-AMP rats. This difference between IC and EC rats was greatest when the discrete cue complex associated with AMP reinforcement was presented. Interestingly, the differences between IC and EC or SAL and AMP are not associated with the AMPA subunits GluA1 or GluA2 or metabotropic mGlur1 or mGlur5 measured after brief or prolonged abstinence. However, after prolonged abstinence, IC-AMP had a significant decrease in the ratio between GluA1:mGlur5. Differential rearing and housing is a robust environmental manipulation that modifies centrally mediated behaviours and can be used to reveal the mechanisms responsible for compulsive drug seeking during early and prolonged abstinence.

Supplementary Material

Supplemental figures

ACKNOWLEDGEMENTS

The authors would like to thank Allison Beesley, Bree Humburg and Thomas Wukitsch, for their contributions in collecting the behavioural data. This work was supported by National Institute on Drug Abuse (DA035435). The western blot and other protein analyses were supported by the Molecular Biology Core, Kansas State University, College of Veterinary Medicine. EJG was supported by Promoting Health Diversity Supplement (DA035435-01S1). EJG was supported by the American Psychological Association Dissertation Research Award 2016.

Abbreviations:

AMP

amphetamine

AMPA

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

EC

enrichment condition

GluA1

AMPA subunit 1

GluA2

AMPA subunit 2

IC

isolated condition

mGlur1

metabotropic glutamate receptor 2

mGlur5

metabotropic glutamate receptor 5

NAc

nucleus accumbens

SUDs

substance use disorders

Footnotes

CONFLICT OF INTEREST

The authors have no conflicts of interest that would alter the interpretation of the data presented in the manuscript.

PEER REVIEW

The peer review history for this article is available at https://publons.com/publon/10.1111/ejn.15441.

SUPPORTING INFORMATION

Additional supporting information may be found in the online version of the article at the publisher’s website.

DATA AVAILABILITY STATEMENT

Data for this manuscript can be found in .csv file format in the public Open Science Framework repository. It can be found using this URL: https://osf.io/r4bzp/(Garcia, 2021). Additionally, the .csv data files are located in the GitHub repository, ‘erikgarcia22/EC_IC_AMP_incubation’. All other associated files including the data, figures and analysis are attainable by contacting Erik J. Garcia at ejgarcia@unomaha.edu.

REFERENCES

  1. Abdolahi A, Acosta G, Breslin FJ, Hemby SE, & Lynch WJ (2010). Incubation of nicotine seeking is associated with enhanced protein kinase A-regulated signaling of dopamine-and cAMP-regulated phosphoprotein of 32 kDa in the insular cortex. The European Journal of Neuroscience, 31, 733–741. 10.1111/j.1460-9568.2010.07114.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adhikary S, Caprioli D, Venniro M, Kallenberger P, Shaham Y, & Bossert JM (2017). Incubation of extinction responding and cue-induced reinstatement, but not context- or drug priming-induced reinstatement, after withdrawal from methamphetamine. Addiction Biology, 22, 977–990. 10.1111/adb.12386 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ahmed SH (2011). Escalation of drug use. In Olmstead MC (Ed.), Animal models of drug addiction (pp. 267–292). Springer. 10.1007/978-1-60761-934-5_10 [DOI] [Google Scholar]
  4. Anastasio NC, Gilbertson SR, Bubar MJ, Agarkov A, Stutz SJ, Jeng Y, Bremer NM, Smith TD, Fox RG, Swinford SE, Seitz PK, Charendoff MN, Craft JW Jr., Laezza FM, Watson CS, Briggs JM, & Cunningham KA (2013). Peptide inhibitors disrupt the serotonin 5-HT2C receptor interaction with phosphatase and tensin homolog to allosterically modulate cellular signaling and behavior. The Journal of Neuroscience, 33, 1615–1630. 10.1523/JNEUROSCI.2656-12.2013 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Anastasio NC, Liu S, Maili L, Swinford SE, Lane SD, Fox RG, Hamon SC, Nielsen DA, Cunningham KA, & Moeller FG (2014). Variation within the serotonin (5-HT) 5-HT2C receptor system aligns with vulnerability to cocaine cue reactivity. Translational Psychiatry, 4, e369. 10.1038/tp.2013.131 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Arndt DL, Garcia EJ, Erickson GR, Ulmer M, & Cain ME (2015). Differential rearing alters amphetamine self-administration: Role of mGluR2/3 activation. Chicago: Society for Neuroscience. [Google Scholar]
  7. Arndt DL, Johns KC, Dietz ZK, & Cain ME (2015). Environmental condition alters amphetamine self-administration: Role of the MGluR5 receptor and schedule of reinforcement. Psychopharmacology, 232, 3741–3752. 10.1007/s00213-015-4031-x [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Arndt DL, Wukitsch TJ, Garcia EJ, & Cain M (2019). Histone deacetylase inhibition differentially attenuates cue-induced reinstatement: An interaction of environment and acH3K9 expression in the dorsal striatum. Behavioral Neuroscience, 133, 478–488. 10.1037/bne0000333 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bardo MT, Klebaur JE, Valone JM, & Deaton C (2001). Environmental enrichment decreases intravenous self-administration of amphetamine in female and male rats. Psychopharmacology, 155, 278–284. 10.1007/s002130100720 [DOI] [PubMed] [Google Scholar]
  10. Bardo MT, Neisewander JL, & Kelly TH (2013). Individual differences and social influences on the neurobehavioral pharmacology of abused drugs. Pharmacological Reviews, 65, 255–290. 10.1124/pr.111.005124 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Becker JB, & Koob GF (2016). Sex differences in animal models: Focus on addiction. Pharmacological Reviews, 68, 242–263. 10.1124/pr.115.011163 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Bossert JM, Ghitza UE, Lu L, Epstein DH, & Shaham Y (2005). Neurobiology of relapse to heroin and cocaine seeking: An update and clinical implications. European Journal of Pharmacology, 526, 36–50. 10.1016/j.ejphar.2005.09.030 [DOI] [PubMed] [Google Scholar]
  13. Cain ME, Green TA, & Bardo MT (2006). Environmental enrichment decreases responding for visual novelty. Behavioural Processes, 73, 360–366. 10.1016/j.beproc.2006.08.007 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Chauvet C, Goldberg SR, Jaber M, & Solinas M (2012). Effects of environmental enrichment on the incubation of cocaine craving. Neuropharmacology, 63, 635–641. 10.1016/j.neuropharm.2012.05.014 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Chauvet C, Lardeux V, Goldberg SR, Jaber M, & Solinas M (2009). Environmental enrichment reduces cocaine seeking and reinstatement induced by cues and stress but not by cocaine. Neuropsychopharmacology, 34, 2767–2778. 10.1038/npp.2009.127 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Conrad KL, Tseng KY, Uejima JL, Reimers JM, Heng LJ, Shaham Y, Marinelli M, & Wolf ME (2008). Formation of accumbens GluR2-lacking AMPA receptors mediates incubation of cocaine craving. Nature, 454, 118–121. 10.1038/nature06995 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fuchs RA, Evans KA, Mehta RH, Case JM, & See RE (2005). Influence of sex and estrous cyclicity on conditioned cue-induced reinstatement of cocaine-seeking behavior in rats. Psychopharmacology, 179, 662–672. 10.1007/s00213-004-2080-7 [DOI] [PubMed] [Google Scholar]
  18. Galaj E, Barrera ED, & Ranaldi R (2020). Therapeutic efficacy of environmental enrichment for substance use disorders. Pharmacology, Biochemistry, and Behavior, 188, 172829. 10.1016/j.pbb.2019.172829 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Garcia EJ (2021). EC_IC_AMP_incubation. Retrieved from https://osf.io/r4bzp/. 10.17605/OSF.IO/R4BZP [DOI]
  20. Garcia EJ, Arndt DL, & Cain ME (2019). Dynamic interactions of ceftriaxone and environmental variables suppress amphetamine seeking. Brain Research, 1712, 63–72. 10.1016/j.brainres.2019.01.044 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Garcia EJ, & Cain ME (2020). Environmental enrichment and a selective metabotropic glutamate receptor2/3 (mGluR2/3) agonist suppress amphetamine self-administration: Characterizing baseline differences. Pharmacology, Biochemistry, and Behavior, 192, 172907. 10.1016/j.pbb.2020.172907 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Garcia EJ, Haddon TN, Saucier DA, & Cain ME (2017). Differential housing and novelty response: Protection and risk from locomotor sensitization. Pharmacology, Biochemistry, and Behavior, 154, 20–30. 10.1016/j.pbb.2017.01.004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Gill MJ, Arnold JC, & Cain ME (2012). Impact of mGluR5 during amphetamine-induced hyperactivity and conditioned hyperactivity in differentially reared rats. Psychopharmacology, 221, 227–237. 10.1007/s00213-011-2565-0 [DOI] [PubMed] [Google Scholar]
  24. Gipson CD, Beckmann JS, El-Maraghi S, Marusich JA, & Bardo MT (2011). Effect of environmental enrichment on escalation of cocaine self-administration in rats. Psychopharmacology, 214, 557–566. 10.1007/s00213-010-2060-z [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Green TA, Gehrke BJ, & Bardo MT (2002). Environmental enrichment decreases intravenous amphetamine self- administration in rats: Dose-response functions for fixed- and progressive-ratio schedules. Psychopharmacology, 162, 373–378. 10.1007/s00213-002-1134-y [DOI] [PubMed] [Google Scholar]
  26. Grimm JW, Hope BT, Wise RA, & Shaham Y (2001). Neuroadaptation. Incubation of cocaine craving after withdrawal. Nature, 412, 141–142. 10.1038/35084134 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Grimm JW, Osincup D, Wells B, Manaois M, Fyall A, Buse C, & Harkness JH (2008). Environmental enrichment attenuates cue-induced reinstatement of sucrose seeking in rats. Behavioural Pharmacology, 19, 777–785. 10.1097/FBP.0b013e32831c3b18 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Halbout B, Bernardi RE, Hansson AC, & Spanagel R (2014). Incubation of cocaine seeking following brief cocaine experience in mice is enhanced by mGluR1 blockade. The Journal of Neuroscience, 34, 1781–1790. 10.1523/JNEUROSCI.1076-13.2014 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hofford RS, Chow JJ, Beckmann JS, & Bardo MT (2017). Effects of environmental enrichment on self-administration of the short-acting opioid remifentanil in male rats. Psychopharmacology, 234, 3499–3506. 10.1007/s00213-017-4734-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Howes SR, Dalley JW, Morrison CH, Robbins TW, & Everitt BJ (2000). Leftward shift in the acquisition of cocaine self-administration in isolation-reared rats: Relationship to extracellular levels of dopamine, serotonin, and glutamate in the nucleus accumbens and amygdala-striatal FOS expression. Psychpharmacology, 151, 55–63. 10.1007/s002130000451 [DOI] [PubMed] [Google Scholar]
  31. Kariisa M, Scholl L, Wilson N, Seth P, & Hoots B (2019). Drug overdose deaths involving cocaine and psychostimulants with abuse potential—United States, 2003–2017. Morbidity and Mortality Weekly Report, 68, 388–395. 10.15585/mmwr.mm6817a3 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kelly A, & Hannan AJ (2019). Therapeutic impacts of environmental enrichment: Neurobiological mechanisms informing molecular targets for enviromimetics. Neuropharmacology, 145, 1–2. 10.1016/j.neuropharm.2018.11.005 [DOI] [PubMed] [Google Scholar]
  33. Kippin TE, Fuchs RA, Mehta RH, Case JM, Parker MP, Bimonte-Nelson HA, & See RE (2005). Potentiation of cocaine-primed reinstatement of drug seeking in female rats during estrus. Psychopharmacology, 182, 245–252. 10.1007/s00213-005-0071-y [DOI] [PubMed] [Google Scholar]
  34. Koob GF, Ahmed SH, Boutrel B, Chen SA, Kenny PJ, Markou A, O’Dell LE, Parsons LH, & Sanna PP (2004). Neurobiological mechanisms in the transition from drug use to drug dependence. Neuroscience & Biobehavioral Reviews, 27, 739–749. 10.1016/j.neubiorev.2003.11.007 [DOI] [PubMed] [Google Scholar]
  35. LaCrosse AL, Hill K, & Knackstedt LA (2016). Ceftriaxone attenuates cocaine relapse after abstinence through modulation of nucleus accumbens AMPA subunit expression. European Neuropsychopharmacology: The Journal of the European College of Neuropsychopharmacology, 26, 186–194. 10.1016/j.euroneuro.2015.12.022 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Li C, & Frantz KJ (2009). Attenuated incubation of cocaine seeking in male rats trained to self-administer cocaine during periadolescence. Psychopharmacology, 204, 725–733. 10.1007/s00213-009-1502-y [DOI] [PubMed] [Google Scholar]
  37. Loweth JA, Reimers JM, Caccamise A, Stefanik MT, Woo KKY, Chauhan NM, Werner CT, & Wolf ME (2019). mGlu1 tonically regulates levels of calcium-permeable AMPA receptors in cultured nucleus accumbens neurons through retinoic acid signaling and protein translation. The European Journal of Neuroscience, 50, 2590–2601. 10.1111/ejn.14151 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Loweth JA, Scheyer AF, Milovanovic M, LaCrosse AL, Flores-Barrera E, Werner CT, Li X, Ford KA, Le T, Olive MF, Szumlinski KK, Tseng KY, & Wolf ME (2014). Synaptic depression via mGluR1 positive allosteric modulation suppresses cue-induced cocaine craving. Nature Neuroscience, 17, 73–80. 10.1038/nn.3590 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Loweth JA, Tseng KY, & Wolf ME (2014). Adaptations in AMPA receptor transmission in the nucleus accumbens contributing to incubation of cocaine craving. Neuropharmacology, 76(Pt B), 287–300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Lynch WJ (2018). Modeling the development of drug addiction in male and female animals. Pharmacology, Biochemistry, and Behavior, 164, 50–61. 10.1016/j.pbb.2017.06.006 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ma YY, Lee BR, Wang X, Guo C, Liu L, Cui R, Lan Y, Balcita-Pedicino JJ, Wolf ME, Sesack SR, Shaham Y, Schluter OM, Huang YH, & Dong Y (2014). Bidirectional modulation of incubation of cocaine craving by silent synapse-based remodeling of prefrontal cortex to accumbens projections. Neuron, 83, 1453–1467. 10.1016/j.neuron.2014.08.023 [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Ma YY, Wang X, Huang Y, Marie H, Nestler EJ, Schluter OM, & Dong Y (2016). Re-silencing of silent synapses unmasks anti-relapse effects of environmental enrichment. Proceedings of the National Academy of Sciences of the United States of America, 113, 5089–5094. 10.1073/pnas.1524739113 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. McCutcheon JE, Wang X, Tseng KY, Wolf ME, & Marinelli M (2011). Calcium-permeable AMPA receptors are present in nucleus accumbens synapses after prolonged withdrawal from cocaine self-administration but not experimenter-administered cocaine. The Journal of Neuroscience, 31, 5737–5743. 10.1523/JNEUROSCI.0350-11.2011 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Murray CH, Loweth JA, Milovanovic M, Stefanik MT, Caccamise AJ, Dolubizno H, Funke JR, Foster Olive M, & Wolf ME (2019). AMPA receptor and metabotropic glutamate receptor 1 adaptations in the nucleus accumbens core during incubation of methamphetamine craving. Neuropsychopharmacology, 44, 1534–1541. 10.1038/s41386-019-0425-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. National Research Council. (1996). Guide for the care and use of laboratory animals. National Academy Press. [Google Scholar]
  46. Pacchioni AM, Gabriele A, & See RE (2011). Dorsal striatum mediation of cocaine-seeking after withdrawal from short or long daily access cocaine self-administration in rats. Behavioural Brain Research, 218, 296–300. 10.1016/j.bbr.2010.12.014 [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Parvaz MA, Moeller SJ, Malaker P, Sinha R, Alia-Klein N, & Goldstein RZ (2017). Abstinence reverses EEG-indexed attention bias between drug-related and pleasant stimuli in cocaine-addicted individuals. Journal of Psychiatry & Neuroscience, 42, 78–86. 10.1503/jpn.150358 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Pickens CL, Airavaara M, Theberge F, Fanous S, Hope BT, & Shaham Y (2011). Neurobiology of the incubation of drug craving. Trends in Neurosciences, 34, 411–420. 10.1016/j.tins.2011.06.001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Roth ME, & Carroll ME (2004). Sex differences in the escalation of intravenous cocaine intake following long- or short-access to cocaine self-administration. Pharmacology, Biochemistry, and Behavior, 78, 199–207. 10.1016/j.pbb.2004.03.018 [DOI] [PubMed] [Google Scholar]
  50. Scheyer AF, Loweth JA, Christian DT, Uejima J, Rabei R, Le T, Dolubizno H, Stefanik MT, Murray CH, Sakas C, & Wolf ME (2016). AMPA receptor plasticity in accumbens core contributes to incubation of methamphetamine craving. Biological Psychiatry, 80, 661–670. 10.1016/j.biopsych.2016.04.003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Shalev U, Morales M, Hope B, Yap J, & Shaham Y (2001). Time-dependent changes in extinction behavior and stress-induced reinstatement of drug seeking following withdrawal from heroin in rats. Psychopharmacology, 156, 98–107. 10.1007/s002130100748 [DOI] [PubMed] [Google Scholar]
  52. Solinas M, Chauvet C, Thiriet N, El Rawas R, & Jaber M (2008). Reversal of cocaine addiction by environmental enrichment. Proceedings of the National Academy of Sciences of the United States of America, 105, 17145–17150. 10.1073/pnas.0806889105 [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Solinas M, Thiriet N, Chauvet C, & Jaber M (2010). Prevention and treatment of drug addiction by environmental enrichment. Progress in Neurobiology, 92, 572–592. 10.1016/j.pneurobio.2010.08.002 [DOI] [PubMed] [Google Scholar]
  54. Stairs DJ, Klein ED, & Bardo MT (2006). Effects of environmental enrichment on extinction and reinstatement of amphetamine self-administration and sucrose-maintained responding. Behavioural Pharmacology, 17, 597–604. 10.1097/01.fbp.0000236271.72300.0e [DOI] [PubMed] [Google Scholar]
  55. Stefanik MT, Milovanovic M, Werner CT, Spainhour JCG, & Wolf ME (2018). Withdrawal from cocaine self-administration alters the regulation of protein translation in the nucleus accumbens. Biological Psychiatry, 84, 223–232. 10.1016/j.biopsych.2018.02.012 [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Swinford-Jackson SE, Anastasio NC, Fox RG, Stutz SJ, & Cunningham KA (2016). Incubation of cocaine cue reactivity associates with neuroadaptations in the cortical serotonin (5-HT) 5-HT2C receptor (5-HT2CR) system. Neuroscience, 324, 50–61. 10.1016/j.neuroscience.2016.02.052 [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Thiel KJ, Painter MR, Pentkowski NS, Mitroi D, Crawford CA, & Neisewander JL (2012). Environmental enrichment counters cocaine abstinence-induced stress and brain reactivity to cocaine cues but fails to prevent the incubation effect. Addiction Biology, 17, 365–377. 10.1111/j.1369-1600.2011.00358.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Thiel KJ, Sanabria F, Pentkowski NS, & Neisewander JL (2009). Anti-craving effects of environmental enrichment. The International Journal of Neuropsychopharmacology, 12, 1151–1156. 10.1017/S1461145709990472 [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Vanderschuren LJ, & Everitt BJ (2004). Drug seeking becomes compulsive after prolonged cocaine self-administration. Science, 305, 1017–1019. 10.1126/science.1098975 [DOI] [PubMed] [Google Scholar]
  60. Venniro M, Zhang M, Shaham Y, & Caprioli D (2017). Incubation of methamphetamine but not heroin craving after voluntary abstinence in male and female rats. Neuropsychopharmacology, 42, 1126–1135. 10.1038/npp.2016.287 [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wang Q, Li D, Bubula N, Campioni MR, McGehee DS, & Vezina P (2017). Sensitizing exposure to amphetamine increases AMPA receptor phosphorylation without increasing cell surface expression in the rat nucleus accumbens. Neuropharmacology, 117, 328–337. 10.1016/j.neuropharm.2017.02.018 [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Wang R, Hausknecht KA, Haj-Dahmane S, Shen R-Y, & Richards JB (2018). Decreased environmental complexity during development impairs habituation of reinforcer effectiveness of sensory stimuli. Behavioural Brain Research, 337, 53–60. 10.1016/j.bbr.2017.09.032 [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Wolf ME (2010a). The Bermuda Triangle of cocaine-induced neuroadaptations. Trends in Neurosciences, 33, 391–398. 10.1016/j.tins.2010.06.003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Wolf ME (2010b). Regulation of AMPA receptor trafficking in the nucleus accumbens by dopamine and cocaine. Neurotoxicity Research, 18, 393–409. 10.1007/s12640-010-9176-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Wolf ME (2016). Synaptic mechanisms underlying persistent cocaine craving. Nature Reviews, 17, 351–365. 10.1038/nrn.2016.39 [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Wolf ME, & Ferrario CR (2010). AMPA receptor plasticity in the nucleus accumbens after repeated exposure to cocaine. Neuroscience & Biobehavioral Reviews, 35, 185–211. 10.1016/j.neubiorev.2010.01.013 [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Yates JR, Bardo MT, & Beckmann JS (2017). Environmental enrichment and drug value: A behavioral economic analysis in male rats. Addiction Biology, 24(1), 65–75. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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

Supplementary Materials

Supplemental figures
NIHMS1863408-supplement-Supplemental_figures.pdf (589.1KB, pdf)

Data Availability Statement

Data for this manuscript can be found in .csv file format in the public Open Science Framework repository. It can be found using this URL: https://osf.io/r4bzp/(Garcia, 2021). Additionally, the .csv data files are located in the GitHub repository, ‘erikgarcia22/EC_IC_AMP_incubation’. All other associated files including the data, figures and analysis are attainable by contacting Erik J. Garcia at ejgarcia@unomaha.edu.

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