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. Author manuscript; available in PMC: 2022 Sep 1.
Published in final edited form as: Drug Alcohol Depend. 2021 Jun 24;226:108852. doi: 10.1016/j.drugalcdep.2021.108852

Environmental enrichment reduces heroin seeking following incubation of craving in both male and female rats

Eddy D Barrera 1, Lacey Loughlin 1, Stephen Greenberger 1, Scott Ewing 1, Robert Ranaldi 1
PMCID: PMC8355213  NIHMSID: NIHMS1718148  PMID: 34225225

Abstract

Background.

Contemporary treatments for heroin use disorder demonstrate only limited efficacy when the goals are long term abstinence and prevention of relapse. We have demonstrated that environmental enrichment (EE) reduces cue-induced heroin reinstatement in male rats. The present study is an attempt to extend the “anti-relapse” effects of EE to female rats and to periods where incubation of craving is hypothesized to occur.

Methods.

This experiment implemented a 3-phase procedure. In Phase 1, male and female rats were trained to self-administer heroin for 15 days. Phase 2 consisted of a 3- or 15-day forced abstinence (FA) period. In Phase 3 half of the rats were placed into EE and the other half in non-EE housing and subsequently tested for responding in extinction (no heroin or cues) for 15 days followed by a cue-induced reinstatement test.

Results.

We found that rats in the 15 days FA condition showed significantly enhanced drug seeking during extinction, irrespective of sex. We also found that EE significantly reduced this effect. During reinstatement, EE significantly reduced drug seeking in male and female rats and in both 3- and 15-day FA groups.

Conclusions.

EE, with or without prolonged FA, effectively reduced heroin seeking in male and female rats. These findings indicate that EE can reduce drug-seeking in males and females and when putative incubation of craving (i.e., prolonged abstinence period) has occurred and suggest that it may aid in the development of future long-term behavioral treatments for individuals at risk for heroin relapse.

Keywords: environmental enrichment, intravenous self-administration, craving incubation, heroin, reinstatement

Introduction

Heroin is a highly addictive opioid whose long-term use can lead to severe negative financial, medical, social and legal consequences (Kronenburg et al., 2014; Jiang et al., 2017; Moses et al., 2018,). Nevertheless, in 2018 an estimated 10.3 million people aged 12 or older abused opioids and close to 526,000 of these individuals met DSM IV criteria for Heroin use disorder (HUD) (The National Survey on Drug Use and Health [NSDUH], 2018). A major hurdle for individuals with HUD attempting to remain abstinent is relapse (Rajita Sinha, 2011), often caused by exposure to drug-associated cues (O’Brien et al., 1984), such as drug paraphernalia, environments and people. Therefore, a major focus of both human and animal research aimed at developing treatment strategies emphasizes how to diminish cue-induced relapse.

Most treatment strategies consist of FDA approved replacement therapies (e.g., methadone, buprenorphine) or a combination of pharmacotherapy and behavior modification (Sofuoglu et al., 2018) such as contingency management therapy (Preston et al., 2000; Kennedy et al., 2013). In such behavior modification therapy desirable behaviors (i.e., abstinence) are reinforced with meaningful rewards (e.g., vouchers, money) (Prendergast et al., 2006). Although somewhat successful, these strategies continue to result in high relapse rates (Joe et al., 1994; Termorshuizen et al., 2005a; Termorshuizen et al., 2005b; Tang et al., 2006; Mannaioni et al., 2018). The inability to maintain long-term abstinence with contingency strategies may be due to the eventual disruption of the reinforcement process when treatment is stopped.

Strategies involving reward/reinforcement that are not contingent on abstinence or drug-taking behaviors may be useful in these situations. For instance, the use of camping retreats and exercise-based video game playing (Bennett et al., 1998; Cutter et al., 2014) has been shown to reduce alcohol relapse and substance use. Other non-contingent treatment strategies such as adjunctive exercise, have been shown to reduce alcohol consumption, enhance mood and reduce stress (Brown et al., 2009; Hallgren et al., 2014) and in substance use disorder patients, improve quality of life (Muller & Clausen, 2015). These findings suggest that strategies involving sensory stimulation, and or physical activity, administered in manners that are non-concurrent with drug reward and non-contingent with drug-taking or abstinence, could be effective long-term treatments for substance use disorder. In animal research, the effects of exposure to non-concurrent and non-contingent sensory, physical and social stimulation, referred to as environmental enrichment (EE), on brain and behavior has long been studied. The findings indicate powerful effects in modifying behavior and in shaping brain circuits, including those implicated in reinforcement and drug addiction (for review see Galaj et al., 2020).

We, and others, have started to investigate EE as a potential treatment strategy in various animal models of addiction. Studies using conditioned place preference (CPP) show the capacity of EE, when administered after drug conditioning, to reduce expression and reinstatement of cocaine (Solinas et al., 2008; Chauvet et al., 2011; Zernig and Pinheiro, 2015; Mustroph et al., 2016), heroin (Galaj et al., 2016), and ethanol (Li et al., 2015) CPP. Similarly, EE administered after self-administration training can effectively facilitate abstinence to cocaine- and heroin-seeking in rats (Peck et al., 2015; Ewing & Ranaldi, 2018), reduce cocaine-seeking after context renewal (Ranaldi et al., 2011) and during cue-induced, but not drug-induced (Chauvet et al., 2009), heroin (Thiel et al., 2010; Galaj et al., 2016; Sikora et al., 2018) and cocaine-seeking (Thiel et al., 2009; Chauvet et al., 2009; Li and Frantz, 2017). However, some major gaps in knowledge exist; we do not know (1) if and how EE is effective in females and (2) if and to what degree EE is effective after prolonged periods of time between drug-taking cessation and beginning of EE treatment—prolonged periods that have been associated with enhanced drug-seeking and have been referred to as periods where “incubation of craving” may occur. Thus, in the present study, we investigated the effects of EE, implemented after heroin self-administration and 3 or 15 days of forced abstinence (FA) on extinction and cue-induced reinstatement of heroin-seeking in male and female rats.

1. Methods and Materials

2.1. Subjects

Subjects were male and female (Ns = 36) Long Evans rats bred within our animal facility, from males and females purchased from Charles River Laboratories (Wilmington, MA). All rats were aged between PND 65–75 at the time of surgery. All rats were individually housed at all phases of the experiment, in a temperature (70° F) -controlled animal facility where they had access to food (LabDiet chow) and water ad libitum. Each rat was housed in a standard laboratory cage on a reverse 12h light:12h dark cycle with lights turning on at 9:00 PM and tested during their active period (dark cycle). All experiments were conducted in accordance with the National Institute of Health guide for the care and use of laboratory animals and approved by the Queens College Institutional Animal Care and Use Committee.

2.2. Catheterization Surgery

Each rat received an intraperitoneal (i.p.) injection of atropine (0.54 mg/0.1 mL dH2O) to reduce bronchial discharge and was subsequently anesthetized under sodium pentobarbital (65 mg/kg, i.p.). Rats were then placed in a stereotaxic apparatus, with the incisor bar set at −3.5 mm. An incision was made on the scalp to expose the skull and 4 stainless steel screws were installed in positions simulating the corners of a rectangular. Next, the rats were removed from the stereotaxic apparatus and placed on the aseptic table. A small incision was made on the ventrolateral neck, approximately 1 cm from the midline. The jugular vein was isolated and opened. A Silastic catheter (Dow Corning, Midland, MI) was inserted into the opened vein and pushed to a depth just short of the right atrium of the heart. The catheter was then secured at this position by tying 3 sutures around it and the adjacent vein. The opposite end of the catheter was routed subcutaneously through the back of the neck to the scalp incision and exited. Here, a bent 22-gauge steel tube (2.5 cm) was inserted into the catheter and mounted to the skull using dental acrylic. The stainless-steel tube served as the junction between a tethered drug-infusion line and the intravenous catheter. After surgery, all rats were housed individually and given 3–4 days to recover, wherein gentamicin (4 mg/kg, i.v.) was administered daily until the final self-administration session to prevent infections. To maintain patency, catheters were filled with a heparin-saline solution (200 U.S.P., i.v.) immediately after surgery and after each self-administration session.

2.3. Self-administration Chambers

All rats were tested in 16 operant conditioning chambers: 8 measuring 26 cm x 26 cm x 30 cm and the other 8 measuring 30 cm x 22 cm x 27 cm. Each chamber was equipped with two retractable levers positioned 10 cm above a steel-rod floor and a white cue light 3 cm above each lever. A polyethylene tube, encased in a metal tether and suspended by a fluid swivel, connected the animal’s catheter assembly to a heroin-filled syringe in a pump (Razel, 3.33 rpm). All chambers were encased within a sound and light attenuating box and programmed through Med Associates interfacing and software (Georgia, VT).

2.4. Drugs

Heroin was a gift from the National Institute on Drug Abuse (NIDA) Drug Supply Program and was dissolved in 0.9% saline. Because females weighed approximately 2/3 of what males did at the start and throughout the experiments, two separate concentrations of heroin were used for males and females, respectively, to achieve a dose of 0.05 mg/kg/infusion (0.125 mL/infusion) for each sex. Methohexital sodium (Brevital; Southern Anesthesia & Surgical, Inc., South Carolina, USA) was administered following the last self-administration session (1 mg in 0.1 ml of saline, i.v.) to verify catheter patency.

2.5. Procedure

The entire procedural timeline can be seen in Fig. 1. The procedure consisted of 3 phases: heroin self-administration, forced abstinence (FA), and extinction/reinstatement. After all of the animals established self-administration (described below), they stayed in their standard home cages with no events or manipulations, for 3 or 15 days (putative “incubation of craving” period). Next, rats were moved to environmentally enriched (EE), or standard housing (non-EE) that was identical to their previous home cage, where they lived for the remainder of the experiment. Three days after placement in the housing, rats underwent 15 sessions of extinction followed by a reinstatement test.

Figure 1:

Figure 1:

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Experimental timeline for all rats in the 3 (a) and 15 (b) day forced abstinence groups.

2.5.1. Heroin self-administration

Each rat was assigned to a single operant conditioning chamber and all subsequent sessions took place in that chamber. During heroin self-administration, rats underwent daily 3-h sessions with access to both levers (left and right levers were programmed as active and inactive in a counterbalanced manner across all chambers). Each press on the active lever produced the illumination of the adjacent light for a 20-s duration and activated the pump, delivering an infusion of heroin for 4.5 s. Subsequent presses on the active lever during the 20-s period were counted but produced no consequences. Presses on the inactive lever were counted but produced no scheduled consequences. All lever presses and infusions were recorded. Four hours after the last self-administration session, catheter patency was tested with a 0.1ml i.v. infusion of Brevital. Immediately following the injection, a tail pinch was done to test sedation; rats who exhibited an escape response to this test were removed from the study.

Each rat was trained to self-administer heroin under a fixed ratio 1 (FR1) schedule of reinforcement. After a minimum of 3 consecutive days of stable heroin intake, all rats proceeded with an additional 15 sessions of self-administration. Stable drug-intake was operationally defined as follows: for each rat daily active lever presses exceeded inactive lever presses and the total number of daily infusions did not exceed ±15 % from their 3-day mean total number of daily infusions. All animals returned to their standard housing between sessions.

2.5.2. Forced Abstinence.

After completing the self-administration phase half of all rats remained in our vivarium for 3- and the other half 15 days in their standard housing where no events occurred (other than daily 2 to 3 min of handling).

2.5.3. Environmental Enrichment.

After 3- or 15-days of FA, half of the males (ns = 9) and females (ns = 9) were randomly assigned to EE housing, and the other halves of each sex (ns = 9 and 9, respectively) were assigned to non-EE housing.

The EE condition consisted of larger than standard cages measuring 82 cm x 49 cm x 35cm. EE cages were equipped with beta chip bedding, a 10-cm diameter PVC tunnel, a running wheel, and seven additional objects that were rotated daily to provide a variety of multidimensional stimulation including bells, rope, toy cars, and bright chew toys. Non-EE housing consisted of standard cages with bedding.

2.5.4. Extinction/Reinstatement.

Three days after the commencement of enrichment conditions, all rats began the extinction/reinstatement phase. For 15 consecutive days, each rat returned to their respective operant chamber for daily 2-h sessions wherein both levers were presented but pressing neither produced any consequences (light was not illuminated, pump was not activated). Presses on both levers were recorded throughout each extinction session. On the day following the last extinction session, the rats underwent a 1-h reinstatement test. This session began with the non-contingent presentation of the heroin-paired cues (illumination of the light, sound of the pump), followed by a second non-contingent presentation 120 s later. Presses on the active lever produced the activation of these cues throughout the session, but heroin was not delivered. Inactive lever presses were counted but produced no scheduled consequences.

2.6. Data Analysis.

2.6.1. Self-administration.

The data consisted of total infusions, active and inactive lever presses for each of the fifteen 3-h self-administration sessions. Lever pressing was analyzed with a 5-way mixed factorial analysis of variance (ANOVA) with lever and session as within subjects factors and housing, sex and FA period as between subjects factors. Infusions were analyzed with a 4-way mixed factorial ANOVA with session as a within-subjects factor and housing, sex and FA period as between-groups factors. For lever pressing, a significant 3-way interaction was followed up by interaction comparisons at each level of sex. Significant 2-way interactions were followed by tests of simple main effects of phase at each level of lever.

2.6.2. Extinction.

The data were comprised of total active and inactive lever presses for each of the fifteen 2-h extinction sessions. Lever pressing was analyzed with a 5-way mixed factorial analysis of variance (ANOVA), with lever and session as within subjects factors and housing, sex and FA period as between subjects factors. A significant 3-way interaction (lever x phase x sex) was followed by interaction comparisons at each level of sex. Significant 2-way interactions were proceeded by tests of simple main effects of phase at each level of lever. A significant 2-way interaction (lever x phase) was followed up by a test of simple effects of phase at each level of lever and a significant FA x housing interaction was followed up by a test of simple effects of FA period at each level of housing.

2.6.3. Cue-induced Reinstatement.

The data consisted of the mean lever presses on the active and inactive levers during the first hour of the last 3 extinction sessions and during the full 1-h reinstatement test session for each rat. The data were analyzed with a 5-way mixed factorial ANOVA with phase and lever as within-subjects factors and housing, sex and FA period as between-groups factors. A significant 3-way interaction (lever x phase x housing) was followed up with interaction comparisons at each level of phase (extinction, reinstatement), significant 2-way interactions were followed up by a test of simple main effect of housing at each level of lever. Separately, a significant 3-way interaction (lever x phase x sex) was proceeded by an interaction comparison at each level of sex. Significant two-way interactions were followed up by a test of simple effect of sex at each level of lever.

3. Results

3.1. Self-administration.

Figs. 2a. and b. show infusions for males and females separated into their eventual EE and non-EE groups in the 3- and 15-day FA groups across 15 sessions. All groups self-administered similar numbers of infusions and all groups showed a similar pattern of increasing the number of total infusions across sessions (see Figs. 2a and b). A 4-way ANOVA on these data revealed a significant effect of session [F(14, 700) = 5.47, p < .05].

Figure 2:

Figure 2:

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Mean (±SEM) number of heroin infusions during 15 consecutive sessions of heroin self-administration for males and females in the 3 (a) and 15 (b) -day forced abstinence groups, separated into eventual EE and non-EE groups. Mean (±SEM) number of presses on the active lever (c, d) and inactive lever (e, f) during 15 consecutive self-administration sessions for males and females separated into eventual EE and non-EE groups in the 3 (c, e)- and 15 (d, f)- day forced abstinence groups.

Figs. 2c–f, show active and inactive lever presses for males and females separated into eventual housing and FA period groups across the 15 self-administration sessions. All groups responded more on the active (Figs 2c and d) than the inactive (Figs 2e and f) levers. Generally, for both the active and inactive levers, all groups showed similar levels of pressing, with slightly lower responding on the active lever by the female 3-day FA non-EE group in sessions 12 to 15. A 5-way ANOVA revealed a significant lever x session x sex interaction [F (14, 700) = 2.78, p < .05]. Subsequent interaction comparisons at each level of sex revealed significant lever x session interactions for males [F (14, 378) = 3.29, p < .05] and females [F (14, 406) = 4.09, p < .05]. Tests of simple effects of session at each level of lever in males revealed a significant effect of session at the active lever [F (14, 378) = 5.05, p < .05], but not the inactive. Similarly, in females, there was a significant effect of session at the active lever [F (14, 420) = 7.33, p < .05], but not at the inactive lever.

3.2. Extinction

Lever presses during extinction for all groups followed a similar pattern of daily decreases but with some differences among groups. In all groups initial responding on the active lever was higher than on the inactive lever for the first few sessions and for the remainder of the sessions responding on both levers was similar (see Fig. 3). Active and inactive lever presses in the 15-day FA group, female and male non-EE groups appeared to remain slightly higher than their corresponding 3-day abstinence groups across 15 sessions (see Fig. 3b and d). Also, generally, females tended to respond on the active lever slightly more than males (see Fig. 3a and b). A 5-way ANOVA revealed a significant session x lever x sex interaction [F (14, 896) = 2.765, p < .05], a significant FA x housing interaction [F (1, 64) = 4.742, p < .05] and significant main effects of session [F (14, 896) = 46.526] sex [F (1, 64) = 11.59, p < .05] and housing [F (1, 64) = 28.78, p < .05]. Following the significant lever x session x sex interaction an interaction comparison of lever x session at each level of sex revealed a significant interaction for males [F (14, 490) = 8.73, p < .05] and females [F (14, 490) = 22.87, p < .05]. Subsequent tests of simple effects of session at each level of lever revealed significant effects at the active lever for males [F (14, 490) = 37.23, p < .05] and females [F (14, 490) = 67.02, p < .05] and at the inactive lever for males [F (14, 490) = 4.52, p < .05] and females [F (14, 490) = 2.72, p < .05]. The significant FA x housing period interaction was followed by a test of simple effect of FA period at each level of housing. This analysis revealed a significant effect of FA in the non-EE groups [F (1, 32) = 159.94, p < .05], but not in the EE groups. These analyses indicate that females pressed significantly more on both levers than males and, that of the non-EE groups, rats exposed to 15 days of FA pressed significantly more than their 3-day counterparts.

Figure 3:

Figure 3:

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Mean (±SEM) number of presses on the active (a, b) and inactive (c, d) lever during 15 consecutive extinction sessions for males and females in EE and non-EE housing in the 3 (a, c) and 15 (b, d) -day forced abstinence groups.

3.4. Cue-induced Reinstatement.

Fig. 4 shows active and inactive lever presses averaged across the first hour of the last 3 days of extinction (left) and during the cue-induced reinstatement test (right) for males (a) and females (b) in EE and non-EE housing in the 3- and 15-day FA period conditions. All groups emitted more presses on the active lever during the reinstatement test than during extinction. In both FA conditions, males and females housed in EE responded less than their non-EE counterparts on the active lever in both extinction and reinstatement phases. A 5-way ANOVA revealed significant lever x phase x housing [F (1, 64) = 17.7, p < .05], lever x phase x sex [F (1, 64) = 7.77, p < .05] and sex x housing [F (1, 64) = 7.15, p < .05] interactions. Following the significant lever x phase x housing interaction, a lever x housing interaction comparison at each level of phase revealed a significant interaction at the level of reinstatement [F (1, 70) = 57.2, p < .05], but not extinction. Following the significant interaction, tests of simple effects of housing at each level of lever in the reinstatement phase revealed a significant effect at the active lever [F (1, 70) = 237.36, p < .05] and the inactive lever [F (1, 70) = 22.19, p < .05]. The lever x phase x sex interaction was followed up by an interaction comparison of lever x sex at each level of phase. These analyses revealed a significant interaction at the level of reinstatement [F (1, 70) = 34.1, p < .05], but not extinction. Subsequent tests of simple effects of sex at each level of lever revealed a significant effect at the active lever [F (1, 70) = 78.7 p < .05], but not the inactive lever. Taken together, these results indicate that EE rats pressed significantly less on both active and inactive levers during reinstatement and generally, females pressed significantly more than males.

Figure 4:

Figure 4:

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Mean (±SEM) number of presses on the active and inactive levers averaged across the first hour of the last 3 extinction sessions and the 1-hour reinstatement test for 3 and 15 day forced abstinence, EE and non-EE males (a) and females (b). * represents significantly less (p < .05) active and inactive lever presses in EE compared to non-EE rats during reinstatement, irrespective of sex.

4. Discussion

During extinction, rats in the EE housing condition responded less than those in the non-EE housing condition. This suggests that EE reduced heroin seeking regardless of sex or duration of FA. Also, females generally responded more than males. This suggests that females show greater heroin seeking than males. Finally, of the groups housed in non-EE, rats in the 15-day FA condition showed significantly more responding than those in the 3-day FA condition, suggesting an “incubation” of drug-seeking (i.e., craving) effect. Interestingly, of the groups housed in EE there were no differences between the 3- and 15-day FA groups, indicating the absence of an incubation effect. Together these data suggest that generally, a time-dependent increase in responding can be observed in our extinction procedure, and EE when administered after prolonged FA, can prevent these changes. These results are in line with previous studies that demonstrate the protective effects of EE on cocaine craving incubation after 30 and 60 days (Chauvet et al., 2012) and 21 days (Thiel et al., 2009).

During cue-induced reinstatement of heroin seeking, females and males housed in EE showed less responding than did groups housed in non-EE for both FA conditions. Again, females responded more than males. These findings indicate that exposure to EE can attenuate cue-induced relapse related behaviors in females and males and that it does so regardless of prolonged periods of non-drug-taking, periods that have been hypothesized to induce ‘incubation’ of craving.

Our main results are consistent with our previous findings that EE, implemented after 15 days of FA, significantly reduced cue-induced reinstatement of heroin seeking in male rats trained in heroin self-administration (Galaj et al., 2016). We have also demonstrated in an animal conflict model, where rats voluntarily abstain from heroin taking and seeking due to negative consequences, that rats housed in EE reached abstinence criteria (3 consecutive days of no active lever presses) from heroin self-administration more rapidly than their non-EE counterparts (Peck et al., 2015). However, these anti-heroin1-seeking effects of EE had not been demonstrated in females. Our present findings, along with the previously mentioned studies, strongly suggest that EE can reduce the drug-seeking effects of heroin cues after prolonged periods without drug-availability in both males and females.

Interestingly, although females tended to self-administer similar numbers of infusions as males during training, they tended to emit more lever presses than males across phases. This was especially true for the non-EE females in the extinction and reinstatement phases. A possible explanation for this finding could be the differential stress hormone response while in periods with no drug availability. Firstly, Brown and Grunberg (1995; 1996) have found that corticosterone (CORT) blood-plasma levels, a peripheral indicator of hypothalamic-pituitary-adrenal (HPA) axis activation, important in stress response modulation, was higher in intact female when compared to male rats. Convergent data shows that basal levels of CORT and adrenocorticotropic hormone (ACTH) are higher in females than males who were singly housed and underwent surgical intravenous catheter placement. In the same study, exposure to EE reduced those basal levels in both males and females (Belz et al., 2003). These differences have been linked to the presence of ovarian hormones, such that estrogen is shown to enhance ACTH and CORT levels in ovariectomized rats (Burgess and Handa, 1992). Moreover, ovarian hormones are known to play a critical role in the acquisition, incubation and extinction/reinstatement of drug-seeking. Interestingly, estrogen is shown to increase motivation for cocaine under a progressive ratio schedule of reinforcement; where females reached higher levels of responding than males, and when in the peak estrous-cycle female rats worked harder to receive cocaine (Roberts et al., 1989). Recent reports demonstrate a similar effect (Johnson et al., 2019) as well as estrous-dependent incubation of cocaine-seeking, seen after 29 and 30 days of forced abstinence (Johnson et al., 2019; Nicolas et al., 2019). Interestingly, enhanced drug seeking is also seen in estrous rats during extinction tests and cue-induced (Fuchs et al., 2005) or as cocaine-primed reinstatement tests (Kippin et al., 2005; Feltenstein & See, 2006). Studies investigating the sex-related effects of EE show that, when administered during development and concurrent with chronic variable stress (CVS) it is protective against CVS dependent anhedonic-like phenotypes, in a forced swim test in females but not males during adulthood (Smith et al., 2018). Interestingly, in the present study the enhanced extinction responding seen in non-EE females appeared more striking in the 15-day abstinence group, than in all other groups. Taken together, it is possible that after periods of prolonged FA, estrogen-dependent stress hormone responses may amplify drug-seeking, and can contribute to enhanced motivation for heroin, whereas EE when implemented during these changes, may blunt the stress-related effects.

Also interestingly, rats in the non-EE condition showed elevated levels of lever pressing during extinction but not during cue-induced reinstatement after 15-days of FA compared to their 3-day counterparts. This FA period-related difference was not seen in the EE groups. The behavioral indications are best seen during the first few extinction sessions, immediately after putative incubation may have occurred. These data are in line with other groups (Chauvet et al., 2012) that demonstrated the protective effect of EE on cocaine craving incubation after 30 and 60 days of forced abstinence. Our data are also in line with more recent studies utilizing alternative forms of enrichment such as social interaction, having demonstrated that operant social reward, can diminish incubation of heroin craving in male and female rats (Venniro et al., 2019). Like others, our data suggest that extended periods of being drug-free may intensify drug-seeking (i.e., craving) and that EE may reduce this effect. Other studies show that cue-induced responding is significantly higher after 6, 11, 12, 25 and 30 days of forced abstinence in rats with a history of heroin-self administration (Shalev et al., 2001; Airavaara et al., 2011; Pickens et al., 2011). These studies did not implement extended extinction learning procedures, rather testing procedures occurred immediately after FA periods. In light of these reports, our findings suggest that extinction learning itself may interrupt the process by which time-dependent increases in drug-seeking occur. This idea is supported by an early incubation study (Niesewander et al., 2001) where rats demonstrated significantly higher cocaine-seeking during an extinction test after 21 days of FA when compared to 21 days of extinction.

Although, during extinction, we observed elevated drug-seeking in rats exposed to a longer duration of FA, and that EE prevented these time-dependent changes, we acknowledge that comparisons to classical incubation studies should be made carefully. Thus, we tested for ‘incubation’ throughout 15 extinction sessions in contrast to many other studies with single drug-seeking test occurring immediately after extended periods of uninterrupted FA (Grimm et al., 2001, Lu et al., 2004, Airavaara et al., 2011; Pickens et al., 2011; Chauvet et al., 2012; Fanous et al., 2012; Theberge et al., 2012). Nevertheless, our incubation effect—induced by 15 days of FA and 3 days of housing—is in line with previous studies where incubation is seen after 11, 12, 14, or 15 days of FA (Grimm et al., 2001; Shalev et al., 2001; Theberge et al., 2012; Fanous et al., 2012). Further, comparisons between late reinstatement tests, days 11 and 30 (Theberge et al., 2012), 15 and 29 (Grimm et al., 2001) seem to lack a robust incubation effect, suggesting that measuring incubation at two later time points may not be optimal. We acknowledge that our reinstatement tests occurring 22 and 34 days after cessation of self-administration may be considered late time points, when compared to other incubation studies (Fanous et al., 2012; Theberge et al., 2012; Venniro et al., 2017), and may not be optimal points for measuring incubation of craving.

In summary, we have demonstrated that EE administered after self-administration training and after periods where incubation of craving may have occurred, can reduce incubation of heroin seeking (seen in early extinction responding) in males and females and can reduce cue-induced reinstatement of heroin seeking in females and males regardless of FA periods. These findings suggest that EE could serve as an effective treatment for heroin use disorder.

Highlights.

  • Environmental enrichment reduces craving incubation in male and female rats.

  • Environmental enrichment reduces cue-induced heroin seeking in male in female rats.

  • Environmental enrichment may have a potential treatment utility for heroin use disorders.

Acknowledgments

This work was supported by National Institute of General Medical Science of the National Institutes of Health under award number 1SC3GM130430-01 to R.R.

Role of Funding

1SC3GM130430-01 to R.R.

Footnotes

Author Disclosures

The authors have no conflict of interest in the manuscript.

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