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. Author manuscript; available in PMC: 2018 Oct 1.
Published in final edited form as: Exp Clin Psychopharmacol. 2017 Oct;25(5):393–401. doi: 10.1037/pha0000137

Effects of Environmental Enrichment on d-Amphetamine Self-Administration Following Nicotine Exposure

Dustin J Stairs 1, Sarah E Ewin 1, Megan M Kangiser 1, Markus N Pfaff 1
PMCID: PMC5654547  NIHMSID: NIHMS882979  PMID: 29048188

Abstract

Adolescent nicotine exposure has been shown to lead to further psychostimulant use in adulthood. Previous preclinical research in rats has shown that environmental enrichment may protect against drug abuse vulnerability. The current study was designed to examine whether environmental enrichment can block the ability of adolescent nicotine exposure to increase d-amphetamine self-administration in adulthood. Male Sprague Dawley rats were raised in either enriched conditions (EC) or isolated conditions (IC) and then injected with saline or nicotine (0.4 mg/kg, s.c.) for seven days during adolescence. In adulthood rats were allowed to self-administer d-amphetamine under fixed ratio (0, 0.006, 0.01, 0.02, 0.06, and 0.1 mg/kg/infusion) and progressive ratio (0, 0.006, 0.06, and 0.1 mg/kg/infusion) schedule of reinforcement. Nicotine-treated IC rats self-administered more d-amphetamine at 0.006, 0.01 and 0.02 mg/kg/infusion doses compared to their saline-treated IC counterparts regardless of the schedule maintaining behavior. This effect of nicotine was reversed in EC rats on a fixed ratio schedule. These findings indicate that environmental enrichment can limit the ability of adolescent nicotine exposure to increase vulnerability to other psychostimulant drugs, such as d-amphetamine.

Keywords: Environmental Enrichment, rats, nicotine exposure, d-Amphetamine, Self-administration

Adolescent cigarette smoking is still a national problem, as 41.1% of high school students reported having tried smoking in 2013 and 15.7% reported use within the past 30 days (Kann et al., 2014). These rates are of concern given that the majority of adult smokers began smoking during their adolescent years (Audrain-McGovern et al., 2004). Research shows that adolescent nicotine exposure is also related to an increase in other drug use in adulthood. In humans, adolescent nicotine exposure is correlated with increased use of other illicit drugs (Degenhardt et al., 2010; Kandel, Yamaguchi, & Chen, 1992; Palmer et al., 2009) use as well as increased craving for cocaine in adulthood (Lambert, 2002; Lambert, McLeod, & Schenk, 2006). Additionally, adolescents and young adults who used tobacco are more likely to use marijuana and, subsequently, cocaine (Wagner & Anthony, 2002). Researchers have also developed a potential molecular mechanism for how nicotine may serve as a gateway drug (Kandel & Kandel, 2014).

In preclinical models, adolescent nicotine exposure in rats is also associated with an increased sensitivity to stimulants in adulthood. Using the conditioned place preference (CPP) procedure, de la Pena et al. (2014) found that rats pre-exposed to nicotine or cigarette smoke during adolescence showed a greater CPP response following conditioning with high dose of nicotine. However, at a low conditioning dose of nicotine, this effect was reversed; nicotine-naïve rats demonstrated stronger CPP than rats pre-exposed to nicotine. McMillen et al. (2005) exposed rats to either nicotine or saline during their periadolescent period and found that rats exposed to nicotine had a stronger cocaine-induced CPP response compared to saline controls.

While conditioned place preference is a widely used model to examine drug reward, it demonstrates less about drug reinforcement compared to the rodent self-administration procedure. Research using drug self-administration indicates that adolescent nicotine exposure can result in increases in stimulant self-administration in adulthood; for example, Dickson et al. (2012) found nicotine exposed mice self-administered more cocaine than saline controls. Pipkin et al. (2014) found that a low dose of nicotine given throughout adolescence increased methamphetamine self-administration in rats, while exposure to high doses of nicotine in adolescence did not. This suggests that nicotine’s cross-sensitizing effects may be dose-dependent. Also, adolescent nicotine exposure may not sensitize the reinforcing effects of nicotine in adulthood as previous research has found that adolescent nicotine exposure did not alter the level of nicotine self-administration in adulthood (de la Pena et al., 2014).

Many preclinical studies show that environmental enrichment may protect against drug abuse vulnerability (Solinas, Thiriet, Chauvet, & Jaber, 2010; Solinas, Thiriet, El Rawas, Lardeux, & Jaber, 2009; Stairs & Bardo, 2009). The environmental enrichment model can be used in rodents (Stairs & Bardo, 2009); however, it is done in varying ways, which can make cross-laboratory comparisons difficult (Simpson & Kelly, 2011). Studies using an enrichment model similar to those used by Bardo and colleagues, have found that rats raised in enriched environments (EC) show a decrease in the sensitivity to the behavioral effects of stimulants compared to rats raised in isolated conditions (IC; (Bardo et al., 1995; Stairs & Bardo, 2009)). In rats, environmental enrichment decreased self-administration of low unit doses of d-amphetamine, methylphenidate and cocaine (Alvers, Marusich, Gipson, Beckmann, & Bardo, 2012; Bardo, Klebaur, Valone, & Deaton, 2001; Gipson, Beckmann, El-Maraghi, Marusich, & Bardo, 2011; Green, Gehrke, & Bardo, 2002).

A previous study from our lab examined the effects of adolescent nicotine exposure on nicotine locomotor sensitization and d-amphetamine cross-sensitization in adulthood between EC and IC rats (Adams, Klug, Quast, & Stairs, 2013). Following exposure to nicotine during periadolescence when animals were challenged with either d-amphetamine or nicotine in adulthood, EC rats did not exhibit locomotor sensitization to nicotine and did not show cross-sensitization to a low dose of d-amphetamine, while IC rats did show nicotine sensitization and cross-sensitization to a low dose of d-amphetamine. This suggests that environmental enrichment can serve as a protectant against the effects of adolescent nicotine exposure, which may cause sensitization to the rewarding effects of future stimulant use (Stairs, Kangiser, Hickle, & Bockman, 2016).

While previous work from our laboratory has shown that enrichment can protect against adolescent nicotine exposure’s sensitizing effects on locomotor activity (Adams et al., 2013), no study to date has examined whether enrichment can affect nicotine exposure-induced changes in stimulant self-administration. The goal of the present experiments was to examine the effects of environmental enrichment on both fixed- and progressive-ratio self-administration of d-amphetamine in adulthood following adolescent nicotine exposure. We also sought to determine if the effects of nicotine and enrichment were specific to drug self-administration by also testing EC and IC animals self-administering food.

Methods

Animals

Forty-eight, male Sprague Dawley rats (Envigo Inc., Indianapolis, IN, USA) were received on PND 21 and used to investigate the effects of nicotine on d-amphetamine self-administration. Twenty-four male Sprague Dawley rats were used to test the effects of nicotine on food-maintained behavior. Rats had ad libitum access to food and water in their home cages, and were maintained on a 12/12 hr light/dark cycle with lights on from 7:00 to 19:00. All protocols were approved by the Creighton University Animal Care and Use Committee and conformed to the NIH Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources (U.S.), 2011).

Environmental Conditions

Upon arrival, rats were randomly assigned to one of two conditions, either the enriched condition (EC) or the isolated condition (IC). Animals stayed in these conditions for the duration of the experiment. The EC rats were housed in stainless steel cages (62 × 62 × 42 cm) with 12 social cohorts per cage. The EC cages also contained 14 hard plastic objects (toys, containers, etc.) placed throughout the cage. Seven of these objects were replaced daily and all objects were reconfigured every day. These rats had brief daily handling during the object change. IC rats were housed in individual stainless steel cages (17 × 24 × 20 cm) with solid stainless steel sides, top and back walls, with wire mesh flooring and front panel. They were handled minimally during weekly cage changing and injections. Animals were housed in their respective conditions throughout the duration of the experiment.

Behavioral Apparatus

Standard operant conditioning chambers (28 × 21 × 21 cm; ENV-001; MED Associates, St. Albans, VT) with alternating aluminum and Plexiglas walls including a metal rod floor were located inside sound-attenuating chambers (ENV-018M; MED Associates, St. Albans, VT) were used in both d-amphetamine and food self-administration sessions. A recessed food tray (5 × 4.2 cm) was located 2 cm above the floor in the center of one of the aluminum walls, and a response lever was located 6 cm above the floor on each side of the food tray. A white stimulus light (28 v; 3 cm diameter) was located 6 cm above each lever. The standard operant chambers were retro-fitted with a counterbalance arm, swivel, and leash (PHM-110, PHM-115I and PHM-120A, respectively; MED Associates, St. Albans, VT). Drug infusions were delivered via a MED Associates infusion pump located outside of the sound-attenuating chamber (PHM-100; MED Associates, St Albans, VT). Responses were recorded and programmed consequences were controlled by a computer in an adjacent room equipped with Med-PC software (Med Associates, St. Albans, VT).

Drugs

S-(−)-nicotine bitartrate was purchased from Sigma/RBI (St. Louis, MO) and dissolved in 0.9% w/v NaCl (saline). The nicotine solutions were adjusted to pH 7.4 using 1M NaOH and injected subcutaneously (s.c.) in a volume of 1ml/kg body weight. D-amphetamine HCl was purchased from Sigma/RBI, dissolved in saline in a volume of 1 ml/kg body weight. Nicotine doses are expressed as free base weight while d-amphetamine doses are expressed as the salt weight.

Procedure

Nicotine Pre-treatment

Following seven days of habituation to their housing conditions, EC and IC rats underwent a nicotine or saline pretreatment period from PND 28–34. Specifically, half of the EC and IC rats received once daily injections of nicotine for seven days (0.4 mg/kg, s.c.) while the other half of EC and IC rats received once daily injections of saline for seven days (1 ml/kg, s.c.). Following the week of pretreatments, all animals were then given a 35-day washout period during which they were maintained in their respective environments. The nicotine dose, dosing regimen and wash out period were selected to match a previous studies from our laboratory and other laboratories which have found effects of nicotine (Adams et al., 2013; Collins & Izenwasser, 2004).

D-Amphetamine Self-Administration

Food Pre-training

Previous experience with the current environmental enrichment procedure indicates that EC rats do not readily acquire d-amphetamine self-administration, so to establish reliable lever pressing in both EC and IC rats prior to d-amphetamine self-administration, rats were initially trained to respond through food reinforcement. Following the 35-day washout period, food access was restricted over a period of 4 days in order to decrease body weights to approximately 85% of free feeding weights. Following food deprivation, rats were exposed to 5 g of food pellets (45 mg pellets; BioServ, Frenchtown, NJ) on one day to alleviate neophobia during training. On the following day, rats were placed into the operant conditioning chamber with both levers extended into the chamber. The rats were allowed to complete a 60-min session in which responses on either lever resulted in delivery of a food pellet on a fixed-ratio 1 (FR1) schedule. The rats had five sessions during which completion of an FR1 on either lever resulted in the delivery of a 45 mg food pellet. The lever from which the animals obtained the majority of reinforcers was then deemed the “active lever” for that rat once d-amphetamine self-administration started. On the last day of food pre-training EC rats had a mean number (±SEM) of active lever presses of 177.3 (±14.06) while IC rats had a mean of 197.1 (±16.97). Following food pre-training, rats were maintained on ad libitum food access for the remainder of the experiment.

Catheterization Surgery

Following return to free-feeding body weights, all rats were implanted with an indwelling catheter in the right jugular vein, which allowed for intravenous drug delivery. Rats were anesthetized with an injection of ketamine (80 mg/kg; i.p.) and midazolam (5 mg/kg; i.p.). The silastic catheter (0.2 mm i.d.; Fisher Scientific) was threaded subcutaneously to exit from a piece of stainless-steel hypodermic tubing (22 ga) embedded in a dental acrylic head cap mounted to the top of the skull with four stainless-steel jeweler’s screws. Catheters were flushed daily with 0.1 mg/ml-heparinized saline (0.25 ml/day) and the antibiotic gentamicin (10 mg/kg, i.v.) to maintain patency during the seven days of recovery. Following recovery only heparinized saline was used to flush catheters.

Amphetamine Self-Administration

Following seven days of recovery from surgery, rats were placed in the operant conditioning chamber and allowed to acquire d-amphetamine self-administration during daily 60-min sessions at a high unit dose of d-amphetamine (0.1 mg/kg/infusion). This dose of d-amphetamine was chosen because it engenders reliable self-administration in both EC and IC rats (Green et al., 2002). D-amphetamine self-administration was established under an FR1 schedule of reinforcement, with a 20-sec signaled timeout (TO). During the TO, the white cue lights above each lever were illuminated and lever presses were recorded but had no programmed consequences. Lever presses on both the active and inactive levers were recorded throughout each daily session. Each infusion was delivered in a volume of 0.06 ml over 5.9 sec, which coincided with the beginning of the 20-sec signaled TO.

Rats were allowed to self-administer d-amphetamine at the 0.1 mg/kg/infusion dose for a seven sessions in order to establish reliable levels of drug intake. Within the seven session all rats met the following criterion of reliable levels of intake: there was less than 20% variability in the number of infusions earned across three consecutive sessions and there was a minimum of 2:1 (active: inactive lever) response ratio. There was no significant differences between any of the groups of animals in the number of earned infusions by the final session. Following the seven sessions of acquisition a full dose effect curve was established using the following doses: 0, 0.006, 0.01, 0.02, 0.06, and 0.1 mg/kg/infusion. Three sessions per dose of d-amphetamine were used for each rat, with the order of d-amphetamine doses and saline given in a Latin square design.

Upon completion of the FR1 dose effect curve, the remaining animals with patent catheters (EC; N=13, IC; N=15) were placed back on the 0.1 mg/kg/infusion dose of d-amphetamine for one session on an FR1. Following this session all animals were switched to a PR schedule of reinforcement. The number of responses required to earn each successive infusion in the PR schedule was determined by the following equation; ratio = [5 × EXP(0.2 × infusion #)] − 5. Due to past literature showing that EC rats maintained low levels of responding on this PR schedule (Green et al., 2002) we shortened the length of the session to 60 min, which previous literature has been shown to increase responding maintained by d-amphetamine in EC rats (Arndt, Johns, Dietz, & Cain, 2015). Breakpoint was defined as the last ratio completed when the session ended. For the first seven PR sessions, the 0.1 mg/kg/infusion dose of d-amphetamine maintained responding. Following those seven sessions at the 0.1 mg/kg/infusion, a dose effect curve was established using the following d-amphetamine doses: 0, 0.006, 0.06, and 0.1 mg/kg/infusion. Three sessions per dose of d-amphetamine were used for each rat, with the order of d-amphetamine doses and saline were given in a Latin square design.

Food Self-Administration

Following the 35-day washout period from the nicotine pretreatment, the rats underwent the same food-training exposure as d-amphetamine self-administration group with the exception that the rats only had three sessions during which both levers were active. Following the three food-training sessions, rats were maintained on ad libitum food access for the remainder of the experiment and were allowed to return to free-feeding weights for the remainder of the study.

The rats were given for seven days in their home cages in order to match the timing of the self-administration animals. Following the seven days in their home cages, the rats were allowed to self-administer 45 mg pellets (BioServ, Frenchtown, NJ) on an FR1 schedule of reinforcement for 60 min daily sessions. The sessions were exactly the same as the self-administration sessions except that food pellets were the reinforcer instead of drug infusions. Following seven sessions in which the animals were self-administering food on an FR1 schedule, the animals were switched to a PR schedule of reinforcement. The PR sessions were identical to those in in Experiment 1 except a single food pellet reinforced responding instead of drug infusions. The animals were given 12 sessions on the PR schedule to stabilize behavior.

Data Analysis

Data for d-amphetamine self-administration were analyzed using mixed-factor analyses of variance (ANOVAs). Environment (EC vs. IC) and adolescent nicotine exposure (nicotine vs. saline) were between-subject factors, whereas d-amphetamine dose and lever (active vs inactive) were within subject factors. Data for food self-administration were analyzed using a two-way ANOVA with nicotine and enrichment as between- subject effects. Post hoc comparisons were conducted using a Tukey’s HSD. For tests of significance, an alpha of 0.05 was used.

Results

A mixed factor ANOVA on the number of infusions earned on the FR1 schedule of reinforcement revealed a significant main effect of d-amphetamine dose [F(5,120)=6.42, p<0.001], lever [F(1,24)=150.56, p<0.001], and environment [F(1,24)=91.88, p<0.001]. There were also significant two-way interactions of dose × environment [F(5,120)=6.58, p<0.001], lever × environment [F(1,24)=49.04, p<0.001], dose × lever [F(5,120)=9.44, p<0.001], and environment x nicotine [F(5,120)=11.83, p<0.01]. There were also significant three-way interactions of lever × environment × nicotine [F(1,24)=6.37, p<0.05] and dose × lever × environment [F(5,120)=8.21, p<0.001].

Post hoc comparisons for the significant three-way interactions revealed a number of significant differences between EC and IC nicotine- and saline-treated animals. Figure 1A illustrates that IC rats, regardless of nicotine pretreatment, self-administered significantly more infusions at 0, 0.006, 0.01 and 0.02 mg/kg doses compared to their EC counterparts. Figure 1B indicates that IC nicotine rats also self-administered significantly more d-amphetamine than the EC nicotine rats at the 0.06 and 0.1 mg/kg/infusion doses. IC nicotine-treated rats also self-administered more infusions at the 0 and 0.1 mg/kg/infusion doses compared to IC saline-treated rats. When the 0.06 and the 0.1 mg/kg/infusion dose of d-amphetamine was available, EC nicotine-treated rats self-administered significantly less d-amphetamine than the EC saline-treated rats. Levels of responding on the inactive lever were low for all groups of animals and there were no systematic effects seen on responding on the inactive lever across doses of d-amphetamine (Figure 1C).

Figure 1.

Figure 1

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Figure 1A. Dose-response function for intravenous d-amphetamine self-administration under an FR1 schedule of reinforcement in EC and IC rats. Data represent mean (±SEM) number of infusions earned on an FR1 schedule for the EC and the IC rats (ignoring nicotine treatment) across the different doses of d-amphetamine. # denotes a statistically significant difference between EC and IC groups (p≤0.05).

Figure 1B. Dose-response function for intravenous d-amphetamine self-administration under an FR1 schedule of reinforcement. Data represent mean (±SEM) number of infusions earned on an FR1 schedule for the EC nicotine- and saline-treated (EC Nic and EC Sal, respectively) and the IC nicotine- and saline-treated (IC Nic and IC Sal, respectively) across the different doses of d-amphetamine. Asterisk (*) denotes a statistically significant difference between saline-treated and nicotine-treated groups (p≤0.05). & denotes a statistically significant difference compared to saline (p≤0.05).

Figure 1C. Mean inactive lever presses (±SEM) in the EC nicotine- and saline-treated rats and IC nicotine- and saline-treated rats across the different doses of d-amphetamine.

A mixed-factor ANOVA on the breakpoints obtained on the PR schedule revealed a significant main effect of dose [F(3,54)=10.15, p<0.001], lever [F(1,18)=36.80, p<0.001], and environment [F(1,18)=38.95, p<0.001], as well as significant interactions of lever × environment [F(1,18)=16.44, p<0.01], dose × lever [F(3,54)=5.38, p<0.01] and dose × environment × nicotine [F(3,54)=3.35, p<0.05]. Post hoc comparisons revealed that, regardless of nicotine pretreatment, IC rats maintained higher breakpoints at all d-amphetamine doses tested compared to their EC counterparts (Figure 2A). Post hoc comparisons revealed a significant difference in breakpoints between nicotine- and saline-treated IC rats at the 0.06 mg/kg/infusion dose of d-amphetamine, and a near-significant difference at the 0.1 mg/kg per infusion dose (p=0.06; Figure 2B). There was no significant differences in breakpoints between nicotine- and saline-treated EC rats. There were no significant differences or trends in the number of inactive lever presses during PR sessions (Figure 2C).

Figure 2.

Figure 2

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Figure 2A. Dose-response function for breakpoints maintained by intravenous d-amphetamine self-administration under a PR schedule of reinforcement in EC and IC rats. Data represent mean (±SEM) mean breakpoints for the EC and the IC rats (ignoring nicotine treatment) across the different doses of d-amphetamine. # denotes a statistically significant difference between EC and IC groups (p≤0.05).

Figure 2B. Dose-response function for breakpoints maintained by intravenous d-amphetamine self-administration under a PR schedule of reinforcement. Data represent mean (±SEM) breakpoints under a PR schedule for the EC nicotine- and saline-treated and the IC nicotine- and saline-treated across the different doses of d-amphetamine. Asterisk (*) denotes a statistically significant difference between saline-treated and nicotine-treated groups (p≤0.05). & denotes a statistically significant difference compared to saline (p≤0.05).

Figure 2C. Mean inactive lever presses (±SEM) in the EC nicotine- and saline treated rats and IC nicotine- and saline-treated rats across the different doses of d-amphetamine.

Finally, when analyzing the effects of adolescent nicotine exposure in EC and IC rats on food self-administration, a two-way ANOVA on the number of responses during the final food self-administration FR session revealed no significant effects of enrichment or nicotine (see Figure 3A). Likewise, a two-way ANOVA on the breakpoint reached on the last day of food-self-administration under a PR schedule indicated there were no significant effects of either enrichment or nicotine (Figure 3B).

Figure 3.

Figure 3

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Figure 3A. Food self-administration maintained under the final FR1 session. Data represent mean (±SEM) number of responses on the active lever for the EC and IC nicotine- and saline-treated rats.

Figure 3B. Breakpoints reached under the final PR food self-administration session. Data represent mean (±SEM) breakpoints on the active lever for EC and IC nicotine- and saline-treated rats.

Discussion

The current results indicate that adolescent nicotine exposure differentially affects d-amphetamine self-administration, depending on housing condition during development. Specifically, we found that IC rats treated with nicotine in adolescence tended to self-administer more d-amphetamine across the majority of doses tested compared to their saline-treated IC counterparts. This effect in IC rats was consistent when self-administration was under a FR or PR schedule of reinforcement. While adolescent nicotine exposure tended to increase drug intake in IC rats, these effects were reversed in EC rats. When EC rats were treated with nicotine in adolescence, self-administration of high doses of d-amphetamine under the FR schedule was decreased compared to saline-treated EC rats. In fact, adolescent nicotine exposure appeared to abolish d-amphetamine self-administration in EC rats, as the level of drug intake at any dose tested was not significantly higher than saline control levels regardless of the reinforcement schedule, which were unexpected findings. While nicotine-treated EC rats did not show significant levels of drug intake, saline-treated EC rats did at the two highest doses of d-amphetamine under both schedules of reinforcement relative to levels of responding maintained by saline substitution.

The effects of adolescent nicotine exposure in IC rats in the current study are congruent with both Dickson et al. (2012) and Pipkin et al. (2014), which found adolescent nicotine pretreatments increased self-administration of psychostimulants in adulthood compared to saline-treated controls. These results are also consistent with Renda and Nashima (2014), which found chronic nicotine exposure in mice leads to an increase in oral nicotine intake. A novel aspect of the present finding is that the ability of adolescent nicotine to increase drug intake can be blocked or reversed by housing the animals in an enriched environment. This protectant effect of enrichment is congruent with a previous study from our laboratory which found that enrichment blocked the ability of adolescent nicotine exposure to lead to nicotine locomotor sensitization or locomotor cross-sensitization to d-amphetamine (Adams et al., 2013). The current study extends that previous finding demonstrating that enrichment can block the ability of adolescent nicotine exposure to increase d-amphetamine self-administration.

The current study’s finding that enrichment can block the ability of adolescent nicotine exposure to increase d-amphetamine self-administration is also congruent with previous literature investigating the effects of environmental enrichment on stimulant self-administration. The significant differences in the saline-treated EC and IC rats in the current study are similar to a previous study by Green et al., (2002) which found that rats tended to self-administer less d-amphetamine at low doses under either FR or PR schedules of reinforcement. Moreover, they found that this effect was not maintained once higher unit doses of the drug were available, which was replicated in the in the current study (see Figure 1A&B). Interestingly enough, nicotine pretreatment in the current study resulted in significant differences between EC and IC nicotine-treated rats at all doses tested under both the FR and PR schedule. This indicates that adolescent nicotine exposure can amplify the enrichment-induced protection in d-amphetamine self-administration compared to what has previously been shown in non-nicotine exposed animals (Bardo et al., 2001; Green et al., 2002).

One possible explanation for the protective effects of enrichment following nicotine exposure may be through alterations in the HPA-axis. Stressors and the activation of the HPA axis are known to interact with the limbic system and increase drug intake and relapse (Brown, Vik, Patterson, Grant, & Schuckit, 1995; Goeders & Guerin, 1994, 1996; Jacobsen, Southwick, & Kosten, 2001; Kosten, Rounsaville, & Kleber, 1986). Previous research using the same enrichment model as the current study have shown that EC rats have lower initial basal free corticosterone levels compared to IC rats (Stairs, Prendergast, & Bardo, 2011). That study also found that IC rats showed greater d-amphetamine-induced increases in free corticosterone (Stairs et al., 2011). It has also been shown that environmental enrichment decreases the HPA-axis activation following acute, repeated nicotine exposure and nicotine withdrawal in adult rats (Skwara, Karwoski, Czambel, Rubin, & Rhodes, 2012). It is possible that EC nicotine-treated rats self-administered less d-amphetamine because their enriched environments deceased the nicotine-induced HPA activity during a crucial developmental period, while the IC nicotine-treated rats experienced the nicotine-induced increases in HPA activity. This protectant effect of enrichment combined with EC rats having decreased HPA activity once d-amphetamine was on board compared to IC rats could explain the decreased levels of d-amphetamine intake and breakpoints in nicotine- and saline-treated EC rats.

The ability of adolescent nicotine exposure to increase d-amphetamine intake in IC rats appears to be specific to drug self-administration as there were no significant differences between IC nicotine- and saline-treated rats in responding maintained through food reinforcement on either a FR or PR schedule of reinforcement. The lack of an effect of environmental enrichment on FR1 responding maintained by food in the current study is somewhat consistent with the limited studies that have looked at enrichment on sucrose-maintained behavior. For instance, Bardo et al. (2001) found only an effect of enrichment during one session under an FR2 schedule of reinforcement (EC rats > IC rats), but this effect was not observed during any other session. Also, in a study where responding was maintained under an FR1 schedule of sucrose reinforcement for 15 sessions, there were no differences between EC and IC rats (Stairs, Klein, & Bardo, 2006). This is the first published study investigating the effects of environmental enrichment on food-maintained PR responding. Whereas in the current study we did not find any effects of enrichment on food-maintained PR responding which was maintained by a single food pellet, future studies may want to look at food-maintained PR responding across various reinforcer magnitudes in EC and IC rats.

While the current study extends the literature on the effect of adolescent nicotine exposure and environmental enrichment on stimulant self-administration, a potential confound between EC and IC rats is the fact that IC rats consistently maintained higher rates of self-administration when saline was substituted for d-amphetamine compared to EC rats. This was true regardless of nicotine exposure or schedule maintaining behavior. While having EC and IC rats not differ when saline is substituted would be ideal, it is not uncommon to see IC rats have higher levels of responding when saline is substituted (Green et al., 2002; Smith et al., 2009; Stairs et al., 2006). We believe the reason for this effect is that in both the current and previous studies saline substitution was done in the presence of the drug-paired cues (either light or tone). Previous research has shown that IC rats will emit greater levels of lever pressing that turns on a visual cue (Cain, Green, & Bardo, 2006). Also enrichment has been shown to reliably decrease d-amphetamine-seeking behavior in a reinstatement model (Stairs et al., 2006), which is similar to how saline substitution was done in the current study.

The current study also has some limitations. One of the limitations is in the manner in which the animals were exposed to nicotine. It is possible that the adolescent nicotine exposure used in the current study does not translate well to the human condition in which the majority of adolescents choose to expose themselves to nicotine in the form of smoking or vaporizing (Singh et al., 2016). Given that stimulant drugs can have very different effects depending on whether the drug is self-administered versus experimentally administered (Dworkin, Mirkis, & Smith, 1995), it would have been preferred to have the rats self-administer nicotine to themselves. Although this would have increased the translational validity of the current study there are a number of technical difficulties that make that form of nicotine exposure difficult using a rodent model. In order to expose the rats to nicotine during critical periods of neural development (Andersen, 2003) they had to be exposed to nicotine at ~ PND 28–34, it would be technically challenging to implant and maintain a catheter in rats that young. Also to get that young of rats to self-administrate significant amounts of nicotine to themselves in the narrow window of seven days would be a challenge given the lower primary reinforcing effects of nicotine (Caggiula et al., 2009; Palmatier et al., 2006). Given these difficulties we decided on the current regimen of nicotine exposure based of past literature that have shown consistent effects of nicotine on stimulant drug effects in adulthood (Adams et al., 2013; Collins & Izenwasser, 2004).

A second limitation of the current study is that we cannot determine whether it is the physical novelty or social novelty of the enriched condition that lead to the protective effects of enrichment on adolescent nicotine exposure. The EC and IC conditions that were used in the current study differ both in the level of physical and social novelty (Stairs & Bardo, 2009). There is considerable debate in the field of environmental enrichment of what constitutes and appropriate social control for the EC condition, i.e. pair-housed vs group-housed (Solinas et al., 2010). In general when labs use a social control condition (SC) in combination with the current EC and IC conditions the SC rats are an intermediate group in terms of the sensitivity to drugs of abuse (Bardo et al., 2001; Bowling & Bardo, 1994; Green et al., 2010; Melendez, Gregory, Bardo, & Kalivas, 2004; Solinas, Chauvet, Thiriet, El Rawas, & Jaber, 2008; Thiel, Sanabria, Pentkowski, & Neisewander, 2009). Given the debate on what constitutes an appropriate SC control group and the intermediate effects seen in SC groups and the nicotine exposure group design we chose not to include an SC group in the current study. Without the inclusion of the SC group we are not able to differentiate whether the physical or social novelty in the EC condition had a greater impact on the protective effects of enrichment. Future studies will need to employ the use of an SC group to tease apart the role of social versus physical enrichment.

Despite these limitations, the current set of experiments indicated that environmental enrichment can block the ability of adolescent nicotine exposure to increase vulnerability to use stimulant drugs in adulthood. Future studies will be needed to investigate the mechanisms of how environmental enrichment is reversing this effect of adolescent nicotine exposure. Future studies may also want to determine if the protective effects of environmental enrichment seen in the current study require the enrichment exposure to be during early development or whether enrichment exposure could be applied later in a more “curative” approach which have been shown previously (Chauvet, Lardeux, Goldberg, Jaber, & Solinas, 2009; Solinas et al., 2008; Thiel et al., 2009; Thiriet et al., 2011).

Public Health Significance.

Adolescence is a particularly vulnerable period of development and insults during this period can lead to increased drug abuse vulnerability. Environmental enrichment (EE) has been shown to protect against this vulnerability, specifically we found that EE can limit the ability of adolescent nicotine exposure to increase psychostimulant drug use in adulthood.

Acknowledgments

Role of Funding Source

This study was also partially funded by Nebraska Cancer and Smoking Disease Research Program and Creighton University Cancer and Smoking Disease Research Program Development Grant to DJS and C. Bockman. Finally, the project described was also supported by Grant Number G20RR024001 from the National Center for Research Resources. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health.

The authors would like to thank Chris Salvatore, Katie Dougherty, Marshall Schroeder and Sonnie Lee for assisting in the collection of the data. The authors would like to thank generous support from the Creighton College of Arts and Science.

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