Effects of environmental enrichment on nicotine-induced sensitization and cross-sensitization to d -amphetamine in rats

Drug and Alcohol Dependence 129 (2013) 247–253

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Effects of environmental enrichment on nicotine-induced sensitization and cross-sensitization to d-amphetamine in rats Emily Adams 1 , Jenna Klug 1 , Michaela Quast, Dustin J. Stairs ∗ Creighton University, Department of Psychology, United States

a r t i c l e

i n f o

Article history: Received 21 August 2012 Received in revised form 15 February 2013 Accepted 16 February 2013 Available online 17 March 2013 Keywords: Environmental enrichment Nicotine d-Amphetamine Sensitization Cross-sensitization Locomotor activity

a b s t r a c t Introduction: Research indicates that adolescent nicotine exposure may predispose individuals to use other psychostimulants later in adulthood, offering support for the incentive-sensitization theory of addiction. Preclinical studies testing the incentive-sensitization theory show that repeated nicotine exposure in adolescent rats can lead to an increased sensitivity to the motor stimulant effects of nicotine and other psychostimulants in adulthood. Although previous nicotine exposure can increase sensitivity to stimulant drugs, rats raised in enriched conditions (EC) show, decreased sensitivity to psychostimulant drugs compared to rats raised in isolation conditions (IC). Methods: We examined whether nicotine sensitization or cross-sensitization to d-amphetamine induced by adolescent nicotine exposure is altered by exposure to environmental enrichment. Adolescent EC and IC male rats received subcutaneous (s.c.) injections of saline or 0.4 mg/kg of nicotine once daily for seven days. Thirty-five days following the last nicotine injection EC and IC animals were challenged with saline, nicotine (0.2 or 0.4 mg/kg) or d-amphetamine (0.5 or 1.0 mg/kg). Results: EC rats failed to show nicotine sensitization at either nicotine dose tested while IC rats showed nicotine sensitization following the 0.4 mg/kg nicotine dose. EC rats also failed to show nicotine-induced cross-sensitization to the 0.5 mg/kg dose of d-amphetamine while IC rats displayed cross-sensitization. However, EC rats did exhibit nicotine-induced cross-sensitization to the 1.0 mg/kg dose of d-amphetamine. Conclusion: These findings indicate that environmental enrichment can alter the ability of adolescent nicotine exposure to induce sensitization and cross-sensitization in adulthood and may be used as a protectant factor against adolescent nicotine exposure. © 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Despite massive efforts from anti-tobacco campaigns, cigarette use is still prevalent, particularly among youth. Nearly 53.5% of students nationwide report having tried a cigarette at least once, and 15.8% report smoking daily (Grunbaum et al., 2004). Although not all users will become addicted, many adult smokers report having started using nicotine in adolescence (Audrain-McGovern et al., 2004). Data also indicates that cigarette use in adolescence is associated with an increased likelihood of cocaine use in adulthood (Kandel and Yamaguchi, 1993; Kandel et al., 1992) and an increased ‘craving’ or ‘wanting’ for cocaine in adulthood (Lambert, 2002; Lambert et al., 2006). Together, these findings suggest nicotine use

∗ Corresponding author at: Creighton University, Department of Psychology, Hixson-Lied, Room 308, 2500 California Plaza, Omaha, NE 68178-0321, United States. Tel.: +1 402 280 2461; fax: +1 402 280 4748. E-mail address: [email protected] (D.J. Stairs). 1 These authors have put equal authorship into this manuscript and are co-first authors. 0376-8716/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.drugalcdep.2013.02.019

during adolescence may predispose individuals to a long-term use of psychostimulants. Lambert et al. (2006) proposed that an increased susceptibility to cocaine in individuals who were adolescent smokers is support for the incentive-sensitization theory of addiction. Specifically, the incentive-sensitization theory proposes that compulsive drug use is a result of repeated drug exposure, which sensitizes important brain structures involved in drug reinforcement (Robinson and Berridge, 1993, 2008). Much of the evidence for the incentivesensitization theory comes from preclinical animal studies looking at behavioral sensitization (Robinson and Berridge, 2008). In a typical behavioral sensitization model, repeated exposure of low doses of a psychomotor stimulant results in a progressive increase in drug-induced locomotor activity. This increase in drug-induced locomotor behavior is due to neuroadaptations in important mesolimbic pathways (Robinson and Berridge, 1993, 2008). Several drugs have been found to result in behavioral sensitization including; cocaine, amphetamine, opiates (Vanderschuren and Kalivas, 2000), and nicotine (DiFranza and Wellman, 2007). Previous studies investigating behavioral sensitization to nicotine found that repeated pretreatments with nicotine in adult rats

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resulted in a sensitized response to nicotine (Benwell and Balfour, 1992; Cadoni and Di Chiara, 2000; Collins and Izenwasser, 2004; Cruz et al., 2005). While nicotine sensitization in adult rats has frequently been replicated, studies investigating nicotine behavioral sensitization in adolescent rats are mixed, with some studies finding behavioral sensitization to occur (Faraday et al., 2003) while others did not (Cruz et al., 2005). These discrepancies, however, may depend on whether the test for sensitization is conducted during adolescence or later in adulthood. That is, Faraday et al. (2003) found sensitization in adolescent rats when they were tested 22 days later in adulthood, whereas, Cruz et al. (2005) did not find nicotine sensitization when animals were tested only three days later still in adolescence. While nicotine exposure during adolescence can potentially result in nicotine sensitization in adulthood, repeated administration of nicotine in adolescence can clearly produce crosssensitization to other stimulants. For instance, periadolescent rats pretreated with nicotine have shown cross-sensitization to the stimulant-induced locomotor effects of amphetamine (Collins et al., 2004; Santos et al., 2009) and cocaine (Collins and Izenwasser, 2004). In the case of amphetamine, sensitization can occur both immediately (Collins et al., 2004) and later, following a “washout” period, in adulthood (Collins et al., 2004; Santos et al., 2009). Given the preclinical data showing that nicotine exposure during adolescence can lead to increased sensitivity to stimulant drugs, it is imperative to identify factors that can protect against this effect. In addition to previous adolescent drug exposure, several environmental factors can influence vulnerability to drug abuse, such as environmental enrichment. Notably, environmental enrichment exposure during adolescence appears to have a protective influence on drug vulnerability (Solinas et al., 2010, 2009). Enrichment exposure can be modeled in the laboratory using rodents (Renner and Rosenzweig, 1987; Solinas et al., 2010; Stairs and Bardo, 2009). There are numerous environmental enrichment paradigms which are used in different laboratories, these paradigms can vary on a number of different dimensions which can make it difficult to compare results across laboratories (see Simpson and Kelly (2011) for review). In the Bardo and colleagues laboratory, they use an environmental enrichment paradigm, which requires animals to be raised from approximately 21 days of age to 51 days of age in one of two environmental conditions: enriched or impoverished (Bardo et al., 1995; Stairs and Bardo, 2009). In these experiments, a typical enriched condition (EC), rats are housed in a large cage with a number of social cohorts and novel objects reconfigured daily. In the impoverished condition (IC), rats are housed individually in hanging stainless-steel cages, without novel objects or social cohorts. Using this enrichment paradigm, previous research has found that environmental enrichment can alter the sensitivity to the locomotor stimulating effects of damphetamine and nicotine (Bardo et al., 1995; Bowling and Bardo, 1994; Bowling et al., 1993; Green et al., 2003). For instance, EC rats have shown to be more sensitive to the locomotor effects of acute d-amphetamine (Bowling and Bardo, 1994; Bowling et al., 1993) compared to their IC counterparts, although following repeated administration of d-amphetamine EC rats showed less locomotor sensitization to d-amphetamine than IC rats (Bardo et al., 1995). Green et al. (2003) found that EC animals were less sensitive to both the acute and repeated locomotor effects of nicotine compared to their IC counterparts. Drug self-administration studies have also found enrichment to exhibit a protectant effect against sensitivity to d-amphetamine. (Bardo et al., 2001; Green et al., 2002; Stairs et al., 2011). While previous research in both humans and rodents indicates that nicotine exposure during adolescence can increase vulnerability to stimulant abuse in adulthood, no studies to date have investigated whether the protectant effects of

environmental enrichment could counteract the increased vulnerability to stimulant drugs due to adolescent nicotine exposure. The goal of the present experiments was to determine if environmental enrichment during development could protect against the ability of nicotine exposure during adolescence to induce either crosssensitization to d-amphetamine or sensitization to nicotine in adult male rats. 2. Methods 2.1. Animals Ninety-five male Sprague-Dawley rats (Harlan Industries, Indianapolis, IN, USA) arrived at approximately postnatal day (PND) 21. Animals had unlimited access to food and water in their home cage, and were maintained on a 12/12 h light/dark cycle with lights on from 07:00 to 19:00 h. All protocols were approved by the Creighton University Institutional 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.), 1996). 2.2. Environmental conditions Upon arrival to the colony, rats were assigned randomly to one of two conditions, either the EC condition or IC condition. EC rats were housed in stainless steel cages (62 cm × 62 cm × 42 cm) with social cohorts (8 per cage) and 14 hard plastic objects (commercial toys, plastic containers, etc.) placed randomly throughout the cage. Seven objects were replaced daily with new objects and all objects were rearranged to create a novel object arrangement every day. EC rats were removed and handled briefly during the daily object change. IC rats were housed in individual hanging stainless steel cages with wire mesh floor and front panel, and solid metal side walls and top (17 cm × 24 cm × 20 cm). IC rats were handled minimally during cage cleaning and injections throughout PND 28–72. All animals remained in these conditions during postnatal days 21–51 and throughout the duration of the experiment. 2.3. Behavioral apparatus Rats were tested in custom-made locomotor chambers for ambulatory activity. Each locomotor chamber was circular with a 12 in. diameter stainless steel floor. Each chamber was equipped with a 2 × 2 array of infrared photobeams that were situated 1.5 in. above the floor of the chamber. Locomotor activity was measured by interruptions in photobeams that were collected and tallied by a computer interface. 2.4. Drugs S(−)-Nicotine bitartrate was purchased from Sigma/RBI (St. Louis, MO) and was dissolved in 0.9% w/v NaCl (saline). All nicotine solutions were adjusted to pH 7.4 using sodium hydroxide (1 M) and injected in a volume of 1 ml/kg body weight. dAmphetamine HCl was purchased from Sigma/RBI, dissolved in saline and injected 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. 2.5. Statistical analysis Data were analyzed using mixed-factor analyses of variance (ANOVAs). Environment (EC vs. IC), adolescent nicotine exposure (nicotine vs. saline), and drug challenge (for cross-sensitization experiment, amphetamine vs. saline; for sensitization experiment, nicotine vs. saline) were between subject factors, whereas the drug challenge doses (for cross-sensitization experiment, amphetamine 0.5 or 1.0 mg/kg; for sensitization experiment, nicotine 0.2 or 0.4 mg/kg) were within subject factors. Tukey’s HSD statistical tests were used for all post hoc comparisons. Results were deemed significant at p ≤ 0.05. Given the design of the study where both amphetamine and nicotine doses were tested using a within subject counterbalanced design we tested for dosing order effects by analyzing the data using mixed-factor ANOVAs using dosing order as a within subject factor (receiving a dose first versus second) with the same between subject factors as described above. The order of the administration had no significant main effects or interactions for the groups receiving saline, nicotine or amphetamine on locomotor test days. Given the absence of a dosing order effect this variable was collapsed and further analysis of nicotine or amphetamine dose was analyzed ignoring order of exposure as described above. 2.6. Procedures 2.6.1. Nicotine exposure. Sixty-three rats were used in Experiment 1. Following their arrival and placement into either EC or IC conditions, the animals were given seven days to habituate to their housing conditions prior to the start of nicotine exposure. After the seven day habituation period, EC and IC rats underwent a nicotine or saline pretreatment period from PND 28–34. Specifically, half of the EC and IC

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2.6.3. Experiment 2. Nicotine sensitization testing. Tests for sensitization to nicotine were conducted following the 35-day washout period with both the EC and IC nicotine- and saline-pretreated rats. All EC and IC rats were tested with nicotine. In order to test more than one dose of nicotine (0.2 and 0.4 mg/kg, s.c.), injections of nicotine were done across two different locomotor sessions (PND 69 and 72). These two sessions were separated by two days in order to alleviate concerns with tolerance of repeated nicotine injections. The order of nicotine doses were counterbalanced so that half of the animals received 0.2 mg/kg doses of nicotine first, while the other half of animals received the 0.4 mg/kg dose of nicotine first. Following the injection with nicotine, rats were immediately placed in locomotor chambers for 45 min sessions. Once the locomotor session finished, animals were returned to their home cages. All behavioral tests were done during the animals’ light phase. With the inclusion of the saline control groups from Experiment 1, this design results in four groups for both the EC and IC rats; (1) rats pretreated with saline and tested with saline (Sal–Sal), (2) rats pretreated with saline and tested with nicotine (Sal–Nic), (3) rats pretreated with nicotine and tested with saline (Nic–Sal) and (4) rats pretreated with nicotine and tested with nicotine (Nic–Nic). See Table 1 for the layout of the different groups.

3. Results 3.1. Experiment 1 – cross-sensitization to D-amphetamine The ability of nicotine to produce cross-sensitization to damphetamine was dependent upon environmental enrichment and the dose of d-amphetamine tested in adulthood. A mixed factor ANOVA revealed that there were main effects of d-amphetamine test dose [F(1,51) = 4.03, p ≤ 0.05] and d-amphetamine vs. saline at test day [F(1,51) = 28.48, p ≤ 0.001]. There were also significant twoway interactions of d-amphetamine test dose (0.5 vs. 1.0 mg/kg) by environment [F(1,51) = 12.26, p ≤ 0.001], and nicotine exposure by d-amphetamine vs. saline at test day [F(1,51) = 7.276, p ≤ 0.01]. Most importantly, we found a significant four-way interaction of d-amphetamine test dose (0.5 vs. 1.0 mg/kg) × environment x nicotine exposure × d-amphetamine vs. saline at test day [F(1,51) = 10.221, p ≤ 0.01]. In order to probe this four-way interaction, the results are graphed according to d-amphetamine test dose as seen in Fig. 1A and B. At the 0.5 mg/kg test dose of d-amphetamine (Fig. 1A), post hoc analysis revealed that only the IC rats showed nicotine crosssensitization. This was evident by the increase in locomotor activity by the Nic–Amp IC rats compared to all other groups (including EC rats) and in particular to the IC Sal–Amp controls. The Sal–Amp IC rats were significantly different from the Nic–Sal IC rats but

7500

Total Beam Breaks

2.6.2. Experiment 1. d-Amphetamine cross-sensitization testing. Tests for crosssensitization to d-amphetamine were conducted following the 35-day washout period with both the EC and IC nicotine- and saline-pretreated rats. Half of the animals from each pretreatment group were injected with d-amphetamine, while the other half received saline injections. In order to test more than one dose of damphetamine, two injections of d-amphetamine were done across two locomotor sessions (PND 69 and 72). These two sessions were separated by two days in order to alleviate concerns with tolerance or repeated d-amphetamine injections. The order of d-amphetamine dose (0.5 and 1.0 mg/kg) was counterbalanced so that half of the d-amphetamine tested animals received 0.5 mg/kg dose first, while the other half received 1.0 mg/kg dose first. The animals tested only with saline were also tested across two different locomotor sessions (PND 69 and 72), receiving saline injections prior to both sessions. Rats were given their respective injections and immediately placed in locomotor chambers for 45 min sessions. Once the locomotor session finished, animals were returned to their home cages. All behavioral tests were done during the animals’ light phase. This design results in four groups for both the EC and IC rats; (1) rats pretreated with saline and tested with saline (Sal–Sal), (2) rats pretreated with saline and tested with amphetamine (Sal–Amp), (3) rats pretreated with nicotine and tested with saline (Nic–Sal) and (4) rats pretreated with nicotine and tested with amphetamine (Nic–Amp). See Table 1 for the layout of the different groups.

0.5 mg/kg Amp

A 6000 4500

*

#

*

*

*

3000 1500 0

EC

IC

1.0 mg/kg Amp

#

7500

Total Beam Breaks

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 given a 35-day washout period during which they were maintained in their respective environments until sensitization testing was started on PND 69. Thirty-two rats were used in Experiment 2. Animals in this experiment were exposed to nicotine or saline as previously described in Experiment 1.

249

6000 4500

*

*

Sal-Sal Sal-Amp Nic-Sal Nic-Amp

*

3000 1500 0

EC

IC

Fig. 1. (A) The mean (±SEM) total beam breaks during the 45-min locomotor sessions for EC and IC rats following injection of the 0.5 mg/kg amphetamine dose (N = 7–8/group). Sal–Sal indicates that the animals were pretreated with saline during adolescence and injected with saline on test day. Sal–Amp indicates that the animals were pretreated with saline during adolescence and injected with d-amphetamine on test day. Nic–Sal indicates that the animals were pretreated with nicotine (0.4 mg/kg) during adolescence and injected with saline on test day. Nic–Amp indicates that the animals were pretreated with nicotine during adolescence and injected with d-amphetamine on test day. *Indicates a significant difference between groups (p ≤ 0.05). #Indicates a significant difference between indicated group and all other EC and IC groups (p ≤ 0.05). (B) The mean (±SEM) total beam breaks during the 45-min locomotor sessions for EC and IC rats following injection of the 1.0 mg/kg amphetamine dose (N = 7–8/group). The groups are the same as described in Fig. 1A. *Indicates a significant difference between groups (p ≤ 0.05). #Indicates a significant difference between indicated group and all other EC and IC groups (p ≤ 0.05).

not the Sal–Sal IC rats. Finally, the nicotine pretreatment in IC rats appeared to blunt locomotor behavior following saline injections in adulthood, as the Nic–Sal group was significantly lower than the Sal–Sal group. In terms of the EC rats, the Nic–Amp group did not show cross-sensitization at the 0.5 mg/kg dose of amphetamine compared to the Sal-Amp controls, the Nic-Amp groups were significantly higher when compared to the EC Nic–Sal and Sal–Sal groups. At the 1.0 mg/kg test dose of d-amphetamine (Fig. 1B), post hoc analysis revealed that the Nic-Amp EC rats had significantly higher levels of locomotor activity than the Sal–Amp EC controls. This was evident by the increase in locomotor activity by the Nic–Amp EC rats compared to all other groups (including IC rats) and in particular to their Sal–Amp controls. The EC Sal–Amp rats were also significantly higher levels of locomotor behavior compared to the Nic–Sal EC rats, although there were no differences between the EC Sal–Amp rats vs. the EC Sal–Sal rats. In terms of IC rats at

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Table 1 The timing and breakdown of the various groups for both Experiments 1 and 2. Environment (PND 21–72)

EC n = 47

Pretreatment (PND 28–34)

NIC n = 24

IC n = 48

Experiment (Ex) 1 or 2 Locomotor Test days

Ex 1 AMP

Ex 2 NIC

Ex 1&2 SAL

Ex 1 AMP

Ex 2 NIC8

Ex 1&2 SAL

Ex 1 AMP

Ex 2 NIC

Ex 1&2 SAL

Ex 1 AMP

Ex 2 NIC

Ex 1&2 SAL

(PND 69 & 72)

n=8

n=8

n=8

n=7

n=8

n=8

n=8

n=8

n=8

n=8

n=8

n=8

SAL n = 23

The ability of adolescent nicotine exposure to produce nicotine sensitization in adulthood was also dependent on environmental enrichment and nicotine test dose. A mixed factor ANOVA revealed only a significant three-way interaction of nicotine test dose x environment x nicotine exposure [F(1,52) = 12.168, p ≤ 0.001]. Again, in order to probe this interaction the results were graphed by nicotine test dose, as seen in Fig. 2A and B. At the 0.2 mg/kg test dose of nicotine (Fig. 2A), post hoc analysis revealed that neither EC nor IC animals showed nicotine sensitization or significant differences between any groups. At the 0.4 mg/kg test dose of nicotine (Fig. 2B) only IC animals displayed nicotine sensitization. This is evident by the increase in locomotor activity in the Nic–Nic IC rats compared to the Sal–Nic controls. The IC Nic–Nic group was also significantly higher than the EC Nic–Nic group. 4. Discussion The results taken together indicate that environmental enrichment can protect against the ability of adolescent nicotine exposure to result in a sensitized response to d-amphetamine or nicotine in adulthood. This protectant effect of enrichment in crosssensitization to d-amphetamine may only occur when the dose of d-amphetamine is low: nicotine-pretreated IC rats showed crosssensitization to the low dose of d-amphetamine while EC rats did not. However, when the higher dose of d-amphetamine was tested, nicotine-pretreated EC rats displayed cross-sensitization to the high dose of d-amphetamine. Although it seems the protectant effect of enrichment in crosssensitization may be restricted to low doses of d-amphetamine, the protectant effect of enrichment was evident in both doses of nicotine tested in the current study. Specifically, in Experiment 2 we found nicotine-pretreated EC rats did not show sensitization when tested with either the low or the high dose of nicotine in adulthood, while nicotine-pretreated IC rats had a sensitized response to the high dose of nicotine relative to their saline-pretreated counterparts. The results of our study are congruent with previous studies investigating nicotine cross-sensitization with other psychostimulant drugs. For example, Santos et al. (2009) found cross-sensitization in adolescent rats pretreated with 0.4 mg/kg nicotine and tested with 1.0 mg/kg d-amphetamine in adulthood. The current study extended these findings, showing that enrichment can alter this effect, in that IC rats displayed crosssensitization at a lower dose of d-amphetamine, while EC rats did not display sensitization until the 1.0 mg/kg dose. The ability of environmental enrichment to alter cross-sensitization to low doses of amphetamine is consistent with the effects of enrichment on low dose amphetamine self-administration (Bardo et al., 2001;

0.2 mg/kg Nic Total Beam Breaks

3.2. Experiment 2 – nicotine sensitization

SAL n = 24

Green et al., 2003). One interesting finding in the current study was that IC rats did not show a typical dose-dependent effect in d-amphetamine cross-sensitization. The lack of cross-sensitization in IC rats with the 1.0 mg/kg dose of d-amphetamine may be due in part to IC rats being more sensitive to d-amphetamine-induced stereotypy. Previous research investigating the effect of barren and restrictive caging in mice, caging that would be analogous to our IC condition, has shown that these animals display significantly higher levels of stereotypy when compared to enriched mice (Gross et al., 2012). In addition, unpublished studies in our laboratory suggest IC rats, when compared to EC rats, display more intense d-amphetamine-induced stereotypy at higher doses, which competes with horizontal activity. The increased likelihood

5000 4000

Sal-Sal Sal-Nic Nic-Sal Nic-Nic

3000 2000 1000 0

EC

IC

0.4 mg/kg Nic Total Beam Breaks

the 1.0 mg/kg dose of d-amphetamine, the Nic-Amp IC rats were not significantly different from their Sal–Amp controls. However, the Sal–Amp IC rats were significantly more active than both the Nic–Sal and Sal–Sal controls.

NIC n = 24

5000

*

4000

Δ

3000 2000 1000 0

EC

IC

Fig. 2. (A) The mean (±SEM) total beam breaks during the 45-min locomotor sessions for EC and IC rats following injection of the 0.2 mg/kg nicotine dose (N = 7–8/group). Sal–Sal indicates that the animals were pretreated with saline during adolescence and injected with saline on test day. Sal–Nic indicates that the animals were pretreated with saline during adolescence and injected with nicotine on test day. Nic–Sal indicates that the animals were pretreated with nicotine (0.4 mg/kg) during adolescence and injected with saline on test day. Nic–Nic indicates that the animals were pretreated with nicotine during adolescence and injected with nicotine on test day. (B) The mean (±SEM) total beam breaks during the 45-min locomotor sessions for EC and IC rats following injection of the 0.4 mg/kg nicotine dose (N = 7–8/group). The groups are the same as described in Fig. 2A. *Indicates a significant difference between groups (p ≤ 0.05).  Indicates a significant difference between the corresponding EC group (p ≤ 0.05).

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of stereotypy in IC rats could mask our ability to find locomotor sensitization in this group of animals when testing the higher doses of d-amphetamine. Finally, the current findings are also congruent with previous studies showing adolescent nicotine exposure results in cross-sensitization to both d-amphetamine in adult male rats (Collins et al., 2004) as well as cocaine (Collins and Izenwasser, 2004). In terms of replicating the effects of environmental enrichment on locomotor behavior found by previous studies (Bardo et al., 1995; Bowling and Bardo, 1994; Bowling et al., 1993; Smith et al., 2009) Experiment 1 fails to replicate all of the previous effects of enrichment on locomotor behavior. One of the more surprising findings of the current study was the lack of a baseline difference in locomotor response between EC and IC in our Sal–Sal groups. While it is fairly well established that IC rats have a higher baseline level of locomotor behavior compared to EC rats (Bardo et al., 1995; Bowling et al., 1993); although see Bowling and Bardo, 1994 for an exception) our study failed to replicate this effect. While we failed to find a statistically significant difference between the EC and IC Sal–Sal groups there was a trend in IC rats having a higher baseline locomotor response compared to EC rats. One potential explanation for the lack of a statistically significant effect could have been due to an increase in variability in the EC Sal–Sal group due one of the EC rat having a higher than normal response to a saline injection. The increase in variability in the EC Sal–Sal could also account for the lack of a significant increase in the response to acute amphetamine (Sal–Amp vs. Sal–Sal) in EC rats. While the lack of a baseline differences in EC and IC rats could be due to statistical variability, we cannot rule out the possibility that the increased handling of the IC rats during development due to injections could have resulted in a lessening of our isolated condition that could have affected their baseline levels of locomotor behavior. The effects of handling in disrupting the baseline behavior in our EC and IC groups given a recent study found that brief handling can partially alter the effects of social isolation (Pritchard et al., 2013). Future studies in our lab using the current nicotine exposure regimen will allow us to determine if this dosing procedure “compromises” the effects of our environmental conditions. While the current study appears to fail to replicate the finding that EC rats are more sensitive to the acute effects of d-amphetamine compared to IC rats, which some previous studies have found (Bowling and Bardo, 1994; Bowling et al., 1993), the data on the acute locomotor effects of psychostimulants in EC and IC rats appears to be more mixed than consistent. For instance, Bardo et al. (1995) found no difference in raw line crosses between EC and IC rats following 1.0 mg/kg amphetamine which would be congruent with the current study. In addition, there was no effect of environmental enrichment in the locomotor response to a low dose of methamphetamine in a recent study by Wooters et al. (2011). How the locomotor data are displayed may be an important aspect that needs to be considered with interpreting the effect of enrichment on acute exposure to psychostimulants. For example, Smith et al. (2009) found that data transformed to percent change from baseline in EC and IC rats can alter the interpretation of the effects of stimulants. In the same study, when raw data are presented, IC rats showed a greater locomotor response compared to EC rats across two doses of cocaine (Smith et al., 2009). Given the inconsistencies in the effects of environmental enrichment in the acute locomotor response to psychostimulants, it may not be surprising that we failed to see a significant difference between our EC and IC Sal–Amp groups. The results from Experiment 2 are congruent with some results from previous literature and incongruent with others. These mixed results may be due to procedural differences across the different studies. For instance, when nicotine sensitization is defined as an increase in locomotor behavior across sessions of nicotine

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exposure, adolescent male rats do not show nicotine sensitization (Collins and Izenwasser, 2004; Collins et al., 2004), which would be incongruent with our finding that IC rats showed nicotine sensitization following the 0.4 mg/kg nicotine challenge dose. Conversely, when nicotine sensitization is defined as administration of a nicotine challenge dose in adulthood following a washout period (Faraday et al., 2003), we found congruent results with our IC rats. This latter point highlights the importance of a washout period between induction and expression of nicotine sensitization and is consistent with research investigating the neurochemical changes that take place during sensitization to psychostimulants (Vanderschuren and Kalivas, 2000; Vezina, 2004; Vezina et al., 2007). Various other differences between the current study and the two studies by Collins et al. could explain the incongruent results, such as, age of the animals or even the differences in housing due to the environmental enrichment manipulation. In terms of the effects of environmental enrichment on nicotine sensitization, the current study extends the findings that enrichment can alter the ability of nicotine to result in locomotor sensitization. Just as a previous study by Green et al. (2003) found that adult EC rats showed less locomotor sensitization than IC rats, the current study, using both a different sensitization procedure and nicotine doses, found that EC rats exposed to nicotine during adolescence do not display locomotor sensitization at the two nicotine doses tested. This protectant effect of enrichment is also consistent with Coolon and Cain (2009), which found EC rats to exhibit less nicotine-induced conditioned hyperactivity than IC rats. The lack of an acute locomotor response following the 0.2 mg/kg dose of nicotine in either EC or IC rats (Sal–Nic vs. Sal–Sal) in the current study is consistent with Green et al. (2003). In addition, the lack of an effect of enrichment on the acute effects of nicotine (Sal–Nic) is consistent with Coolon and Cain (2009) which found the EC and IC rats did not differ in their locomotor response following an acute injection of 0.4 mg/kg of nicotine. The results from the current study indicating that IC rats showed nicotine sensitization while EC rats do not at first appears to be incongruent with a recent study by Gomez et al. (2012) which reported EC rats to have a greater locomotor sensitization follow repeated nicotine. Although comparisons between the two studies are difficult considering the Gomez et al. (2012) study did not begin nicotine pretreatments until the animals were adults. More importantly, Gomez et al. (2012) discusses how EC rats appeared to be more sensitive to nicotine locomotor sensitization when the data are presented as a percent change from saline controls, but IC rats appeared more sensitive when raw locomotor data were examined. The protectant effects of enrichment on both nicotine sensitization and cross-sensitization to d-amphetamine, found in the current study, most likely take place through different neural mechanisms. For instance, nicotine exposure during adolescence has been shown to result in long-lasting up regulation of nicotinic cholinergic receptors (nAChR) in midbrain areas (Trauth et al., 1999), which are believed to be responsible for the sensitized locomotor response to nicotine (Ksir et al., 1987). Our lab, using the current enrichment paradigm, has unpublished autoradiography data using [125 I]-epibatidine binding which indicate EC rats have a lower density of nAChR in the ventral tegmental area (VTA) compared to their IC counterparts. Given this data, perhaps EC rats do not show nicotine sensitization because the enriched environment alters the expression nAChR in the VTA decreasing the amount of dopamine (DA) release in the nucleus accumbens (NAcc), which has been implicated in nicotine-induced locomotor sensitization (Birrell and Balfour, 1998). While the protectant effect of enrichment for nicotine sensitization may involve changes in nAChRs and DA levels in the striatum, the decreased sensitivity to d-amphetamine in EC rats may involve alterations in response to DA in the prefrontal cortex

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(PFC). Previous studies have shown that the ability of nicotine to produce a cross-sensitized locomotor response to d-amphetamine is not dependent on DA overflow in the NAcc (Birrell and Balfour, 1998). The ability of nicotine to result in a sensitized locomotor response to d-amphetamine may involve the PFC since previous research has shown that repeated nicotine exposure can alter d-amphetamine-induced DA release by altering dopamine transporter (DAT) function in the PFC (Drew et al., 2000; Drew and Werling, 2001, 2003). Enrichment may protect against nicotine cross-sensitization to d-amphetamine by altering the DAT response in the PFC. This hypothesis is supported by previous research which shows that EC rats have differential DAT levels in the PFC at baseline compared to IC rats (Zhu et al., 2005), and also an increase in DAT function specific to the PFC following acute nicotine exposure (Zhu et al., 2007). Finally, these changes in the functioning of the mesocorticolimbic system caused by enrichment may take place through changes in the phosphorylation of DARPP-32 and CREB in the PFC; given the recent finding that EC rats show greater phosphorylation of these proteins following repeated nicotine pretreatments compared to IC rats (Gomez et al., 2012). More studies will be required to uncover the neural mechanisms responsible for the protectant effects of enrichment against adolescent nicotine exposure. Although future studies will help us gain a better understanding of the underlying neural mechanisms for this effect of environmental enrichment, the current behavioral studies may have important clinical implications. The results from the current experiments indicate that environmental enrichment during adolescence not only alters future drug abuse vulnerability (Stairs and Bardo, 2009), but may also alter or block the ability of adolescent nicotine exposure to predispose individuals to a long-term use of psychostimulants. The use of an environmental enrichment manipulation once a young individual has exposed himself or herself to nicotine may allow for the prevention of future or long-term psychostimulant use.

Role for funding source Funding was kindly provided by Creighton University College of Arts and Sciences. The funding source had no involvement in study design, collection, analysis and interpretation of the data or writing of the reports. Contributors If allowed by the journal both Jenna Klug and Emily Adams should be considered first authors on the manuscript as they both put in equal effort in writing the manuscript and data collection, analysis and interpretation of the data. Michaela Quast was involved in data collection and assisted in writing of the manuscript. The data were collected in the research laboratory of Dustin J. Stairs who was involved in the design of these studies, data analysis, data interpretation and assisted the first authors in the writing of the manuscripts. All authors have contributed to and approved the final manuscript. Conflict of interests The authors of the manuscript have no financial, personal or other relationships with people or organizations with could result in a conflict of interests. Acknowledgements We have no further acknowledgments.

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