Behav Brain Res. Author manuscript; available in PMC 2015 Jun 5.
Published in final edited form as:
Behav Brain Res. 2014 Apr 1; 262: 101–108.
Published online 2014 Jan 7. doi: 10.1016/j.bbr.2013.12.014
This study determined the effects of adolescent nicotine administration on adult alcohol preference in rats exhibiting high or low behavioral reactivity to a novel environment, and ascertained whether nicotine altered ΔFosB in the ventral striatum (vStr) and prefrontal cortex (PFC) immediately after drug administration or after rats matured to adulthood.
Animals were characterized as exhibiting high (HLA) or low (LLA) locomotor activity in the novel open field on postnatal day (PND) 31 and received injections of saline (0.9%) or nicotine (0.56 mg free base/kg) from PND 35–42. Ethanol-induced conditioned place preference (CPP) was assessed on PND 68 following 8 days conditioning in a biased paradigm; ΔFosB was measured on PND 43 or PND 68. Following adolescent nicotine exposure, HLA animals demonstrated a CPP when conditioned with ethanol; LLA animals were unaffected. Further, adolescent nicotine exposure for 8 days increased levels of ΔFosB in limbic regions in both HLA and LLA rats, but this increase persisted into adulthood only in LLA animals.
Results indicate that adolescent nicotine exposure facilitates the establishment of an ethanol CPP in HLA rats, and that sustained elevations in ΔFosB are not necessary or sufficient for the establishment of an ethanol CPP in adulthood. These studies underscore the importance of assessing behavioral phenotype when determining the behavioral and cellular effects of adolescent nicotine exposure.
Numerous studies have indicated that high novelty-seeking and exploration are associated with increased sensitivity to drug reward [1–8]. Adolescents have been shown to exhibit greater novelty-seeking and exploration than adults [9–11], and several reports demonstrate that adolescents are more likely than adults to progress to addiction when initiating drug use [12–18]. Thus, adolescents may be more susceptible to the reinforcing and rewarding effects of abused drugs, and adolescents with a high sensation-seeking profile may represent the most vulnerable population.
The two drugs most commonly used by adolescents are nicotine and alcohol [19, 20], and evidence suggests that the use of nicotine affects alcohol consumption. Smoking and drinking behaviors often occur together, with the frequency of either behavior associated with the frequency of the other . Grant  reported that nearly 29% of individuals who begin smoking before the age of 14 become alcohol dependent and 8% progress to alcohol abuse during their lifetime. Further, 19% of those who initiate smoking between 14 and 16 become alcohol dependent, with 7% of these individuals progressing to alcohol abuse. Interestingly, individuals who do not initiate smoking until 17 years of age are half as likely to become alcohol dependent or progress to addiction. Thus, early onset smoking is a strong predictor of lifetime drinking, and alcohol dependence and abuse .
Adolescent nicotine exposure has been shown to increase the rewarding effects of several drugs in adult laboratory animals, including nicotine, cocaine and diazepam [23–26]. Further, Riley et al.  demonstrated that the administration of nicotine to mice during adolescence, but not adulthood, increases sensitivity to ethanol withdrawal when measured in adulthood, and suggested that adolescence represents a critical period of sensitivity to nicotine that results in changes in the brain that persist into adulthood. This idea is supported by several studies demonstrating that adolescent exposure to nicotine leads to an anxiogenic state in adulthood [28–30]. It is possible that enduring alterations following adolescent nicotine exposure involve the transcription factor ΔFosB, which has been shown to produce persistent sensitization of the mesolimbic pathway and to heighten sensitivity to the motivational properties of several drugs of abuse, including alcohol [31–34], and whose overexpression in the limbic system enhances drug preferences [31, 35]. Interestingly, adolescent animals exhibit greater increases than adults in ΔFosB in the nucleus accumbens (NAcc) in response to the administration of cocaine or amphetamine ; the effect of nicotine administration during adolescence on ΔFosB has not been examined. Because adolescent animals exhibit enhanced up regulation of ΔFosB relative to adults in response to abused drugs, they may be more sensitive to rewarding stimuli following repeated exposure than similarly exposed adults. This idea is supported by studies indicating that adolescent rats that establish a nicotine-induced conditioned place preference (CPP) following 4 injections exhibit an increase in FosB immunoreactivity (the ΔFosB splice variant was not specifically measured) in the ventral tegmental area (VTA), NAcc and prefrontal cortex (PFC) immediately following behavioral testing .
Despite evidence that adolescence is a period of increased sensation-seeking and first time drug use, that nicotine use is linked with increased ethanol use, and that an increased sensitivity to drugs of abuse is associated with ΔFosB accumulation , the impact of adolescent nicotine exposure on ΔFosB levels and its long term consequences on ethanol reward are unclear. Therefore, this study: 1) determined the effects of adolescent nicotine administration on adult alcohol preference in rats characterized during adolescence by their behavioral reactivity to a novel environment, viz., exhibiting high or low locomotor activity; and 2) ascertained whether nicotine altered ΔFosB in the ventral striatum (vStr) and PFC of these animals immediately after administration in adolescence or after rats matured to adulthood.
Ethanol was obtained from AAPER Alcohol and Chemical Company (Shelbyville, KY). All other reagents were purchased from Sigma-Aldrich Life Sciences (St. Louis, MO) unless otherwise noted.
The male and female offspring (n=89) of timed pregnant rats (n=10) were used as subjects; the day of birth was defined as postnatal day 0 (PND 0). To assure similar development across litters, all litters were culled to 10–12 pups (5–6 males/5–6 females) on PND 1, and remained housed with their respective dams until PND 21, at which time animals were weaned and housed in same sex groups of 3 in standard polypropylene cages with corncob bedding. All animals were housed at the University of South Florida in a temperature and humidity-controlled vivarium on a 12:12-hr light–dark cycle (7 a.m./7 p.m.). Experiments were conducted during the light phase, and the care and use of animals was in accordance with guidelines set by the Institutional Animal Care and Use Committee and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. In accordance with these guidelines, experiments utilized the fewest number of animals per group necessary to obtain meaningful data.
2.3 Characterization of Behavioral Reactivity to a Novel Environment
Locomotor activity was used to characterize the behavioral reactivity of rats to a novel environment. To accomplish this, on PND 31, animals were removed from their home cage and placed in a circular arena (100 cm diameter) under moderate illumination (20 lux) for 5 min. The total distance moved (TDM) was recorded automatically with a video camera and analyzed using EthoVision software (Noldus Information Technology, Leesburg, VA) as described . Animals were classified as exhibiting either high (HLA) or low (LLA) locomotor activity in the novel open field using a median split strategy, with the former exhibiting activity in the upper 50%, and the latter in the lower 50% relative to their littermates .
2.4 Nicotine Injections
Animals received injections (s.c.) of either phosphate-buffered saline (PBS, 0.9%), or nicotine hydrogen bitartrate in PBS (0.56 mg free base nicotine/kg) once daily for 4 or 8 days beginning on PND 35. This dose of nicotine has been demonstrated to increase responding for conditioned stimuli [39, 40] and increase breakpoints for reinforced responding  indicating that it is rewarding and reinforcing, and was used in a prior study of adolescents . For each injection, animals were transported in their home cage to a dimly lit procedure room, placed in a new cage lined with fresh bedding, injected, and returned to their home cage.
2.5 Conditioned Place Preference (CPP)
For measures of CPP, rats received injections of nicotine from PND 35–42 and 18 days following the last injection of nicotine, on PND 60, animals (n=40; 4–5 per group) were allowed free access to two interconnected Plexiglas chambers (each chamber: 21 cm wide × 18 cm long × 21 cm high) containing distinct visual (vertical or horizontal black and white stripes) and tactile cues (rubberized or sandpaper flooring) for three 5 min intervals. The mean time spent on each side of the apparatus was used to determine baseline chamber preference for each animal. Although each animal exhibited a side preference at baseline, there was no tendency within the population for a particular chamber to be preferred. Over the next 8 days, from PND 61 to 68, a biased conditioning paradigm was used wherein animals were trained to associate the non-preferred chamber with the subjective effects of ethanol. For conditioning, each animal received an injection of ethanol (17%; 1.0 g/kg, i.p.) and was subsequently confined to the initially non-preferred chamber for 15 min. This dose and concentration of ethanol has been shown to establish a CPP during late adolescence  and to significantly elevate dopamine in the NAcc of adolescent and young adult animals [43, 44]. Control animals were confined for 15 min to the initially non-preferred chamber following an injection of saline (0.9%, i.p.). Both ethanol-conditioned and control animals received saline injections prior to being confined to the initially preferred chamber for 15 min each day. Thus, each animal received 2 training sessions per day, one for the initially non-preferred and one for the preferred chamber. The order of these sessions was alternated on each day and occurred in the morning and afternoon, separated by at least 5 hours. On PND 69, approximately 16–18 hours after the last training session, animals were allowed free access to both chambers for 5 min and the time spent in each chamber was measured to assess the CPP. A preference score was calculated by subtracting the time spent in the initially preferred chamber from the time spent in the initially non-preferred chamber.
2.6 Western Blot Analyses
For immunoblot analyses, rats were decapitated rapidly and the vStr and PFC isolated 24 hrs after either the 4th or 8th nicotine injection on PND 39 or 43, respectively, (n=32; 4 per group) or 26 days following the 8th injection on PND 69 (n=16; 4 per group), corresponding to the day that CPP was assessed in a separate group of animals. Tissue was quick frozen on dry ice and stored at −80°C until homogenized as described . Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (10% polyacrylamide) and transferred electrophoretically to polyvinylidene fluoride membranes. The membranes were blocked for 1 hour in Tris-buffered saline containing 0.1% Tween 20 and 5% nonfat dry milk. Subsequently, primary antibody [FosB (5G4) #2251, 1:4000; Cell Signaling, Danvers, MA], which produces robust labeling of ΔFosB , was added in blocking solution and the membranes were incubated overnight at 4°C. Sixteen hours later, the membranes were washed and incubated with secondary antibody [goat anti-rabbit IgG-HRP, 1:2000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA] in blocking solution for 1 hour at room temperature, and signals visualized using enhanced chemiluminescence. After immunodetection, blots were stripped, blocked and incubated with a primary antibody directed against β-tubulin [H-235, Santa Cruz Biotechnology, Inc., 1:16,000] as a loading control. The 35/37 kDa band representing ΔFosB and the 50 kDa band corresponding to β-tubulin were quantified on each blot using a densitometer and Un-Scan-It gel digitizing software (Silk Scientific Inc., Orem, Utah). The optical density of the former was normalized to the latter for each sample, and results are expressed as percent of corresponding saline controls on each blot to eliminate variability across blots.
2.7 Statistical Analyses
A 4 factor analysis of variance (ANOVA) was used to determine effects on CPP [(male or female) × (HLA or LLA) × (saline or nicotine exposure) × (saline or ethanol conditioning)] and Tukey’s test was used post hoc to ascertain significant differences between groups. A 3 factor ANOVA was used to determine differences in ΔFosB between male and female HLA and LLA animals [(male or female) × (HLA or LLA) × (saline or nicotine)] with the Student’s t-test performed post hoc to ascertain significant differences between groups. A level of p<0.05 was accepted as evidence of a significant effect. Because the sample size in these studies was small, leading to reduced statistical power, effect size
3.1 Behavioral Reactivity to a Novel Environment
The locomotor activity exhibited by adolescent rats in a novel open field for 5 min is shown in Figure 1. The TDM was normally distributed (Kolmogorov-Smirnov D = 0.083, p > 0.05), with animals exhibiting a range of movement between 4339 and 7739 cm/5 min. The median TDM was 5936 cm/5 min with one animal at the median (shown in the grey circle), which was removed from further study. The TDM for HLA and LLA groups was significantly different [t(86) = 12.15, p<0.05; Cohen’s D =2.56] with a TDM of 6621 TDM ± 71 cm/5 min for HLA animals and 5499 ± 59 cm/5 min for LLA animals. Animals were systematically assigned to experimental groups according to behavioral reactivity to the novel environment to ensure that all groups exhibited equivalence in novel open field activity, and contained equal numbers of HLA and LLA animals (Table 1). Further, no more than 1 male and 1 female from a given litter were assigned to each group.
3.2 Ethanol CPP in Adulthood Following Nicotine Exposure During Adolescence
The first set of experiments determined whether nicotine exposure during adolescence increased vulnerability to the rewarding effects of alcohol in adulthood, and ascertained whether responses were dependent on the behavioral reactivity of the rats to a novel environment. Following classification of rats as HLA or LLA, animals received injections of saline or nicotine from PND 35–42, and CPP to ethanol was determined when rats were young adults on PND 69. Results are shown in Figure 2. ANOVA indicated a significant 3-way interaction among novel open field activity (HLA or LLA), nicotine exposure, and ethanol conditioning [F (1,19) = 5.165, p < 0.05], with an observed power of 0.578 and an estimated effect size
3.3 ΔFosB in Adolescence During Repeated Nicotine Exposure
Because increases in ΔFosB in limbic structures enhance drug preference [15,16], experiments determined whether adolescent nicotine exposure had a differential effect on levels of this transcription factor in vStr and PFC from HLA and LLA rats. Following behavioral classification, male and female rats received injections of either saline or nicotine for 4 or 8 days beginning on PND 35. Brain samples were isolated 24 hours after the final injection on PND 39 or 43, respectively, and subjected to Western immunoblot analyses. Results of ΔFosB measurements in the vStr (Figure 3) indicated a significant main effect of both the number of days of injections [F(1, 16) = 4.542, p<0.05;
3.4 ΔFosB in Adulthood Following Nicotine Exposure During Adolescence
To determine whether the nicotine-induced elevations in ΔFosB observed in adolescence persisted through young adulthood, following the behavioral classification of rats, animals received injections of saline or nicotine for 8 days from PND 35–42, and 27 days later, on PND 69, the vStr and PFC were isolated and ΔFosB quantified. Results of ΔFosB measurements in the vStr (Figure 4) indicated a significant main effect of both phenotype [F(1, 16) = 14.349, p< 0.05;
The present study demonstrates that exposure to nicotine during adolescence has differential effects on ethanol CPP and alterations in ΔFosB in limbic regions from rats with different behavioral reactivities to a novel environment. Adolescent nicotine exposure facilitated the establishment of an ethanol CPP in adulthood only in animals who exhibited high locomotor activity in the novel environment in adolescence. Further, although adolescent nicotine exposure increased levels of ΔFosB in the vStr and PFC following 8 days of administration, this increase persisted into adulthood only in animals who exhibited low locomotor activity in a novel environment.
Thus, results indicate that the effects of adolescent nicotine exposure on ethanol CPP in adulthood depend on the behavioral phenotype of the animals, and suggest that sustained elevations in ΔFosB in limbic regions are not necessary or sufficient to facilitate an ethanol CPP in adulthood.
The finding that adolescent nicotine exposure facilitates a CPP to ethanol in adulthood in HLA animals agrees with findings that individuals with increased behavioral reactivity to novel stimuli exhibit a greater sensitivity to the rewarding effects of abused compounds than individuals with lower reactivity [1–8]. However, it should be noted that a CPP can be produced by the reinforcement of specific behaviors during conditioning or result from conditioned drug effects , and thus, caution should be used when interpreting CPP results as indicative of heightened drug reward. Indeed, Smith et al.  did not observe increased ethanol intake in adult Sprague-Dawley rats following adolescent nicotine exposure, suggesting that the rewarding properties of ethanol were not changed by prior experience with nicotine. However, these authors used a continuous exposure paradigm over 21 days and did not distinguish animals based on locomotor activity in a novel environment. The results of the present study suggest that the consequences of daily injections of nicotine may differ from those produced by continuous nicotine exposure and demonstrate the importance of distinguishing between HLA and LLA rats, a distinction that may be particularly important when studying adolescents. Although many investigators have reported that the adolescent population may be more sensitive to the rewarding and reinforcing effects of drugs [49–51], this observation likely reflects the developmental tendency for adolescents to possess characteristics of HLA animals . Indeed, studies in the human population have demonstrated that sensation-seeking peaks during adolescence and declines thereafter, with those maintaining adolescent-like sensation-seeking most likely to escalate alcohol use .
Results indicating a differential effect of adolescent nicotine exposure on ΔFosB in brain from HLA and LLA rats underscore inherent differences between these groups of animals. Results show a clear increase in ΔFosB levels in the vStr and PFC from both groups of rats following 8 days of adolescent nicotine exposure, but this effect persisted into adulthood only in brain from LLA rats. Soderstrom et al.  demonstrated that 10 days of nicotine exposure (0.4 mg/kg, i.p.) from PND 34–43 increased FosB immunoreactivity in the NAcc at 37 days following the last nicotine injection, but these authors did not specifically measure ΔFosB or characterize the behavioral phenotype of the animals. Results indicating that prolonged elevations in ΔFosB following adolescent nicotine exposure occur only in LLA adolescents suggest that LLA adolescents are more “adult-like” than their HLA counterparts. Indeed, a prolonged elevation of ΔFosB following drug administration has been demonstrated repeatedly in adult animals [31, 33, 34].
It was expected that HLA animals exposed to nicotine during adolescence would demonstrate both an ethanol-induced CPP in adulthood and a sustained elevation of ΔFosB that presumably sensitized the reward pathways. However, results indicate that persistent elevations in ΔFosB following adolescent nicotine exposure are neither necessary nor sufficient for the establishment of an ethanol CPP in adulthood. Because the biased CPP paradigm used in this study is sensitive to the anxiolytic effects of ethanol [54, 55], the ethanol-induced CPP observed following adolescent nicotine exposure may be mediated by changes in sensitivity to ethanol’s anxiolytic effects, rather than the result of a sensitized reward pathway. Adult animals exposed to nicotine during adolescence exhibit increased sensitivity to stress and anxiety in adulthood, as evidenced by elevated corticosterone , decreased exploration of the novel open field and decreased time in the open arms of the elevated plus maze [29, 30]. Thus, it seems likely that adult animals exposed to nicotine as adolescents may exhibit an ethanol CPP in a biased paradigm as a consequence of the anxiolytic properties of ethanol. Interestingly, animals exhibiting elevated ΔFosB expression may be less sensitive to stress and anxiety as indicated by increased time spent in the open arms of the elevated plus maze , increase swim time in the Porsolt forced swim test , increased resilience following social defeat stress  and a diminished corticosterone response to restraint stress . Thus, nicotine exposed LLA animals, who exhibit sustained ΔFosB expression as adults, may not find the anxiolytic effects of ethanol rewarding, and as a consequence, fail to exhibit a CPP in the biased paradigm. Indeed, ethanol-injected LLA animals exhibited a large reduction (D = 0.80) in time spent on the ethanol-paired side when compared to saline-injected LLA animals, suggestive of an ethanol-induced conditioned place aversion. Further studies are necessary to confirm differences between HLA and LLA animals in anxious behavior and stress sensitivity following adolescent nicotine exposure.
Although no statistically significant differences were observed between male and female animals, some moderate to large sex-related effects were present. ΔFosB measurements in the PFC were about 25% lower in male adolescents than their female counterparts after 4 saline injections, and about 19% higher in male than female adolescents following 4 nicotine injections, suggesting that adolescent males may exhibit an increase in ΔFosB following fewer exposures to nicotine than adolescent females. Additionally, ΔFosB measurements were 15–17% higher in the vStr and PFC of adult males than observed in adult females regardless of whether these animals were exposed to saline or nicotine as adolescents. The latter finding is consistent with a report demonstrating that adult males exhibit slightly higher levels of ΔFosB in the nucleus accumbens core and shell regions than their female counterparts and that this difference is present in animals injected with either saline or cocaine (15 mg/kg) for 2 weeks indicating that this difference is independent of drug exposure . To our knowledge, no studies of adolescent or adult animals have examined sex differences in ΔFosB expression following nicotine exposure; these findings warrant further investigation.
In sum, adolescent animals demonstrating differences in behavioral reactivity to a novel environment also exhibit differences in: 1) the long term consequences of nicotine exposure on sensitivity to ethanol’s effects in adulthood; 2) the induction of ΔFosB during repeated exposure to nicotine; and 3) the persistence of ΔFosB following repeated nicotine exposure. These findings provide a foundation for investigating differences in the inherent vulnerabilities of adolescent animals, characteristics that can be screened using relatively simple behavioral measures.
Adolescent nicotine exposure results in an alcohol CPP in high sensation seeking adults
Adolescent nicotine exposure increases ΔFosB expression
ΔFosB expression following adolescent nicotine persists into adulthood in low sensation seekers
Research was supported by the State of Florida and NIAAA of the National Institutes of Health under award number F32AA016449. The content is solely the responsibility of the authors and does not necessarily represent the official views of the State of Florida or National Institutes of Health.
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