Exposure to fruit-flavoring during adolescence increases nicotine consumption and promotes dose escalation

Abstract

The rise of e-cigarette popularity has sparked interest in the role of palatable flavors on nicotine use. Despite growing evidence that sweet flavorants enhance nicotine reward, their influence on nicotine consumption has not been studied extensively. In addition, the impact that flavored nicotine use in adolescence could have on nicotine reward and dependence in adulthood remains unclear. This study examined the role of flavored nicotine access on nicotine preference and consumption longitudinally, from adolescence to adulthood. Male and female adolescent mice preferred a fruit-flavored nicotine solution over an unflavored nicotine solution. However, only adolescent female mice with access to flavored nicotine consumed higher doses. Furthermore, while adolescent male mice escalated consumption of both flavored and unflavored nicotine, female mice only escalated nicotine consumption when given access to flavored nicotine. As mice matured into adulthood, there was no evidence that a history of flavored-nicotine access altered preference for unflavored nicotine compared to a nicotine-free control in a classic two-bottle choice design. However, when the nicotine concentration was progressively reduced, mice that had consumed strawberry-flavored nicotine in adolescence maintained baseline nicotine consumption levels longer than mice that initiated nicotine use without flavor in adolescence. Finally, addition of fruit-flavorants into the nicotine solution during adulthood led to nicotine preference and increased levels of nicotine consumption, regardless of previous flavored-nicotine access or of familiarity with the selected flavorant. These results indicate that flavorants increase nicotine consumption independent of life stage, possibly posing a disproportionate risk to adolescent females. Our results also point to an effect of adolescent flavored-nicotine use on nicotine dose maintenance in adulthood, which could have implications for the success of future quit attempts.

Keywords: Adolescence; Devaluation; Flavorants; Nicotine; Nicotine reward; Sex differences.

Figure 1.. Timeline showing the sequence of events, drug treatments, and behavioral testing.

Mice were divided into groups at two points in the experimental timeline. First, at the start of the “Limited Access (2hr) Nicotine 2 Bottle Choice (2BC) Test” mice were separated into either the “UNFLAV” or “CHOICE” groups. Data from the “Initial Strawberry Preference Test” ensured that animals in each group had approximately equal baseline strawberry preference. All mice then followed the same treatment pattern, until “Nicotine Fading and Flavor Re-introduction”, when groups were further divided into one of three nicotine flavors: No Flavor, Strawberry Flavor, or Grape Flavor.

Figure 2.. Male and female adolescent mice prefer strawberry-flavored nicotine over a nicotine-free strawberry and an unflavored nicotine solution.

A-B) Before and after plots display the individual values of preference for a bottle containing strawberry flavor only and a bottle containing nicotine + strawberry flavor in males and females, respectively (paired t-test or Wilcoxon signed rank test). C) The preferences for bottle #2 (unflavored in the UNFLAV group but strawberry-flavored in the CHOICE group) are displayed by sex and according to treatment groups. Lines and error bars represent summary data (mean +/− SEM). (Two-way ANOVA, F_sex(1,66) = 2.616, p=0.111, F_treatment(1,66) = 32.07,**** p<0.0001, F_interaction(1,66) = 3.015, p=0.0872).(Male, UNFLAV: n=18, CHOICE: n= 18; Female, UNFLAV: n=17, CHOICE: n=17).

Figure 3.. Adolescent female mice escalate nicotine consumption and consume higher doses of nicotine when given access to a strawberry flavored nicotine solution.

A,C) Line graphs display the summary data (mean +/− SEM) of the total dose consumed by adolescent mice during a 2-hour nicotine access period over the course of 2-weeks in adolescence. A line of best-fit is overlayed for each data set. The dose consumed by mice that had access to two bottles of unflavored nicotine (UNFLAV) is compared to mice with access to one bottle of unflavored nicotine and one bottle of strawberry-flavored nicotine (CHOICE). (2-way RMANOVA, followed by Dunnett’s multiple comparison relative to session 1 consumption). B,D) Points display the average nicotine consumption of each mouse during the last week of adolescence (Sessions 4–7) (Unpaired t-tests). *P < 0.05. **P < 0.01, and ****P < 0.0001 for all comparisons. (Male, UNFLAV: n=18, CHOICE: n= 18; Female, UNFLAV: n=17, CHOICE: n=17).

Figure 4.. Access to strawberry-flavored nicotine in adolescence does not increase preference for or consumption of unflavored nicotine during maturation.

Data points represent the response of individual animals and lines and error bars represent summary data (mean +/− SEM). In panel B, the inlayed graph represents an average of the volume consumed by mice of both sexes during 24-hours across all maturation drinking sessions. (Male, UNFLAV: n=18, CHOICE: n= 17; Female, UNFLAV: n=17, CHOICE: n=17).

Figure 5.. Mice that initiate nicotine use in adolescence with flavored nicotine maintain baseline levels of nicotine consumption for longer than counterparts who initiate with unflavored nicotine and are more sensitive to increased consumption of flavored nicotine in adulthood.

Data from mice that only had access to unflavored nicotine in adolescence (UNFLAV) (B,E) and mice that had access to strawberry flavored nicotine in adolescence (CHOICE) (C, F) are displayed vertically. A) In adulthood (starting at ~PND95), the concentration of nicotine was reduced sequentially, and mice were either left with an unflavored nicotine, had strawberry Kool-Aid® added to the nicotine bottle, or had Grape Kool-Aid® added to the nicotine bottle. All mice had access to a second bottle that did not contain flavor or nicotine. B-C) Line graphs display the dose of nicotine consumed (mg/kg) during the baseline nicotine consumption phase and during the nicotine fading experiment of UNFLAV and of CHOICE mice, respectively (mean +/− SEM). +P < 0.05. ++P < 0.01, and ++++P < 0.0001 represent a significant change from each group’s baseline consumption (Two-way RMANOVA, Sidak’s multiple comparisons test). D) Data displayed is the % change in nicotine consumption from baseline for each mouse when the nicotine solution contained 0.075 mg/ml nicotine. A significant increase in % change of dose consumed by animals that received flavored solutions compared to unflavored solution is indicated by **P < 0.01 near the figure legend (Two way ANOVA, Sidak’s multiple comparisons test). On the graph, ++P < 0.01 indicates a significant change in % consumed of each group from their own baseline consumption (one sample t-test or Wilcoxon test, depending on distribution of data). E-F) Line graphs display the preference for the nicotine bottle (%) during the baseline nicotine consumption phase and during the nicotine fading experiment of UNFLAV and of CHOICE mice, respectively (mean +/− SEM). Preference for the nicotine bottle in UNFLAV group depended on nicotine concentration, flavor and an interaction (Two-way ANOVA, Tukey’s test for multiple comparisons). *P<0.05 and **P<0.01. Whereas, preference for the nicotine bottle in the CHOICE group only depended on nicotine concentration (Repeated Measures Mixed Effects Model). One sample t-tests and Wilcoxon tests were used to measure either aversion to or preference for the nicotine bottle at each concentration (one sample t-tests) # P < 0.05. ## P < 0.01, and ### P < 0.001 represent a significant difference from theoretical chance (i.e., 50% preference) (UNFLAV in adolescence, ‘No Flavor’: n=12, ‘Strawberry’: n= 11, ‘Grape’: n=12; CHOICE in adolescence, ‘No Flavor’: n=10, ‘Strawberry’: n= 11, ‘Grape’: n=12).