Effects of menthol and its interaction with nicotine-conditioned cue on nicotine-seeking behavior in rats

Abstract

Rationale: Increasing clinical evidence suggests that menthol, a significant flavoring additive in tobacco products, may contribute to smoking and nicotine dependence. Relapse to smoking behavior presents a formidable challenge for the treatment of tobacco addiction. An unresolved issue is whether the mentholation of tobacco products precipitates relapse to tobacco use in abstinent smokers.

Objectives: The present study examined the effects of menthol on the perseverance and relapse of nicotine-seeking behavior in rats.

Methods: Male Sprague-Dawley rats were trained to press a lever for intravenous nicotine self-administration (0.03 mg/kg/infusion) under a fixed-ratio five schedule of reinforcement. Each nicotine infusion was signaled by the presentation of a sensory stimulus that was established as a discrete nicotine-conditioned cue. Five minutes prior to the sessions, the rats received an intraperitoneal injection of menthol (0.1 mg/kg) or vehicle. In the subsequent extinction test sessions, nicotine was unavailable with or without menthol and/or the nicotine-conditioned cue. The reinstatement tests were performed the following day after the extinction criterion was met. Menthol was also tested on food-seeking responses. In a subset of nicotine-trained rats, a transient receptor potential melastatin 8 (TRPM8) antagonist RQ-00203078 was given prior to menthol administration.

Results: Continued administration of menthol sustained responses on the previously active and nicotine-reinforced lever in the extinction tests. The readministration of menthol after extinction reinstated active lever responses. In both the extinction and the reinstatement tests, a combination of pre-session menthol administration and cue representation during the session produced a more robust behavioral effect than either menthol or the cue alone. No such effects of menthol was observed in food trained rats. RQ-00203078 did not change menthol effect on nicotine seeking.

Conclusion: These data demonstrated that menthol specifically sustained and reinstated nicotine-seeking behavior, and this effect was independent of TRPM8 activity. These findings suggest that menthol in most tobacco products, even not menthol labeled, may contribute to the perseverance of and relapse to tobacco-seeking behavior.

Keywords: Conditioned stimulus; Cue; Discriminative stimulus; Extinction; Food seeking; Menthol; Nicotine seeking; Reinstatement; Self-administration; TRPM8.

Figure 1

Top: Extinction profiles of responses on the active lever (nicotine-seeking behavior) in different test conditions in rats (n = 10/group). The animals were trained to self-administer nicotine with pre-session menthol administration. SA indicates lever responses averaged across the final three self-administration sessions. Vehicle/- represents the pre-session vehicle without in-session cue presentation condition. Menthol/- represents the pre-session menthol without in-session cue presentation condition. Vehicle/Cue represents the pre-session vehicle with in-session cue presentation condition. Menthol/Cue represents the pre-session menthol with in-session cue presentation condition. For the sake of clarity, the means are presented without the SEM. Below: The mean ± SEM number of active lever responses pooled across the final three extinction sessions. *p < 0.05, **p < 0.01, significant difference from Vehicle/- group.

Figure 2

Lever responses in the reinstatement tests in rats (n = 12) that were trained to self-administer nicotine with pre-session menthol administration. Menthol represents the pre-session menthol without in-session cue presentation condition. Cue represents the pre-session vehicle with in-session cue presentation condition. Menthol+Cue represents the pre-session menthol with in-session cue presentation condition. The data are expressed as the mean ± SEM number of lever responses. *p < 0.05, ****p < 0.0001, significant difference from extinction baseline; ⁺⁺p < 0.01, significant difference from menthol; ^{^}p < 0.05, significant difference from cue.

Figure 3

Effect of TRPM8 antagonist RQ-00203078 on menthol-induced reinstatement of nicotine-seeking responses in the rats (n = 10) trained to self-administer nicotine with pre-session menthol administration. The data are expressed as the mean ± SEM number of lever responses.

Figure 4

Lever responses in the reinstatement tests in menthol-naive rats (n = 12). Menthol represents the pre-session menthol without in-session cue presentation condition. Cue represents the pre-session vehicle with in-session cue presentation condition. Menthol+Cue represents the pre-session menthol with in-session cue presentation condition. The data are expressed as the mean ± SEM number of lever responses. **p < 0.01, significant difference from extinction or menthol condition.

Figure 5

Lever responses in the reinstatement tests in the rats (n = 10) that were trained to self-administer food pellets with pre-session administration of menthol. The data are expressed as the mean ± SEM number of lever responses. **p < 0.01, significant difference from extinction or menthol condition.