Nicotine e-cigarette vapor inhalation and self-administration in a rodent model: Sex- and nicotine delivery-specific effects on metabolism and behavior

Abstract

E-cigarettes, which deliver vaporized nicotine, have dramatically risen in popularity in recent years, despite many unanswered questions about safety, efficacy in reducing dependence, and overall impact on public health. Other factors, such as sex, also play an important role in determining behavioral and neurochemical responses to drugs of abuse. In these studies, we sought to develop a protocol for vaporized e-cigarette nicotine self-administration in rats, as a foundation to better understand the differing effects of nicotine exposure routes on behavior and physiological function. We report a novel method that elicits robust nicotine vapor self-administration in male and female rats. Our findings indicate that 5-mg/ml nicotine vape solution provides a high level of consistency in lever-pressing behavior for both males and females. Moreover, in male rats, we find that such e-cigarette nicotine vapor induces similar blood levels of nicotine's main metabolite, cotinine, as that found with intravenous nicotine self-administration. Therefore, the breathing pattern during vapor exposure in males leads to similar levels of titrated nicotine intake as with intravenous nicotine self-administration. Interestingly, a differential effect was found in the females, in which the same conditions of vapor exposure led to decreased cotinine levels with vapor compared to intravenous self-administration. Finally, differences in nicotine-mediated locomotion provide further support of the physiological effects of e-cigarette vapor inhalation. Taken together, our findings reveal important sex differences in nicotine intake based on the route of exposure, and we further establish a protocol for nicotine vapor self-administration in rats.

Keywords: e-cigarette nicotine vape; nicotine self-administration; sex specific effects.

Figure 1.. Nicotine vapor self-administration in male and female rats.

Subjects were examined for self-administration behavior at baseline (0 mg/ml, vehicle) and across a range of nicotine doses, which progressively increased from 2.5 to 5.0 to 7.5 mg/ml. (A) Male rats (n=8/group) self-administered a significantly increased number of vapor puffs at the 5 and 7.5 mg/ml doses compared to vehicle vapor. *p<0.05, **p<0.01 vs. vehicle control. (B) Female rats (n=5-8/group) exhibited increased responding for the delivery of nicotine vapor puffs at the 5.0 mg/kg dose compared to vehicle control. *p<0.05 vs. vehicle control. (C-D) When the number of presses on the active and inactive levers were examined, both males (C) and females (D) exhibited a clear dissociation between the active (colored circles) and inactive (grey circles) levers. This preference in responding on the active lever was present for all nicotine doses examined, but not present for the vehicle control. ****p<0.0001 active vs. inactive lever. (E-F) Blood samples were examined for cotinine levels 30 min after the final session of vapor treatment at each dose. (E) Cotinine levels progressively increased in male rats as the unit dose increased. *p<0.05, ***p<0.001, ****p<0.0001 vs. vehicle control. (F) In females, significant levels of cotinine were found in rats self-administering at the 5 and 7.5 mg/ml concentrations of nicotine. *p<0.05, **p<0.01 vs. vehicle control. (G) When rats (n=13 males/females combined) were only provided access to vehicle vapor, their behavioral lever pressing decreased ~3 puffs per hour session, indicating a low baseline level of responding. (H) When initially provided access to the higher 5 mg/ml dose, subjects (n=8 males/females combined) decreased their responding across sessions to near baseline levels, indicative of an aversive behavioral response when presented with this higher dose initially. Data are presented as mean ± SEM.

Figure 2.. Comparison of nicotine metabolite levels following passive vapor or intravenous nicotine self-administration (IVSA) in male rats.

Blood samples were collected at baseline (pre-nicotine) or following 1 hr sessions of either nicotine vapor (5 mg/ml) or IVSA (0.03 mg/ml/infusion) from male rats. (A) Cotinine levels (n=8-16/group) were decreased with passive vapor exposure 30 min after the first session, as compared to IVSA subjects. However, these differences were not maintained at the later Day 1 time-point of 6 hr, or on Day 7 at the 30 min post-session time-point. *p<0.05 passive vapor vs. IVSA. (B) Male rats (n=8/group) exposed to nicotine vapor did not differ in their cotinine levels if placed in the chamber individually (single exposure) or with a cagemate (dual exposure) at all time-points assessed. (C) During the nicotine IVSA session (n=8), the rats self-administered a greater net amount of nicotine on Day 1 than on Day 7, which is consistent with the differences found in blood cotinine levels. *p<0.05 Day 1 vs. Day 7. (D) When IVSA nicotine intake was examined in 15-min intervals across the 1 hr session, it was found that the males exhibited a significant increase in responding only during the first 15 min interval, and thereafter, they maintained a consistent level of responding. *p<0.05 Day 1 vs. Day 7. Data are presented as mean ± SEM.

Figure 3.. Comparison of nicotine metabolite levels following passive vapor or intravenous nicotine self-administration (IVSA) in female rats.

Blood samples were collected at baseline (pre-nicotine) or following 1 hr sessions of either nicotine vapor (5 mg/ml) or IVSA (0.03 mg/ml/infusion) from female rats (n=6/group). (A) Female cotinine levels were decreased following passive nicotine vapor exposure for all time-points, as compared to IVSA nicotine. *p<0.05, **p<0.01, ***p<0.001 passive vapor vs. IVSA. (B) Females did not differ in the amount of nicotine self-administered between Day 1 and Day 7. Data are presented as mean ± SEM.

Figure 4.. In male rats, nicotine vapor exposure and intravenous selfadministration (IVSA) both induce a hyperlocomotor effect in male rats.

Male rats (n=6/group) were tested for nicotine-mediated locomotor effects in an open field chamber at baseline (pre-nicotine) and immediately after each nicotine sessions. (A) Passive nicotine vapor induced a significant increase in locomotor activity on Day 7 of exposure, whereas nicotine IVSA induced a significant increase on Day 1 of exposure, as compared to baseline levels. *p<0.05, **p<0.01, ****p<0.0001. (B-D) Locomotor behavior was analyzed in 5 min intervals for each session to compare the level of responding between nicotine passive vapor and IVSA. (B) At baseline, there were no differences between groups in distance traveled. (C) On Day 1, the IVSA nicotine group exhibited an increase in their distance traveled across the first two 5 min epochs. **p<0.01, ***p<0.001. (D) No significant differences were found between groups during the last day of treatment (Day 7). Data are presented as mean ± SEM.

Figure 5.. In female rats, intravenous nicotine self-administration (IVSA), but not passive vapor exposure, induces a hyperlocomotor effect.

Female rats (n=5-6/group) were tested for nicotine-mediated locomotor effects in an open field chamber at baseline (pre-nicotine) and immediately after the nicotine sessions. (A) Passive nicotine vapor did not significantly alter locomotion, whereas nicotine IVSA induced a significant increase on Day 1 and Day 7 of exposure, as compared to baseline levels. **p<0.01. (B-D) Locomotor behavior was analyzed in 5 min intervals for each session to compare the level of responding between nicotine passive vapor and IVSA. (B) At baseline, there were no differences between groups in distance traveled. (C) On Day 1, the IVSA nicotine group exhibited an increase in their distance traveled across the first two 5 min epochs. *p<0.05, **p<0.01. (D) No significant differences were found between groups during the last day of treatment (Day 7). Data are presented as mean ± SEM.

Figure 6.. No effect of nicotine on anxiety-related behaviors in the open field.

Male (n=6/group) and female (n=5-6/group) subjects were further examined for indicators of behaviors associated with anxiety on Day 0 (baseline, no nicotine), and immediately after nicotine sessions on Day 1 and Day 7. (A-B) Time in the center of the open field did not differ across all exposures or time-points for both males (A) and females (B). (C-D) Freezing time was examined but no differences were found across all exposures or time-points for both males (C) and females (D). Data are presented as mean ± SEM.