Article Figures & Data
Figures
- Figure 1.
Nicotine delivery via e-cigarette vapor results in measurable plasma cotinine concentrations, and the rewarding properties of e-cigarette vapors can be evaluated using conditioned place preference. A, A vapor exposure setup allows for the simultaneous delivery of e-cigarette vapor to four mice, each in its own airtight chamber. B, Scatter plots of plasma cotinine concentrations detectable in adolescent mice (mean ± SEM) 30 min after nicotine exposure via an intraperitoneal injection (yellow diamonds, top x-axis, n = 7, 4, 7, 5) or e-cigarette vapor exposure as depicted in A (blue boxes, bottom x-axis, n = 27, 28, 4, 46). C, Schematic of the vapor-conditioned place preference paradigm in which mice receive experimental vapor (nicotine) once per day for 5 d in the CS+ compartment and vehicle once per day for 5 d in the CS– compartment. D, Before and after plots show the time spent in the CS+ environment before and after conditioning with nicotine vapor (n = 28, 20, 24, 25). E, Before and after plots show the time spent in the CS+ environment before and after conditioning with strawberry vapor of various concentrations (n = 28, 20, 13). In D and E, bars show the mean ± SEM, and the vehicle data (i.e., “0”) are the same in both panels. Two-way repeated-measures ANOVA and multiple comparisons: ***p < 0.001, relative to time spent in CS+ before conditioning. For all charts, circles represent individual male mice and triangles represent female mice. NIC, Nicotine; VEH, vehicle.
- Figure 2.
Strawberry additive does not affect nicotine CPP. Adolescent mice were conditioned to nicotine vapor with (2.5% strawberry flavorant) or without a strawberry additive using a CPP-biased protocol (nicotine only, n = 20, 24, 25; nicotine + additive, n = 16, 29, 30). Scatter plots represent individual mice, and bars represent the mean ± SEM. One-sample t tests (vs 0 s change in time spent in CS+): +p < 0.05, +++p < 0.001. Two-way ANOVA and multiple comparisons: *p < 0.05, **p < 0.01. For all charts, circles represent individual male mice and triangles represent female mice.
- Figure 3.
Increased plasma cotinine levels in mice exposed to strawberry nicotine vapors, a proxy for increased nicotine exposure. A, Mice exposed to vapor containing a strawberry-additive and nicotine e-liquid (10 or 50 mg/ml) had higher plasma cotinine concentrations relative to their body weight (in ng/ml/g) than mice exposed to nicotine-only vapor (10 mg/ml nicotine: n = 28; +additive, 34; 50 mg/ml nicotine: n = 46; +additive, 60). B, Adolescent mice were interperitoneally injected with 1.0 mg/kg nicotine in PBS with or without 2.5% strawberry additive. The presence of strawberry additive in the injection vehicle did not influence plasma cotinine concentrations 30 min postinjection; however, there was a significant effect of sex where, overall, male mice had higher plasma cotinine concentrations 30 min post-nicotine injection (females: n = 7, 7; males: n = 6, 5). **p < 0.01, ****p < 0.0001. Circles represent individual male mice, and triangles represent female mice.
- Figure 4.
Paradigm for monitoring the sniffing of mice to e-cigarette odors. A, Design of the system. Openings of computer-controlled valves (a) allows for the flow of air as regulated by a flowmeter (b) to pass through its respective valve into the connected odor vial (c). Airflow continues for 10 s, flowing from the headspace of the vial containing liquid odor through the connected tubing to ultimately combine with constant airflow in a manifold (d) and then to the mouse in the plethysmograph (e). Odor enters from the base of the plethysmograph, beneath the perforated floor. Changes in air pressure within the plethysmograph (indicating respiration) are detected by a flow transducer and digitized (f). Image was made with BioRender. B, The average of 5 trails of 10 s presentations of 2.5% strawberry (Str) e-liquid odor acquired via a photoionization detector (Aurora Scientific) illustrating onset and evacuation of odor from within the plethysmograph. Timing of strawberry odor delivery is indicated by gray box. C, Representative respiratory traces from a single mouse throughout presentations of PG/VG. Rasters indicate sniff peaks. Timing of PG/VG delivery is indicated by gray box.
- Figure 5.
The addition of strawberry e-cigarette additive promotes the inhalation of nicotine vapor. A, Example respiratory traces from one mouse showing its sniffing in response to pseudorandom presentations of 50 mg/ml nicotine (50N; left) and 50 mg/ml nicotine with the strawberry additive (50N+Str; right). Rasters indicate sniff peaks. B, Sniffing dynamics of all mice during the first and fifth presentation of all e-cigarette odors. 2-D histograms depict the average sniffing frequency across the 10 s of odor presentation. Data are organized in descending order based on average sniff frequency during odor presentation. Dashed line indicates odor onset. C, Percentage of time spent sniffing by mice during presentation with flavored and unflavored nicotine vapors. Two-way ANOVA detected a main effect of flavor, and multiple-comparisons test determined that mice spent significantly more time in investigatory sniffing when presented with 50N+Str. N = 25, **p < 0.01 and ***p < 0.001 for all comparisons. In C, circles represent individual male mice and triangles represent female mice.
Extended Data
Figure 1-1
GC/MS detects chemical volatiles in strawberry e-liquid. The chromatogram from GC/MS analysis of 0.1% “Strawberry Flavor Concentrate” (Liquid Barn) in water shows the following chemicals dominate in the commercial e-liquid headspace in order from highest to lowest chromatographic peak responses: (a) ethyl butyrate, (b) 2-methyl-ethyl butyrate, (c) benzyl acetate, (d) propyl butyrate, (e) 3-hexen-1-ol, (f) 3-hexenyl acetate, (g) linalool, (h) menthyl acetate, and (i) benzyl butyrate. Download Figure 1-1, TIF file.
Figure 1-2
pH of the e-liquids utilized in this study estimated by assuming [H+] in aqueous solution. Briefly, e-liquids were made in a 50:50 VG/PG blend with nicotine dissolved in the VG portion and additive dissolved in the PG portion. To reduce the viscosity of the e-liquid and in order to mimic the protocol used in the study by St. Helen et al. (2017), a 1:10 e-liquid-to-Milli-Q water dilution of each e-liquid was prepared. pH test strips were then used to estimate the pH. Download Figure 1-2, DOC file.
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