Green Apple e-Cigarette Flavorant Farnesene Triggers Reward-Related Behavior by Promoting High-Sensitivity nAChRs in the Ventral Tegmental Area

Abstract

While combustible cigarette smoking has declined, the use of electronic nicotine delivery systems (ENDS) has increased. ENDS are popular among adolescents, and chemical flavorants are an increasing concern because of the growing use of zero-nicotine flavored e-liquids. Despite this, little is known regarding the effects of ENDS flavorants on vaping-related behavior. Following previous studies demonstrating the green apple flavorant, farnesol, enhances nicotine reward and exhibits rewarding properties without nicotine, this work focuses on the green apple flavorant, farnesene, for its impact on vaping-related behaviors. Using adult C57BL/6J mice, genetically modified to contain fluorescent nicotinic acetylcholine receptors (nAChRs), and farnesene doses of 0.1, 1.0, and 10 mg/kg, we observed farnesene-alone produces reward-related behavior in both male and female mice. We then performed whole-cell patch-clamp electrophysiology and observed farnesene-induced inward currents in ventral tegmental area (VTA) putative dopamine (pDA) neurons that were blocked by the nAChR antagonist, DhβE. While the amplitudes of farnesene-induced currents are ∼30% of nicotine's efficacy, this indicates the potential for some ENDS flavorants to stimulate nAChR function. Additionally, farnesene enhances nicotine's potency for activating nAChRs on VTA dopamine neurons. This may be because of changes in nAChR stoichiometry as our data suggest a shift toward high-sensitivity α4β2 nAChRs. Consequently, these data show that the green apple flavorant, farnesene, causes reward-related behavior without nicotine through changes in nAChR stoichiometry that results in an enhanced effect of nicotine on VTA dopamine neurons. These results demonstrate the importance of future investigations into ENDS flavorants and their effects on vaping-related behaviors.

Keywords: electrophysiology; flavorants; microscopy; nicotinic receptors; reward-related behavior.

Figure 1.

Farnesene-alone produces reward-related behavior in male and female mice. A_1,2, Male and female mice were administered saline or farnesene at doses of 0.1, 1.0, or 10 mg/kg in a CPP assay. B_1,2, Male and female mice were administered saline or 0.1 mg/kg farnesene in an open field locomotor assay. All data are mean ± SEM; *p < 0.05, **p < 0.01, ****p < 0.0001; one-way ANOVA with post hoc Tukey (A) or unpaired t test (B). Exact p values are given in Results. Number of mice for each treatment group in CPP assays is indicated in parenthesis. Dots within bars represent the CPP scores or locomotor activity from individual mice within the designated treatment groups.

Figure 2.

Farnesene (0.1 mg/kg) enhances nicotine reward-related behavior in both sexes. A, B, Male and female mice were administered saline, nicotine (0.5 mg/kg), or nicotine (0.5 mg/kg) plus farnesene (0.1 mg/kg) in a CPP assay. All data are mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.005; one-way ANOVA with post hoc Tukey. Exact p values are given in Results. Number of mice for each treatment group is indicated in parenthesis, and dots within bars represent the CPP scores from individual mice within the designated treatment group.

Figure 3.

Farnesene treatment has no effect on nAChR number in the midbrain. A₁, Schematic of target mouse brain region (bregma −3.1 mm). A₂, Sample 10× image of a mouse coronal brain section at approximately bregma −3.1 mm. Scale bar, 250 μm. B, Sample images of saline and farnesene treated VTA dopamine neurons. Scale bar, 15 μm. C, D, RID of α4*, α6*, and α4α6* nAChRs of VTA dopamine neurons (C₁, D₁), α4* nAChRs of VTA GABA neurons (C₂, D₂), and α4* nAChRs of SNr GABA neurons (C₃, D₃) in saline-treated and farnesene-treated (0.1 mg/kg) male (C) and female (D) mice. All data are mean ± SEM. Unpaired t test. Dots indicate the RID values from individual mice.

Figure 4.

Farnesene alters the stoichiometry of α4α6β2* nAChRs in VTA DA neurons. Mean NFRET percentage (A₁, B₁,), mean NFRET pixel count (A₂, B₂), and mean pixels/neuron histograms (A₃, B₃) for saline-treated and farnesene-treated (0.1 mg/kg) VTA dopamine neurons in male (A) and female (B) mice. All data are mean ± SEM; *p < 0.05; unpaired t test. Exact p values are given in Results. Dots within bars represent the values from individual mice within the designated treatment group; n > 40 neurons per mouse per treatment group.

Figure 5.

Farnesene favors high-sensitivity nAChRs in neuro-2a cells. Representative neuro-2a cells transfected with α4-mCherry, α4-GFP or α6-GFP, and β2wt nAChR subunits to produce (A) α4-mCherryα6-GFPβ2 nAChRs or (C) α4-mCherryα4-GFPβ2 nAChRs. Scale bar, 10 μm. Mean NFRET pixel count (B₁, D₁) and NFRET percentage (B₂, D₂) treated as control or with 0.5 μm farnesene for (A) α4-mCherryα6-GFPβ2 nAChRs or (C) α4-mCherryα4-GFPβ2 nAChRs. All data are mean ± SEM; *p < 0.05, ****p < 0.001; unpaired t test. Exact p values are given in Results. Dots within bars represent the values from individual cells within the designated treatment group; n > 30 cells per condition.

Figure 6.

Farnesene favors high-sensitivity nAChRs in neuro-2a cells. A, α4β2 nAChRs assemble in two stoichiometries, and we observed that farnesene treatment shifts a mixed population of HS and LS α4β2 nAChRs to a majority of HS α4β2 nAChRs. B, In examining α4α6β2 nAChRs, under control treatments, ∼65% of the population are α4α6β2 nAChRs while the remainder is likely α4β2 nAChRs. Following treatment with farnesene, <14% of the nAChRs are α4α6β2 nAChRs.

Figure 7.

Farnesene enhances the affinity and potency of nicotine. Representative images of VTA pDA neurons in a brain slice (bregma −3.1) were identified through the presence of α6-GFP nAChRs in IR-DIC (A₁) and GFP fluorescence (A₂) imaging modes. Scale bars, 20 μm. B, Representative inward currents from VTA pDA neurons (α6-GFP-positive) with 10-s applications of 500 nm (B₁) or 10 μm (B₂) nicotine in voltage-clamp mode. Arrows indicate start of nicotine puff application and dotted red lines indicate baseline before puff and the duration of nicotine application. C, Average nicotine concentration response of peak-current amplitude of VTA pDA neurons (n = 7 neurons/4 mice and 5 neurons/3 mice per nicotine concentration for saline-treated and farnesene-treated mice, respectively). D, Representative waveforms of sEPSCs from VTA pDA neurons recorded from saline-treated or farnesene-treated mice in the presence of 30 μm picrotoxin. E, Mean sEPSC frequency (E₁) and amplitude (E₂) in saline-treated and farnesene-treated mouse brain slices (n = 9 neurons/4 mice and 9 neurons/3 mice for saline-treated and farnesene-treated mice, respectively). For all assays, drug treatments were consistent with the CPP assay paradigm using 0.1 mg/kg farnesene. C, E_I,2, Data are mean ± SEM *p < 0.05, ****p < 0.0001; unpaired t test. Exact p values are given in Results. Dots within bars represent the values from individual neurons within the designated treatment group.

Figure 8.

Farnesene acts as a partial agonist on nAChRs. A, B, Voltage-clamp recordings from putative VTA dopamine neurons. A, Five and 500 μm farnesene and 100 μm nicotine were applied to putative VTA dopamine neurons. The β2* nAChR antagonist, DhβE (0.3 μm) blocked inward currents stimulated by 500 μm farnesene. B, Mean peak current amplitude for farnesene and nicotine applications on pDA neurons in the VTA. C–E, Voltage-clamp recordings from neuro-2a cells transiently transfected to contain α4-GFPβ2 and α6-GFPβ2β3 nAChRs. C, Representative images of neuro-2a cells that contain α4-GFPβ2 or α6-GFPβ2β3 nAChRs. D, Representative inward currents stimulated by 300-ms applications of 500 μm farnesene on neuro-2a cells containing α4-GFPβ2 or α6-GFPβ2β3 nAChRs. E_1,2, Mean peak current amplitude of 500 μm farnesene and nicotine applications (3 and 100 μm nicotine for α6-GFPβ2β3 and α4-GFPβ2 nAChRs, respectively) on neuro-2a cells containing nAChRs. B, E_1,2, Data are mean ± SEM; **p < 0.01, ****p < 0.0001; one-way ANOVA with post hoc Tukey (B) or unpaired t test (E). Dots represent data from individual neurons or cells. Exact p values are given in Results.