Table 2. In vitro and in vivo studies showing the effects of flavors and their flavoring agents on different organ systems.

e-C: electronic cigarette; e-liquid: electronic cigarette liquid; e-vapor: electronic cigarette vapor; ROS: reactive oxygen species; IL: interleukin; TEER: transepithelial electrical resistance; PGE2: prostaglandin E2α; LPS: lipopolysaccharide; RAGE: receptor of advanced glycation products; HMGB1: high mobility group box 1 protein; TNFα: tumor necrosis factor alpha; CXCL2: chemokine (C-X-C motif) ligand 2; COL1A1: collagen type I alpha 1 chain; MCP-1: monocyte chemoattractant protein-1; cGMP: cyclic guanosine monophosphate; VEGF: vascular endothelial growth factor; COX-2: cyclooxygenase-2; MUC5AC: mucin 5AC, oligomeric mucus/gel-forming gene; LC3: microtubule-associated protein light chain 3; NO: nitric oxide; NET: neutrophil extracellular trap; APD75: action potential duration at 75% repolarization; AP: action potential; CPP: conditioned plate preference; CPA: conditioned place aversion; pDA: putative dopamine neurons; pGABA: putative gamma-aminobutyric acid releasing neurons; nAChR: nicotinic acetylcholine receptors; VTA: ventral tegmental area; SNr: substantia nigra pars reticulata; TH+/DA: tyrosine hydroxylase dopamine neurons; TH-/non-DA: tyrosine hydroxylase non-dopamine neurons; hERG: human ether-a-go-go-related gene; I_Kr: delayed rectifier potassium channel; DhβE: dihydro-β-erythroidine hydrobromide; ↑: increase(d), ↓: decrease(d); conc.: concentration; % v/v: volume per volume percent; WT: wild type; GFP: green florescent protein; eGFP: enhanced GFP; BAC: bacterial artificial chromosome

Variables	Study design	Flavor or flavoring agent used	Nicotine	Main findings	Reference
Endothelial dysfunction	Bronchial (BEAS-2B) and alveolar (A549) epithelial cells exposed to favoring agents and their propylene glycol (PG) acetals (0.3-10 mM) for 24 h	Benzaldehyde, vanillin, ethyl vanillin, and their corresponding PG acetals	Nicotine free	Benzaldehyde PG acetal and vanillin PG acetal showed conc. dependent ↓ in mitochondrial functions (oxygen consumption rate, ATP production, spare respiratory capacity, and maximal respiration). ↑ Cytotoxicity and ↓ cell proliferation	[28]
	Endothelial cells isolated by venous biopsy from non-smokers (n = 9, 5 female, 4 male, age 25-32 years), non-menthol (n = 6, 2 female, 4 male, age 30-50 years), and menthol cigarette smokers (n = 6, 2 female, 4 male, age 25-53 years) treated with 0.01 mmol/L menthol or eugenol; commercially available human aortic endothelial cells were treated with flavoring compounds (0.001-100 mmol/L) for 90 minutes	Vanillin, menthol, cinnamaldehyde, eugenol, dimethylpyrazine, diacetyl, isoamyl acetate, eucalyptol, and acetylpyrazine	Nicotine free	Impaired NO production was seen in endothelial cells from healthy participants. ↑ Cell death and ROS were induced only at high conc., and ↑ inflammation and NO at lower conc. of selected flavors (vanillin, menthol, cinnamaldehyde, eugenol, and acetylpyridine) in human aortic endothelial cells	[18]
	Human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) exposed to flavored e-liquids and 10% serum of e-C users (combined sole e-C users and dual e-C plus combustible cigarette users, average age 29 years) for 48 h	Fruit-flavored Rainier, sweet tobacco with caramel and vanilla-flavored RY4, tobacco-flavored Red Oak Tennessee Cured, sweet-flavored butterscotch, cinnamon-flavored Marcado, and menthol tobacco-flavored Tundra	0, 6, and 18 mg/mL	↑ ROS, caspase 3/7 activity, and low-density lipoprotein uptake, ↓ cell viability, impaired tube formation, and migration, ↑ IL-1β and IL-6 levels (macrophage polarization into a pro-inflammatory M1 state), ↑ ROS and impaired tube formation were seen in iPSC-ECs treated with serum from e-C users	[29]
	Porcine aortic endothelial cells were incubated with flavoring agent (1 mM each) for 10-75 min; isolated Sprague Dawley rat aortic rings precontracted with U-46619 were incubated with the flavoring compounds (0.1-1 mM each) for 30 min	Acetylpyridine, cinnamaldehyde, diacetyl, dimethylpyrazine, eucalyptol, eugenol, isoamyl acetate, menthol, and vanillin	Nicotine free	Cinnamaldehyde-inhibited Ca2+-stimulated endothelial cGMP accumulation, NO synthase activation, and soluble guanylate cyclase activity were seen in porcine aortic endothelial cells. Relaxation of pre-contracted aortic rings with flavoring compounds (most potent- eugenol and cinnamaldehyde) was not mediated by the endothelial NO synthase/cGMP pathway	[30]
	Bovine aortic endothelial cells exposed to 10% flavored e-C smoke extract (e-CSE) for 24 h; male C57BL/6 mice (10-12 weeks) skin wound treated with recombinant human VEGF + flavored e-CSE for 11 days	Classic tobacco, mint, menthol, vanilla/fruit from BLU or JUUL	0, 1.2%, and, 2.4% (BLU), and 3% (JUUL)	↑ Caspase 3 activity, endothelial superoxide, and ↓ NO bioavailability, and impaired monolayer wound closure were seen in bovine aortic endothelial cells. Impaired VEGF-dependent wound healing in diabetic mice after e-CSE treatment	[31]
Cardiac electrophysiological and functional alterations	Human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CM) treated with either (1-100 µM) parent cinnamaldehyde (no heating) or 100 µM heated cinnamaldehyde (prepared from low (200±50°C) and high (700±50°C) heat generated aerosol) solution for 6, 24, 48 h	Cinnamaldehyde	Nicotine free	Parent cinnamaldehyde induces time- and concentration-dependent ↓ in beating rate, impedance amplitude, beating frequency, and cell index. Longer exposure of parent cinnamaldehyde (≥20 min) at 100 µM conc. ↑ depolarization of resting membrane potential, loss of rhythmic action potential generation, and irregular patterns of AP triggering and repolarization. These changes were abolished with heated 100 mM cinnamaldehyde treatment.	[32]
	HL-1 mouse atrial cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) treated with 0.075-0.75 puffs/mL flavored vapor extract for 24 and 48 h; C57BL/6 mice (5 months old, both sexes) were exposed to 4.7 s puffs of vanilla custard flavored e-vapor puff at 1.4 L/min, every 2 min for total of 60 puffs, 110 mL puff volume delivered over a 2 h period, 5 days a week, for a period of 10 weeks	Hawaiian passion fruit, orange, and guava (POG), vanilla custard (vanillin), and Apple Jax (cinnamaldehyde)	6 mg/mL	Dose-dependent ↓ HL-1 cell viability and ↑ apoptosis with vanilla custard and Apple Jax e-vapor. Both vanilla custard and apple jax e-vapor reduced beating rate and prolonged corrected field potential duration of hiPSC-CM via blockage of hERG encoded potassium current (IKr). 10-week inhalational exposure to vanilla custard e-vapor resulted in ↓ parasympathetic activity seen by decreased pNN06 (percentage of adjacent NN intervals that differ from each other > 6 ms) and lower high-frequency component of heart rate variability, longer ventricular tachycardia episodes and prolonged APD75 at 15 Hz in e-vapor exposed mice.	[20]
	Female apolipoprotein E-deficient (ApoE-/-) mice were exposed to aerosols from 3 different e-vapor formulations as follows: carrier (propylene glycol and vegetable glycerol); base (carrier + nicotine); test (carrier + nicotine + flavor) and to cigarette smoke (CS) from 3R4F reference cigarettes for up to 6 months. The animals were exposed to 3R4F CS or e-vapor aerosols for 3 h/day, 5 days/week, in whole-body exposure chambers	Guaiacol (precursor of eugenol and vanillin)	4% nicotine	↑ Aortic arch area covered by atherosclerotic plaque formation was seen with CS but no such effect was found for any of the three e-vapor aerosols. ↑ Pulse wave velocity and arterial stiffness were significantly lower with base and test e-vapor exposure compared to CS. Ejection fraction, fractional shortening, cardiac output, and isovolumic contraction time remained unchanged following e-vapor exposure, but base and test e-vapor exposure caused an increase in isovolumic relaxation time (index of diastolic function) similar to CS	[33]
Inflammation	C57BL/6 female mice (6-8 weeks) exposed to flavored aerosol from JUUL pods in a 5-L whole-body exposure chamber for 20 min, 3 times/day, i.e., a total of 60 min/day for 1 or 3 months	Mango, mint	5% nicotine salts (59 mg/mL)	In the brain, ↑ inflammatory cytokines (TNFα, ILβ, IL6) were seen in the nucleus accumbens (NAc)-core or shell post 1 and 3 months exposure, significantly ↑ RAGE and its ligand HMGB1 were seen in the NAc-shell post 3-month exposure. In the cardiac tissue, ↓ TNFα, IL6, CXCL2, and COL1A1 with mint-flavor and ↓ CXCL2 with mango-flavor post 1-month exposure was observed but there was an increase in cytokines (TNFα, IL1β, IL6) and chemokines (CCL2, CCL3, CXCL1, CXCL2) with LPS treatment after 1 and 3-month mint-flavor exposure. In the colon, mango-flavor induced TNFα, IL6, and IL8 post-1 month exposure but levels ↓ post 3-month exposure compared to air or mint-flavor	[34]
	Human lung epithelial (NCI-H292) cell line exposed to flavored aerosol for 30 min. The puff regimen was 3 s puff duration, every 30 s, with a 55 mL puff volume for 30 min resulting in a total of 55 puffs	Tobacco, piña colada, menthol, coffee, strawberry	24 mg/mL	↓ Cell viability and metabolic activity and ↑ cytokine levels (IL1β, IL6, IL10, CXCL1, CXCL2, and CXCL10) showed differential effect with different flavors and devices with higher voltage. Coffee and strawberry were most toxic	[15]
	Human bronchial airway epithelial cells (H292) and human fetal lung fibroblasts (HFL1) were treated with 1-5% flavored e-liquid or 0.5-2.5% cigarette smoke extract (CSE) from 3R4F reference cigarettes with or without nicotine for 24 h; eight weeks old C57BL/6J mice were exposed to BLU e-C (classic tobacco flavored containing 16 mg nicotine) aerosol (200 mg/m3 TPM) for 5 h exposures per day for 3 successive days	Tobacco, cinnamon roll, grape vape, menthol, berry, melon, peach, pineapple, coconut, Mountain Dew, cotton candy, strawberry, marbo	0-24 mg	Fibroblast treated with either 1 or 5% tobacco-flavored e-liquid containing 0 or 24 mg nicotine or 1% CSE showed morphological alterations (enlarged cells with spindle formation and vacuolization) and decreased cell viability, which was exacerbated with nicotine. Cinnamon roll flavored e-liquid significantly ↑ IL-8 in fibroblast; tobacco flavor ↑ IL-6 and IL8 in the culture media of lung epithelial cells; ↑ MCP-1, IL-6, IL-1α, and IL-13 in the bronchoalveolar lavage of exposed mice; and reduced glutathione levels in the lung lysates of exposed mice	[35]
	Human bronchial epithelial cells (16-HBE and BEAS2B cell lines) and monocytes (U937 cell line) were exposed to aerosolized favors for 3 sessions of 30 min each at 12 h interval. For each session, a cell culture plate was placed inside the Enzyscreen chamber and the vapors were released (66 puffs during 22 min with a 3 s puff duration at 1.6 L/min flow rate and an inter-puff interval of approximately 17 s). The aerosols were allowed to equilibrate for 8 min post-exposure resulting in a total of 30 min per session before returning the cell culture plate to the incubator	Fruit Medley, Virginia tobacco, cool mint, crème brulee, cool cucumber, mango, classic menthol; other pod favors - just mango (strawberry coconut) and café latte	JUUL pod (5%) other pods (6%)	↑ Mitochondrial ROS, cell death, DNA fragmentation, IL-8, PGE2, IL-15, and other pro-inflammatory cytokines. ↓ TEER and voltage	[14]
	Human middle ear epithelial cells treated with flavored e-liquids (3.3% tobacco and 1.5% menthol) for 24 h	Tobacco and menthol	9.9 mg/mL	↓ Cell viability, ↑ inflammatory mediators COX-2 and TNFα levels, ↑ expression of MUC5AC (mucin production), and ↓ in epithelial sodium channels genes and ↑ aquaporin expression resulting in mucosal membrane expansion and fluid accumulation. ↑ Apoptotic marker, ↓ BCL-2, and ↑ autophagy marker LC3 were seen and more marked with menthol flavor	[36]
Developmental changes	Wild-type zebrafish embryos treated with cinnamon and chocolate-flavored e-vapor were added into dechlorinated water at 0.6, 12, and 25 puffs/L for 24 h	Cinnamon and chocolate	Nicotine free	Cinnamon-flavored e-vapor at 25 puffs/L significantly decreased end-diastolic and end-systolic volumes, stroke volume, heart rate, cardiac output, and red blood cell density except for blood vessel diameter	[37]
	Pregnant BALB/c mice (10 weeks) were exposed to cinnamon-flavored with nicotine e-C aerosols (3 s puff duration, and a 55 mL puff volume every 30 s) or HEPA-filtered air for 14-31 days, which included exposures for either 12 days before mating plus 19 days during gestation (preconception groups) or only during 14 days of gestation (prenatal groups). All preconception-exposed offspring were euthanized at birth and prenatal offspring were euthanized at birth or at 4 weeks of age	Cinnamaldehyde	36 mg/mL (nicotine salts)	↓ Body length and weight in offspring of both preconception and prenatally exposed dams. ↑ Tissue fraction in lung morphometric analysis, altered Notch 2 (epithelial cell differentiation), Shh and Wnt (lung organogenesis) pathways in offspring of both groups. Higher maximum respiratory elastance (elastic stiffness to expand) in preconception group dams. ↓ Maternal serum placental growth factor, increased 17β-estradiol and dysregulated maternal-fetal inflammatory genes (IL1β, IL4, IL6, STAT5A) in both groups	[38]
	Zebrafish embryos were exposed to e-C flavor dilution made in 1:1000 propylene glycol: embryo media from 6 hours post-fertilization (hpf) to 120 hpf	Bubble gum (ethyl acetate, ethyl butyrate, cinnamaldehyde), grape (ethyl acetate, ethyl butyrate), coffee, cotton candy, french vanilla, 555 (maltol, vanillin, ethyl vanillin), nicotine, unflavored	12 mg/mL and 24 mg/mL (present in nicotine flavor only), no nicotine in unflavored	Hyperactive behavior was seen in larval zebrafish at 120 hpf with all flavors, except for nicotine. Yolk sac and pericardial edemas, malformation of eye, jaw, cranial and cardiac regions, craniofacial deformity, bent body axis seen with all flavors at 120 hpf, most prominent effect seen with cinnamaldehyde containing flavor including increase in mortality at earlier time point, i.e., 24 hpf	[17]
	Fetal, neonatal, and adult ovine primary pulmonary artery smooth muscle cells (PASMC) were exposed to flavored e-liquid (1:100, 1:1000, and 1:10000) for 24 h; neonatal and adult ovine intrapulmonary arteries and the 5th generation bronchial rings treated with 1:1000 dilution of flavored e-liquid	Menthol, strawberry, tobacco, vanilla	Nicotine free	Menthol and strawberry flavors induced maximal cell death in both immature and adult PASMC with marked menthol toxicity in neonatal cells. Bronchodilation was more pronounced in the neonatal bronchial rings with menthol and tobacco flavors whereas adult rings only showed slight relaxation with menthol. Adult pulmonary artery relaxation was seen with menthol flavor	[16]
Epithelial barrier dysfunction	Human bronchial epithelial cells (16HBE14o) treated with flavored e-liquids (at conc. representing the % v/v present in e-C cartridges) for 24 h; Primary mouse tracheal epithelial (MTE) cells treated with 2,5-dimethylpyrazine (0.2% v/v present in e-C cartridge)	2,5-dimethylpyrazine (dark chocolate flavor), damascenone, linalool, α-ionone, ethyl maltol, furaneol and vanillin	Nicotine free	Loss of cell index (↓ in impedance, cytotoxicity) seen in flavored treated 16-HBE14o cells except with furaneol and vanillin due to solubility issues. 2,5-Dimethylprazine showed significantly reduced cell impedance in 16-HBE14o cells. ↑ Apical Cl- ion conductance (short circuit current {Isc}) and ↓ transepithelial resistance in MTE cells	[21]
	Human lung epithelial and endothelial cells were exposed to flavored e-C condensed vapor for 24 h. The puff regime was set to 55 mL puff volume, 3 s puff duration, 60 s puff interval, and 200 puff (estimated maximum of puffs per day 235)	Cinnamon, tobacco, menthol	18 mg/mL	Cinnamon flavor ↑ cytotoxicity and pro-inflammatory response by ↑ IL-8 and MCP-1. ↓ TEER with both cinnamon and menthol-flavored aerosol	[22]
Immune dysfunction	Human alveolar macrophages (from bronchoalveolar lavage), neutrophils, and natural killer cells (from venous blood collected from healthy participants) were treated with flavored e-liquids (unheated) and vapored e-liquid condensate (VEC) (0.25, 0.5 and 1% dilutions) for 30 min. Vaped aerosol (1 puff of aerosol, every 30 s for 10 min resulting in 20 puffs total using an output setting of 60 watts) was allowed to cool and condense to generate VEC	Menthol (menthol tobacco and solid menthol), cinnamaldehyde (hot cinnamon candies, kola, and sinicide), isoamyl acetate (banana pudding and banana)	Nicotine free	Cinnamon-flavored e-liquids significantly ↓ phagocytic activity of both alveolar macrophages and neutrophils and suppressed NK cells' ability to eliminate target leukemia cells. ↑ IL-6 in macrophages, ↑ IL-8 production, and enhanced NET formation (anti-bacterial function) in neutrophils. Restoration of phagocytic activity of macrophages with small-molecule reducing agent 1,4-dithiothreitol. Cinnamaldehyde-flavored VEC did not significantly alter neutrophil phagocytosis compared to the unheated e-liquids	[39]
	Neutrophils isolated from venous blood of healthy human subjects were treated with 0-5 mM of flavoring agent for 1 h at 37°C	Cinnamaldehyde (cinnamon), ethyl vanillin (vanilla), benzaldehyde (almond or cherry), and isoamyl acetate (banana)	Nicotine free	Cinnamaldehyde and ethyl vanillin decreased total oxygen consumption in a dose-dependent manner. Cinnamaldehyde, ethyl vanillin, benzaldehyde, and benzaldehyde PG acetal significantly decreased neutrophil phagocytosis while isoamyl acetate had no effect	[40]
	Adult female C57BL/6 mice (8 weeks) were exposed to 70% vegetable glycerin and 30% propylene glycol (VG/PG) with or without French vanilla flavored e-C aerosol for 2 h/day, 7 days/week for 6 weeks in 5 L whole body exposure chamber. Vaping was conducted under a topography profile of 3 s puff duration, and a 55 mL puff volume every 30 s. E-C aerosols were sampled in the airstream exiting the chamber at a flow rate of 1 L/min throughout the experiment	French vanilla	Nicotine free	Greater tidal and minute volumes and ↑ lung tissue resistance were seen in vanilla-flavored exposed mice. Slight significant ↑ in NK cells and dendritic cell populations (CD11b+, CD11c+) exposed to flavored e-C aerosol. Increased T helper cell (CD4+) and B-cell (CD19+) populations in the lungs of mice treated with VG/PG regardless of flavor. Significant increase in IgG1 levels in the bronchoalveolar lavage fluid of mice exposed to VG/PG with vanilla-flavored e-C aerosol	[41]
Neurobehavioral effects	Eight-week-old male C57BL/6J mice were given drinking water containing nicotine (5 conc. - 30, 50, 75, 100, and 200 µg/mL) or nicotine-free (diluted to match the same fluid amounts) fruit- or tobacco-flavored e-liquids for one week in sequential order	Fruit or tobacco-flavored e-liquid (the chemical composition of fruit and tobacco flavoring in the e-liquids used was not mentioned in the present study)	18 mg/mL	Increased average daily consumption and proclivity for nicotine-containing fruit-flavored e-liquid but this increase was not due to flavor itself, average daily consumption of nicotine-free fruit and tobacco-flavored e-liquids increased compared to nicotine-containing flavored e-liquid and nicotine alone at the 200 µg/mL concentration. Total fluid intake (average total fluid consumption per week) and peripheral place conditioning assays (CPP and CPA) showed no difference with both flavored e-liquids with and without nicotine	[42]
	Adult C57/BL6J male mice (3-6 months) were used in (e-vape) vapor self-administration assays with e-liquids containing menthol ~15 mg/mL or green apple flavor 5-15 mg/mL with or without nicotine. Mice were placed in an air-tight chamber with 2 nose-pokes (one active and one inactive). Active nose-pokes resulted in a 3 s delivery of vaporized e-liquid with a 60 s timeout. A yellow cue light remained on during the timeout. For fixed-ratio 1 (FR1) schedule, mice began vapor self-administration on a Monday for 10 daily 3 h sessions for 5 days with weekend abstinence). Following FR1 escalation, mice transitioned to 3 h fixed-ratio 3 (FR3) sessions where they were maintained on their assigned e-liquid for 4 consecutive days to reach stable responding and re-baselined to their original FR1 assigned e-liquid on day 5. For progressive ratio (PR) schedule (after FR3 sessions and a weekend abstinence), the following equation was used (PR = 2(2n/9), where “n” is the number of earned e-vape deliveries within the session) to determine the number of active nose-pokes required for e-vape delivery for each 3 h session for 4 days and then re-baselined for 2 days. During PR sessions, the active and inactive nose-poke was set to match FR1 and FR3 sessions for the first 2 days and then reversed on the next 2 days to examine presence of side bias; DhβE, an α4β2 nAChR antagonist, was intraperitoneally injected (2 mg/kg) prior to self-administration sessions (on day 1 and 2 with saline, day 3 and 4 with DhβE) when used	Menthol and green apple	6 mg/mL	Menthol + nicotine and green apple alone produced the highest number of FR1 e-vape deliveries followed by green apple + nicotine and nicotine alone on FR1 escalation schedule. Nicotine, green apple, green apple + nicotine, and menthol + nicotine produced significantly more FR3 deliveries, marked effect seen with both flavors plus nicotine compared to nicotine alone. DhβE significantly reduced FR3 responses in mice assigned to nicotine, green apple + nicotine, and menthol + nicotine. PR sessions showed no side bias in self-administration behavior, similar trend was observed in both PR and FR3 e-vape deliveries	[25]
	Adult male and female C57BL/6J mice (3 - 6 months) genetically modified to contain fluorescent nicotinic acetylcholine receptors (nAChRs) (α4-mCherryα6-GFP mice) were implanted with menthol osmotic pumps (2 mg/kg/h) with or without nicotine (2 mg/kg/h) for 10 days (for in vivo upregulation assays and chronic treatment). For CPP assay, non-transgenic (WT) littermates of α6-GFP BAC transgenic adult mice were administered intraperitoneal injection of 1 mg/kg menthol with or without 0.5 mg/kg nicotine on alternating days for a total of 8 days. For electrophysiological assay cultured midbrain neurons from tyrosine hydroxylase (TH)-eGFP mice were treated with 500 nM menthol with or without 200 nM nicotine for 10 days. For all experiments, 3-6 months adult mice were used; mouse neuroblastoma 2a (neuro-2a) cells were transiently transfected with α4-GFPβ2 or α6-GFPβ2β3 nAChR subunits and treated with nicotine (50 or 100 nM) or menthol (500 nM) plus nicotine for 24 h	Menthol	0.5 mg/kg	↑ CPP (reward-related behavior) in nicotine + menthol and nicotine alone treated mice. ↓ Baseline firing frequency in TH+/DA and ↑ in TH-/non-DA (pGABA) neurons after menthol (500 nM) + nicotine (200 nM) treatment was more than nicotine alone. TH+/DA neurons treated with menthol plus nicotine exhibited an ~ 8-fold increase in firing frequency following nAChR activation. Menthol enhances only α4 containing nAChR upregulation in VTA DA and SNr GABA neurons and does not enhance α6 containing nAChR upregulation. In the transfected neuro-2a cells, menthol alone increases the number of low-sensitivity α4(3)β2(2) nAChRs and low-sensitivity α6β2 (non-β3) nAChRs	[43]
	Adult male and female C57BL/6J mice (3-6 months) genetically modified to contain fluorescent nicotinic acetylcholine receptors (nAChRs) (α4-mCherryα6-GFP mice) were administered intraperitoneal injection of farnesol (0.1-10 mg/kg) with or without nicotine (0.5 mg/kg); mouse neuroblastoma 2a (neuro-2a) cells transfected with α4-GFPβ2 and β2WT nAChR subunits and treated with 500 nM farnesol for 24 h	Farnesol (green apple)	0.5 mg/kg	Reward-related behavior in CPP assay was observed with 1 mg/kg farnesol-alone and additive effect with nicotine in male mice only. ↑ Locomotor activity in both sexes. ↑ Firing frequency of pDA due to ↓ pGABA firing frequency in farnesol (1 mg/kg) treated neurons from male mice due to upregulation of α6 and α4α6 containing nAChR on VTA pDA neurons and ↓ in α4 containing nAChR in SNr GABA neurons. Farnesol + nicotine has no additive or enhancing effect on nAChR upregulation, it counteracts both the effect of farnesol alone to upregulate α6 containing nAChR on VTA pDA neurons and the effect of nicotine alone on α4 containing nAChR upregulation on SNr GABA neurons. In the transfected neuro-2a cells, farnesol showed concentration-dependent (0-300 µM) inhibition of α4β2 nAChR function and long-term farnesol treatment (500 nM, 24 h) facilitated α4β2 nAChR recovery following desensitization	[23]
	Adult male and female C57BL/6J mice (3-6 months) genetically modified to contain fluorescent nicotinic acetylcholine receptors (nAChRs) (α4-mCherryα6-GFP mice) were administered intraperitoneal injection of farnesene (0.1-10 mg/kg) with or without nicotine (0.5 mg/kg); mouse neuroblastoma 2a (neuro-2a) cells transfected with α4-mCherry, α4-GFP, and β2WT or α4-mCherry, α6-GFP, and β2WT) nAChR subunits and treated with 500 nM farnesene for 24 h to examine changes in α4α6β2 and α4β2 nAChR stoichiometry	Farnesene (green apple)	0.5 mg/kg	Lowest dose of farnesene (0.1 mg/kg) produced a significant rewarding effect in CPP assay in both sexes, higher doses showed no change from baseline in male mice and female mice exhibited a significant change from baseline with all farnesene doses when compared with saline. No significant change in locomotory activity with farnesene in both male and female mice. Both sexes exhibited enhanced rewarding effects in the nicotine + farnesene group compared with nicotine alone, but only females exhibited a significant enhancement. Farnesene treatment produced no significant changes in nAChR number within pDA or GABA neurons in the VTA or SNr. Farnesene enhances nicotine’s potency for activating nAChRs on VTA dopamine neurons by altering nAChR stoichiometry on putative VTA dopamine neurons toward high-sensitivity α4β2 nAChRs. Farnesene treatment promotes high-sensitivity α4(2)β2(3) nAChRs by decreasing the number of low-sensitivity α4(3)β2(2) and α4α6β2 nAChRs in neuro-2a transfected cells	[24]