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Published in final edited form as: Neurosci Lett. 2022 Dec 5;793:137008. doi: 10.1016/j.neulet.2022.137008

Chronic oral nicotine exposure decreases aversive taste of nicotine, increases nicotine withdrawal and reinstatement, but cherry flavor does not alter nicotine’s effects in adolescent rats

Deniz Bagdas 1,2, Lilley Harris 1, Nii A Addy 1,2,3,4,5
PMCID: PMC9948648  NIHMSID: NIHMS1856215  PMID: 36476758

Abstract

Although e-cigarette use among youth is recognized as an epidemic, there is limited understanding regarding nicotine’s orosensory and chronic use effects in youth, and how fruit e-cigarette flavorings may influence nicotine’s effects. We aimed to characterize the orosensory and chronic use effects of nicotine in adolescent rats. We also determined the acute and chronic effects of benzaldehyde, a cherry/berry/almond flavoring, on nicotine’s taste, consumption, withdrawal, and reinstatement. Rats were examined for their acute taste responses to the different nicotine concentrations. The effects of chronic exposure on nicotine’s taste, withdrawal, and reinstatement were also determined. In addition, impact of benzaldehyde on these nicotine use behaviors was evaluated.

While taste responses to low nicotine concentrations did not differ from water, high nicotine concentrations induced aversion. Aversive responses to nicotine that were observed in naïve animals vanished after chronic nicotine exposure, indicating the development of tolerance to nicotine’s aversive taste. Additionally, nicotine abstinence after chronic exposure induced withdrawal. Following abstinence, animals reinstated nicotine use. Further, animals showed higher preference to nicotine after reinstatement, compared to preference values before nicotine withdrawal. Benzaldehyde did not alter nicotine’s taste reactivity, withdrawal, and reinstatement experiments. Some sex differences were found in benzaldehyde’s taste response and choice behavior experiments.

Keywords: nicotine, benzaldehyde, adolescent, taste reactivity, addiction, cherry

1. Introduction

E-cigarette use among U.S. youth is a recognized epidemic [1] and the 2021 National Youth Tobacco Survey showed that 11.3% of high school students and 2.8% of middle school students are current e-cigarette users [2]. Furthermore, 84.7% of current youth e-cigarette users use flavored e-cigarettes, including 85.8% of high school users and 79.2% of middle school users [2]. Although a federal flavor ban (with exceptions for menthol and tobacco flavors) was issued for cartridge-based e-cigarettes in the US (FDA, 2020), flavored e-cigarettes remain widely available in disposable and personalized e-cigarettes. Other tobacco products, such as dissolvable tobacco products and snus, may also contain flavor additives. Flavorants may modulate the aversive sensory properties of nicotine, as well as may increase the appeal of tobacco products [3–5]. Therefore, understanding adolescent nicotine use and dissecting the interaction between flavors and nicotine are critically important.

We previously reported that intraoral nicotine induces both reward and aversive orosensory properties in sex- and concentration-dependent manners in adult rats [6]. Moreover, benzaldehyde (primary chemical of cherry, red berry, and almond flavorings) increased nicotine’s hedonic value and decreased aversive value. Here, we investigated the previously unknown orosensory effects of nicotine in adolescent rats and we examined acute and chronic effects of benzaldehyde on nicotine’s orosensory and rewarding effects.

2. Materials and Methods

2.1. Animals

Adolescent (postnatal day 21; PND21) female and male Sprague Dawley rats (Charles River Laboratories, Wilmington, MA, USA) were housed individually in a temperature-, humidity-, and light-controlled (12-h) husbandry room, with ad libitum food and water. The Yale University Institutional Animal Care and Use Committee (IACUC) approved the study and experiments were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All animal experiments are reported in compliance with the ARRIVE guidelines 2.0 [7].

2.2. Drugs and chemicals

Free base nicotine (#N3876) and benzaldehyde (#B1334, Sigma-Aldrich, St. Louis, MO) were dissolved in tap water, pH adjusted to 7.

2.3. Intraoral catheter implantation and taste reactivity test (TRT)

Rats were implanted with an oral handmade Polyethylene-100 catheter under anesthesia (intraperitoneal 80 mg/kg ketamine HCl and 20 mg/kg xylazine) at PND24. A sterile catheter was inserted lateral to the first maxillary molar and protruded dorsally through the skin, between the ears [8]. The end of the catheter was exteriorized and secured in the back. Post-operative analgesia was provided via daily subcutaneous carprofen (5 mg/kg) injections for two days.

Taste responses to oral infusions were determined via TRT, as previously described [6]. Each TRT session included 20 infusions (100 μl/infusion over 2s) over a 45 min duration. Experiments and analyses were conducted blind to the condition. Tongue protrusions, lateral tongue protrusions, and rhythmic mouth movements were quantified as ingestive responses; head shakes, forelimb flails, and gapes were quantified as aversive responses for 6s after infusions [6]. The summation of ingestive or aversive responses for each individual infusion was determined, and graphed as the average of these summations over 20 infusions. If the mouth was not visible during scoring, data from that infusion was excluded from analysis. Rats with incorrect catheter placements were also excluded.

2.3.1. Characterization of taste responses to oral nicotine in adolescent rats

A wide range of nicotine concentrations (1–100 μg/ml) were examined using a within-subject design, as previously described [6]. Rats were counterbalanced across test solutions and examined between PND28 and PND40, with one day intervals.

2.3.2. Evaluation of benzaldehyde’s acute effects on taste responses to nicotine

Rats were administered nicotine (10 μg/ml) with or without benzaldehyde (100 μg/ml) in the TRT. Rats were counterbalanced across test solutions and examined between PND28 and PND40, with a one-day interval between tests, in a within-subject design.

2.3.3. Evaluation of benzaldehyde’s effects on taste responses to chronic nicotine

Three consecutive experiments were conducted on adolescent rats between PND21 and PND53 (Figure 4). Rats were randomly divided into four groups - water, benzaldehyde (100 μg/ml), nicotine (10 μg/ml), and benzaldehyde plus nicotine, and tested for their taste responses before (PND28–PND34) and after (PND47–PND49) chronic exposure to test solutions using a between-subject design. Fourteen days of chronic exposure was provided via voluntarily intake.

Figure 4: Timeline of chronic exposure study.

Figure 4:

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PND: Postnatal day, TRT: taste reactive test, 2BC: two-bottle choice test.

2.4. Two-bottle free-choice test (2BC)

Rats were given unlimited access to two bottles for 24h, containing either test solution or water. Fluid intake was recorded, and the bottle positions were alternated daily to avoid a side preference. Test solutions were provided to rats in dark bottles and refreshed every three days. Preference to the test solution was calculated as follows: preference (%)=test solution intake/(test solution intake + water intake)x100. A control cage with two bottles, and no rat, was used to determine possible leakage.

2.4.1. Characterization of benzaldehyde and nicotine 2BC in adolescent rats

To determine a concentration of benzaldehyde, rats were given water vs. either water, 20, 100, or 500 μg/ml benzaldehyde in 2BC for three days. Experiments were conducted using a within-subject design between PND28 and rats were counterbalanced across solutions. To assess nicotine’s brain reward effects through withdrawal and reinstatement studies, we aimed to choose a nicotine concentration that could induce stable nicotine consumption during chronic exposure. To determine this concentration, rats were given water vs. either water, 5, 10, or 20 μg/ml nicotine in 2BC for three days with a between-subject design between PND28 and PND31.

2.4.2. Effects of benzaldehyde on nicotine preference in 2BC

In chronic exposure experiments, rats received test solution (water, 100 μg/ml benzaldehyde, 10 μg/ml nicotine, or benzaldehyde plus nicotine) vs water in their home cages between PND35 and PND49. The data were represented the average of 12 days (PND35–PND47).

2.4.3. Evaluation of nicotine withdrawal

On PND51, rats were evaluated for somatic signs of withdrawal following 24 hours of nicotine abstinence in 2BC. Rats were observed for somatic signs - paw and body tremors, head shakes, body shakes, backing, jumps, curls, foot licks, writhes, teeth chattering, cheek tremors, yawns, and ptosis – for 20 min. Next, rats were examined for anxiety-like behaviors in the elevated plus maze (EPM) test AnyMaze (Stoelting Company, Wooddale, IL, USA). Rats were placed in the center of plus-shaped maze with two open and two enclosed arms for a 5 min session. Time spent in the open arms, number of open arm entries, total immobility duration, and number of head dips were quantified.

2.4.4. Evaluation of nicotine reinstatement

To evaluate whether benzaldehyde enhanced nicotine taking behavior when access to nicotine was reinstated, all groups were given water vs. nicotine (10 μg/ml) in 2BC at the end of 48 hours of nicotine abstinence (PND52). Nicotine preference in 2BC during reinstatement was determined on PND53.

2.5. Statistical analyses

All graphing and statistical analyses were performed using GraphPad Prism 8 (Dotmatics, San Diego). Data were evaluated by 2-way repeated measures (RM) or ordinary ANOVA with a post hoc Bonferroni correction, where appropriate. In the TRT nicotine dose response analysis, a 2-way RM ANOVA with Sidak comparison was used, and main effects were corrected by Greenhouse-Giesser (alpha 0.05). A student t test was used for individual comparisons in the reinstatement study. Significance was considered for p < 0.05. Data were expressed as mean ± standard error of the mean (SEM).

3. Results

3.1. Characterization of TRT responses to acute oral nicotine

Intraoral nicotine elicited both ingestive and aversive responses (Figure 1). There was a significant effect of nicotine concentration on ingestive reactions (F2.73,35.5=7.700; p<0.001, Geisser-Greenhouse’s epsilon: 0.547), but no effect of sex (F1,16=1.210; p=0.287, Figure 1A). When nicotine concentrations were increased, ingestive responses decreased. Nicotine at 30 and 100 μg/ml showed significantly lower ingestive responses compared to water, in both females (p=0.044) and males (p=0.015). Increased nicotine concentrations also led to increased aversive taste responses (F3.08,49.9=6.490; p<0.001, Geisser-Greenhouse’s epsilon: 0.616, Figure 1B). Nicotine at 30 μg/ml and 100 μg/ml in females (p=0.001), and at only 100 μg/ml in males, led to significantly higher aversive responses compared to water (p = 0.004).

Figure 1. Orosensory taste responses to nicotine.

Figure 1.

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Rats were evaluated for their ingestive (A) and aversive (B) taste responses to nicotine (1–100 μg/ml) in the taste reactivity test. n=6–9/sex. *p < 0.05 vs. water control (0 μg/ml).

3.2. Characterization of preferences to benzaldehyde and nicotine in 2BC

Dose response analysis of benzaldehyde (20–500 μg/ml) in 2BC test revealed a significant effect of concentration (F3,90=4.10; p=0.008) and a non-significant effect of sex (F1,30=3.12; p=0.087) (Figure 2A). In females, 20 μg/ml benzaldehyde led to a slight, but non-significant increase in preference (p>0.05). In males, all benzaldehyde concentrations resulted in significantly higher preferences compared to the water group (p<0. 05).

Figure 2: Oral benzaldehyde and nicotine consumption.

Figure 2:

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Rats were evaluated for their preference (%) to benzaldehyde (20–500 μg/ml). (A) and nicotine (5–20 μg/ml) (B) in two-bottle choice paradigm. n=16/group/sex (benzaldehyde) and n=10/group/sex (nicotine). *p < 0.05 vs. water control (0 μg/ml).

Dose response analysis of nicotine (5–20 μg/ml) revealed a significant effect of nicotine concentration (F3,72=25.5; p< 0.001) and a non-significant effect of sex (F1,72=2.95; p=0.089) (Figure 2B). Nicotine at 20 μg/ml concentration was significantly less preferred compared to water (p<0.05).

3.3. Effects of acute benzaldehyde exposure on nicotine’s taste responses

Next, we determined the impact of acute benzaldehyde on taste responses to oral nicotine in the TRT. A significant effect of sex (F1,48=21.600; p<0.001), and a non-significant effect of treatment (F3,48=2.100; p=0.113) on ingestive TRT responses were found (Figure 3A). Overall, benzaldehyde did not alter nicotine’s ingestive responses in females (p>0.05). In males, benzaldehyde + nicotine animals showed a non-significant increase (p>0.05) in ingestive responses compared to nicotine alone. Males showed a higher number of ingestive responses compared to females and this reached significance for both the benzaldehyde alone and benzaldehyde plus nicotine groups (p<0.05). For aversive TRT responses, a significant effect of sex (F1,48=6.90; p=0.012) was found, with no-significant effect of treatment (F3,48=1.44; p=0.243) (Figure 3B). Although, females seemed to have higher responses than males, post hoc analyses show no significant differences between sexes (p>0.05, Figure 3B).

Figure 3: Effects of acute benzaldehyde exposure on nicotine’s taste responses.

Figure 3:

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Ingestive (A) and aversive (B) taste responses to oral nicotine (10 μg/ml) in the presence and absence of benzaldehyde (100 μg/ml) in rats. n=6–9/group/sex. *p < 0.05 vs. corresponding sex.

3.4. Effects of chronic benzaldehyde exposure on nicotine taste responses and preference

To test the impact of chronic exposure on TRT, taste responses to test solutions were determined before (pre) and after (post) two weeks of voluntarily intake. In females, ingestive taste responses to water, nicotine (10 μg/ml), benzaldehyde (100 μg/ml) and their combination did not differ from each other before and after chronic exposure (treatment [F3,27=1.46, p=0.248], time [F1,27=1.60, p=0.216]; Figure 5A). However, chronic exposure in females resulted in significant changes in aversive taste responses as seen in treatment (F3,27=3.96, p=0.018) and time (F1,27 = 110.8, p=0.002), with no interaction (F3,27=1.62, p=0.208; Figure 5B). Nicotine and benzaldehyde plus nicotine groups showed a trend toward increases in aversive responses, compared to water, when they were examined before chronic exposure. Aversive taste responses to nicotine were significantly higher than water (p<0.05), but the benzaldehyde plus nicotine combination did not statistically different from water (p>0.05, t=2.18, df=54.0) or nicotine alone (p>0.05, t=1.01, df=54.0) groups (Figure 5B). After chronic exposure, aversive taste response to nicotine were significantly decreased, compared to taste response before chronic exposure (p<0.05). Similarly, aversive taste responses were significantly reduced after chronic exposure in the benzaldehyde plus nicotine group (p<0.05).

Figure 5: Effects of chronic benzaldehyde exposure on nicotine’s taste responses.

Figure 5:

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Ingestive (A, C) and aversive (B, D) taste responses to oral nicotine (10 μg/ml) were tested in the presence and absence of benzaldehyde (100 μg/ml) before and after chronic exposure to the test solutions. n=6–10/group/sex. *p < 0.05 vs. water control. # p < 0.05 vs. corresponding pre-exposure control.

Although chronic exposure did not alter ingestive taste responses of male rats (time [F1,26=0.915, p=0.347]; Figure 5C), a significant effect of treatment was found (F3,26=5.30, p=0.005; Figure 5C). Ingestive taste responses to nicotine were significantly lower than ingestive responses in the water group (p<0.05), before chronic exposure. After chronic exposure, ingestive taste responses to nicotine did not differ from water (p > 0.05). In males, chronic exposure resulted in significant changes in aversive taste responses (treatment [F3,26=1.97, p=0.143], time [F1,26=12.2, p=0.001], with no interaction [F3,26=2.020, p=0.136]; Figure 5D). Although aversive taste responses to nicotine were higher compared to water groups, there were no significant differences before chronic exposure (p>0.05, Figure 5D). After chronic exposure, aversive taste responses to nicotine alone and benzaldehyde plus nicotine solutions were significantly reduced (p<0.05), Figure 5D).

Since chronic exposure was provided via 2BC, the average preference over the first 12 days of exposure was also determined. A significant effect of treatment (F3,54=49.1; p<0.001) and sex (F1,54=4.04; p=0.049), as well as a significant interaction (F3,54=49.1; p<0.001) were found (Figure 6). Nicotine and benzaldehyde plus nicotine combination groups showed lower preferences, compared to water (p<0.05). Male rats also showed greater benzaldehyde consumption compared to their female counterparts (p<0.05).

Figure 6: Effects of chronic benzaldehyde exposure on oral nicotine consumption.

Figure 6:

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Oral consumption of water, benzaldehyde (100 μg/ml), nicotine (10 μg/ml), and benzaldehyde plus nicotine combination were examined in two-bottle choice paradigm. Preference (%) to the solutions were calculated as average of 12 days of consumption. n=6–10/group/sex. *p < 0.05 vs. water control. # p < 0.05 vs. corresponding sex.

3.5. Impact of chronic exposure on nicotine withdrawal

First, physical withdrawal symptoms in female and male rats were determined by quantifying and evaluating somatic signs. A significant effect of treatment (F3,53=44.600; p<0.001) but no effect of sex (F1,53=0.004; p=0.948, Figure 7A) was found. Nicotine alone and benzaldehyde plus nicotine groups showed significantly higher somatic signs (p<0.05) (Figure 7A). Moreover, the number of somatic signs did not differ between females and males (p>0.05, Figure 7A).

Figure 7: Effects of chronic benzaldehyde exposure on nicotine withdrawal.

Figure 7:

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At the end of the chronic exposure, test solutions were replaced with water to determine spontaneous nicotine withdrawal following 24h water exposure. Somatic signs (A) were evaluated as physical signs of withdrawal. The time spent in open arms (B), head dips (C), and immobility time (D) of EPM were determined as anxiety-like behavior associated to withdrawal. n=6–10/group/sex. *p < 0.05 vs. water control.

Next, rats were examined for anxiety-like behaviors in the EPM. Statistical analysis showed a significant effect of treatment (F3,53=10.400; p<0.001) and no effect of sex (F1,53=0.098; p=0.754, Figure 7B). Open arm time decreased in nicotine alone and benzaldehyde plus nicotine groups (p<0.05), however, open arm time did not differ between females and males (p>0.05, Figure 7B). The number of head dips was also altered by treatment (F3,53=25.800; p<0.001), but not by sex (F1,53=0.997; p=0.322, Figure 7C). Number of head dips decreased in nicotine alone and benzaldehyde plus nicotine groups (p<0.05), without any sex difference between females and males (p>0.05). Moreover, total immobility time in the EPM differed by treatment (F3,53=14.800; p<0.001) and sex (F1,53=9.700; p=0.003, Figure 7D). Rats with nicotine alone and or combination groups showed significantly higher immobility time compared to water controls (p<0.05, Figure 7D). Although a statistically significant main effect of sex was found, post hoc analysis showed no difference between females and males (p>0.05, Figure 7D).

3.6. Impact of chronic exposure on nicotine reinstatement

After nicotine withdrawal assessments, rats were given water and nicotine solution in 2BC for reinstatement. Since water and benzaldehyde groups were nicotine naive, we were able to assess the effects of earlier benzaldehyde exposure on nicotine intake. Moreover, the water group represented an acute nicotine exposure in reinstatement study.

In females, when nicotine preference was compared between water and benzaldehyde, no difference was found (t=1.320, df=12, p>0.05; Figure 8A). Further, the impact of chronic exposure on nicotine reinstatement was tested in nicotine alone and in combination groups. Chronic nicotine exposure resulted in significantly greater nicotine preference when rats were given a choice to reinstate (t=3.832, df=18, p=0.001; Figure 8A). Nicotine preference in the nicotine group was also significantly higher than in water group (t=3.556, df=15, p=0.003). Similarly, chronic benzaldehyde plus nicotine exposure led to a higher nicotine preference, compared to the nicotine preference before nicotine withdrawal (t=3.146, df=12, p=0.008; Figure 8A). Nicotine preference in the benzaldehyde plus nicotine combination group was also significantly higher than in water group (t=4.544, df=12, p=0.008; Figure 8A).

Figure 8: Effects of chronic benzaldehyde exposure on nicotine reinstatement.

Figure 8:

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After completion of withdrawal experiments, all rats were given water vs nicotine (10 μg/ml) in two-bottle choice paradigm for 24h. Preference (%) to the nicotine solution in female (A) and male (B) rats was shown and compared to the test solution preference on the last day of chronic exposure. n=6–10/group/sex. *p<0.05 vs. preference to pre-reinstatement solution. # p<0.05 vs. nicotine preference in water group.

In males given a choice of water and nicotine, rats in water and benzaldehyde groups showed similar nicotine preference (t=0.599, df=11, p>0.05; Figure 8B). On the other hand, nicotine alone (t=3.369, df=14, p=0.004; Figure 8B) and benzaldehyde plus nicotine (t=2.657, df=16, p=0.017; Figure 8B) showed higher nicotine preferences after withdrawal, compared the preference before the nicotine withdrawal. Nicotine preference between nicotine and water (t=2.738, df=13, p=0.017), as well as between combination and water (t=2.598, df=14, p=0.021) were also significantly different (Figure 8B). Nicotine preference increased after chronic exposure.

4. Discussion

Here, we found that the orosensory properties of nicotine in adolescent rats were concentration dependent. After chronic nicotine exposure, aversive taste responses to nicotine were no longer present. Benzaldehyde was more preferred by male rats. In contrast to previous findings in adults, neither acute nor chronic benzaldehyde exposure altered nicotine’s orosensory properties, nicotine withdrawal, nor nicotine reinstatement in adolescent rats. These studies represent the first investigation of oral nicotine taste responses in adolescents, revealing effects that differ from those previously observed in our adult investigation [6].

In adult rats, oral nicotine induced both reward and aversion in a sex and concentration dependent manner in TRT [6]. However, intraoral nicotine did not induce reward properties in adolescents. Females were more sensitive, than males, to nicotine’s aversive taste responses at 30 μg/ml. Our adolescent TRT findings are consistent with another study, where only aversive responses were observed using 50 and 100 μg/ml nicotine in male rats [9]. However, the age of rats was not described in that prior study [9]. Previously, nicotine was found to have dose related aversive properties in taste avoidance [10]. Further, nicotine’s irritant and/or olfactory qualities were found to be aversive and its orosensory taste was reported as bitter in mice [11]. Overall, our current adolescent study shows both similarities (as seen in aversive responses) and differences (as seen concentration sensitivity and no sex dependency) to prior nicotine TRT studies in adults.

We also found concentration-dependent aversion to nicotine in 2BC, as reflected in the decreased preference compared to water in both sexes. Previous studies showed low nicotine preferences in adult mice and rats [12–14]. Here in adolescent, and previously in adult [12] rats, 20 μg/ml nicotine was aversive in 2BC. Although sex differences in nicotine consumption have been observed through an interaction between sex and nicotine concentration [15,16], female and male rats showed no differences in their nicotine preferences at any concentrations in this study, consistent with adult rat nicotine consumption studies [12].

We found that adolescent male, but not female, rats preferred benzaldehyde over water. Male rats showed higher benzaldehyde preference, irrespective of concentration. Our results are consistent with prior research indicating that benzaldehyde is preferred and self-administered by C. elegans [17]. A recent study using commercial cherry e-liquid showed that male rats preferred cherry e-liquid at 50% of total fluid intake in 2BC, indicating that cherry flavoring did not induce either reward or an aversion [18] in contrast to our benzaldehyde findings. We note that cherry e-liquids also included other chemicals such as propylene glycol, which could contribute to different outcomes between two studies.

In examining effects of benzaldehyde on nicotine’s taste responses, we found that benzaldehyde did not alter the taste responses to nicotine in adolescent females. However, benzaldehyde did slightly increased the ingestive responses in adolescent males. In adults, benzaldehyde increased ingestive responses in females but not in males, and was able to block nicotine’s aversive responses in both sexes [6]. Together, these results demonstrate that age contributes to effects of benzaldehyde on nicotine’s orosensory properties. Aversive responses to intraoral nicotine, in naïve animals, vanished after chronic nicotine exposure in both sexes, indicating the development of tolerance. Such tolerance is consistent with what has been observed with nicotine’s various effects [10,19,20]. A recent human study showed that liking increased, and disliking decreased, overtime compared to first puff in e-cigarette usage [21].

Little is known about the impact of benzaldehyde on acute and long-term effects of adolescent nicotine use behaviors. Benzaldehyde-containing cherry e-liquids provide a sweet and fruity taste [3,22]. The benzaldehyde + nicotine combination, showed similar results to nicotine alone in 2BC, TRT, withdrawal, and reinstatement experiments. Similar to our findings, a recent study showed that commercial cherry e-liquid did not alter the nicotine preference of male rats in 2BC [18]. Moreover, hedonic rates of cherry flavoring without nicotine were higher than cherry flavoring with nicotine in female and male (aged 21–35) e-cigarette users [22]; indeed, cherry flavoring was recognized as sweetest regardless of the nicotine content in female and male (aged 18–45) e-cigarette users [3]. Our 2BC findings are consistent with these studies.

However, human studies investigating the impact of cherry flavoring on nicotine’s orosensory properties also showed two distinct findings. While some studies showed that cherry flavoring increased nicotine’s liking behaviors [21], other studies found no effect in humans [3,22,23]. Differences in commercial product use, puff topography, device characteristics may underly the differences in human studies. Although our findings revealed that benzaldehyde did not change nicotine’s orosensory properties and nicotine use behaviors, they are consistent with above mentioned studies. Of note, benzaldehyde does not increase ingestive responses in males (Figure 3) despite increased preference for benzaldehyde (Figure 2). Differences may result from olfactory sense involvement: while TRT uses retronasal administration (taste), 2BC uses orthonasal administration (smell and taste). We also note that we used one concentration of benzaldehyde and nicotine, based on our dose response studies. However, different concentrations could reveal differential interactions.

Our study also provides preclinical insights related to clinical oral smokeless tobacco product use. Benzaldehyde didn’t alter nicotine’s orosensory properties or addiction potential in adolescent rats at the concentrations studied here. However, clinical experiments in youth have revealed higher preference for flavored tobacco product use, particularly fruit flavors [5]. It should be noted that we used the primary chemical alone. Flavored commercial tobacco products include several other chemicals, such as sweeteners, rather than the primary chemical alone [24]. Therefore, the total flavor variety, concentration range, and sweetness value may alter liking/disliking behaviors and modulate nicotine’s hedonic responses which can also contribute nicotine’s abuse liability. Future studies, with a range of benzaldehyde/nicotine concentrations, administration routes, or the use of commercial products could reveal additional components impacting benzaldehyde effects on nicotine use behaviors.

Conclusion

Intraoral nicotine at high concentrations was aversive and did not induce reward at any concentrations in adolescent rats. Benzaldehyde did not alter orosensory and central rewarding properties of nicotine. Our recent and previous studies reveal differential effects of benzaldehyde dependent on age. Orosensory properties of nicotine and flavorings clearly exert an important contribution to nicotine addiction in a sex- and age-related fashion.

Highlights.

  • Intraoral nicotine at high concentrations was aversive in adolescent rats

  • Chronic use of nicotine decreased aversive sensory reactions

  • Benzaldehyde, cherry, did not alter orosensory and central properties of nicotine

  • Chronic exposure decreased aversive taste reactions of benzaldehyde plus nicotine

  • Effects of benzaldehyde on nicotine’s taste responses are age dependent

Acknowledgement

Research was supported by grant number [U54DA0361519] from the National Institute on Drug Abuse (NIDA) of the National Institutes of Health (NIH) and Food and Drug Administration (FDA) Center for Tobacco Products (CTP). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or the FDA. The authors have no financial interests or conflict of interests to disclose.

Footnotes

‘Declarations of interest: none’.

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