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Research ArticleBehavioral Pharmacology

Assessment of Abuse-Related Discriminative Stimulus Effects of Nicotine Aerosol in Rodents

Yasmin Alkhlaif and Keith L. Shelton
Journal of Pharmacology and Experimental Therapeutics June 2023, 385 (3) 171-179; DOI: https://doi.org/10.1124/jpet.122.001520

Abstract

The rapid increase in e-cigarette use highlights the importance of developing relevant, predictive animal models exploring their potential health implications. The goal of the present study was to examine the abuse-related effects of brief, repeated e-cigarette aerosol exposures in rodents modeling human e-cigarette user behavior. We evaluated the discriminative stimulus effects of brief, repeated puffs of inhaled nicotine in rats that had been trained to discriminate injected nicotine from saline. Locomotor activity measurement following exposure to injected and aerosolized nicotine was also assessed as an additional behavioral outcome. We hypothesized that the stimulus effects of nicotine aerosol were central nervous system (CNS)-mediated and comparable to that produced by an injected nicotine training stimulus. We further hypothesized that number of aerosol puffs and the e-liquid nicotine concentration which was aerosolized would impact the substitution of nicotine aerosol for injected nicotine. Both nicotine injections and exposures to nicotine aerosol produced a dose-dependent effect on locomotor activity. Nicotine aerosol under our puffing conditions produced e-liquid nicotine concentration-dependent and puff-number-dependent complete substitution for the injected nicotine training condition. The nicotinic antagonist, mecamylamine, completely blocked nicotine-appropriate responding produce by the training dose of 0.3 mg/kg injected nicotine as well as that resulting from exposure to aerosol puffs generated by e-liquid containing 3 mg/ml nicotine, demonstrating that the stimulus of inhaled nicotine was most likely CNS-mediated and not due to olfactory stimulus properties. Overall, the results support the hypothesis that an aerosol exposure drug discrimination model in rodents has applicability to studying the abuse-related effects of e-cigarettes.

SIGNIFICANCE STATEMENT Animal models of nicotine aerosol exposure using testing conditions resembling human e-cigarette use are lacking. In this study, we test a novel preclinical model of nicotine vaping in rodents which allows for the exploration of the abuse-related effects of e-cigarettes. This model has the potential to contribute both to our understanding of the abuse-related pharmacological effects of e-cigarettes as well as aid in the development of rationale, evidence-based e-cigarette regulatory policies.

Introduction

The use of electronic nicotine delivery systems (ENDS or e-cigarettes) continues to escalate, presenting challenges for the development of policies regulating their advertising, promotion, and sales. E-cigarettes are battery-powered devices that produce aerosol from e-liquid, typically containing propylene glycol, vegetable glycerin, flavorings, and nicotine. In 2019, approximately 5.7 million adults in the United States and 4.1 million high school students reported using ENDS (https://www.cdc.gov/mmwr/volumes/69/wr/mm6946a4.htm?s_cid=mm6946a4_w). ENDS are often promoted as a smoking cessation aid (Wang et al., 2021). Unfortunately, ENDS have become a very popular form of nicotine delivery among youths (Singh et al., 2016), including many who have never used combustible cigarettes (https://www.cdc.gov/tobacco/sgr/e-cigarettes/index.htm). Additionally, ENDS use may accelerate conventional cigarette smoking in adults (Chaffee et al., 2018; Berry et al., 2019). While ENDS e-liquids may not contain the same harmful toxins as conventional cigarettes, they do include several chemicals (Sleiman et al., 2016; Hess et al., 2017), the safety of which have not been extensively examined following inhalation (Kitzen et al., 2019). Given these concerns, it is essential that we establish relevant animal models to study the consequences of e-cigarette exposure.

Preclinical rodent models are an important tool in evaluating the health impacts of nicotine and tobacco use (FDA, 2013). The behavioral effects of nicotine in rodents have been assessed using both enteral and parenteral routes of administration. Data have shown that route of administration can have a significant impact on the behavioral and biologic characteristics of nicotine (Matta et al., 2007). Animal models for exploring the abuse-related effects and health consequences of ENDS exposure using parameters which approximate human ENDS use are sparse and have primarily focused on exploring the reinforcing effects of ENDS (Lallai et al., 2021). A second aspect of the abuse-related effects of drugs, including nicotine, are their interoceptive subjective effects. In clinical studies, ENDS users restricted from overnight ENDS use report positive subjective effects, such as a reduction in withdrawal symptoms, reduced craving, relaxation, satisfaction, and euphoria (Dawkins et al., 2016; Mantey et al., 2017; Mantey et al., 2021) as well as negative subjective effects, including dizziness, craving for nicotine, and nausea (Mantey et al., 2021). While it is not possible to model the entire spectrum of human subjective effects in animals, the centrally mediated discriminative stimulus effects can be explored in rodents. The stimulus effects of drugs are pharmacologically selective, dose-dependent, and highly correlated with the abuse-related human subjective effects (Bolin et al., 2016; Perkins et al., 1999). As such, the drug discrimination procedure may be an extremely valuable approach to generating valuable information to help shape data-driven regulatory policy decisions with regard to nicotine delivery devices, including ENDS (Shoaib and Perkins, 2020).

At the present time, we are aware of only one preclinical drug discrimination study examining nicotine aerosol delivered using ENDS (Lefever et al., 2019). The authors demonstrated in mice that exposure to aerosolized nicotine presented in five repeated 1-minute exposure periods, each separated by 2 minutes, only partially substituted for a subcutaneously injected nicotine training stimulus. The authors speculated that some aspect of their aerosol exposure procedure was responsible for the lack of full substitution of aerosolized nicotine for the injected training dose but did not experimentally identify which variables were responsible for this unexpected outcome.

Human clinical studies exploring the physiologic and subjective effects of ENDS typically employ protocols in which a larger number of brief puffs from the ENDS device are self-administered by the subject, which may not be entirely analogous to the fewer but more extended exposures used in the prior rodent study (Spindle et al., 2017). The goal of the present experiment was to more fully examine the stimulus effects of aerosolized nicotine using shorter but more frequent aerosol exposures to more closely mimic these human studies. We hypothesized that the number of puffs as well as the concentration of nicotine in the aerosolized e-liquid solution would modulate the level of substitution of nicotine aerosol for the subcutaneously administered training dose and that the stimulus effects of nicotine aerosol would fully substitute for an injected nicotine training stimulus under the appropriate conditions. To demonstrate that the delivery system of aerosolized nicotine were central nervous system (CNS)-mediated, we examined if substitution of aerosolized nicotine could be blocked by the nicotinic acetylcholine receptor antagonist mecamylamine. Lastly, we explored the time course of the delivery system of aerosolized nicotine to assess both speed of onset of nicotine aerosol stimulus effects as well as their duration.

Materials and Methods

Subjects

Four male and four female Sprague Dawley rats approximately 60 days of age were obtained from Charles River Laboratories (Frederick, MD, USA). Rats were housed individually in polycarbonate microisolator cages on corncob bedding. To promote responding for food reinforcers and prevent obesity, feeding was regulated to maintain a healthy weight of approximately 90% of free-feeding weight. Water was available ad libitum except during experimental sessions. All rats were housed in a temperature- and humidity-controlled room and were maintained on a 12-hour reversed light/dark cycle (lights on from 6:00 PM to 6:00 AM). Experiments were conducted during the dark phase. The animal facilities at Virginia Commonwealth University are fully accredited by the American Association for the Accreditation of Laboratory Animal Care, and all experiments were approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University.

Compounds

(−)-Nicotine hydrogen tartrate salt (Sigma-Aldrich, St. Louis, MO) for injection was dissolved in physiologic saline (ICU Medical, Inc., Lake Forest, Illinois). The pH of nicotine solutions given by injection were adjusted to 7.4 with dilute NaOH. (−)-Nicotine free base (Sigma-Aldrich, St. Louis, MO) used for aerosol administration was mixed in an e-liquid vehicle of 50:50 USP propylene glycol and glycerin (Sigma-Aldrich). Mecamylamine hydrochloride (Thermo Fisher Scientific, Ward Hill, MA) was dissolved in sterile 0.9% NaCl solution. Doses of nicotine and mecamylamine for subcutaneous injection are expressed as mg/kg of the salt. Nicotine was injected subcutaneously at a volume of 1 ml/kg of body weight 10 minutes before the start of the operant session. Mecamylamine injections were given 15 minutes before nicotine administration in a volume of 1 ml/kg of body weight. Concentrations for aerosol administration are expressed as mg/ml of the e-liquid used for aerosolization.

E-cigarette Aerosol Generation and Exposure System

Aerosol exposures were conducted using an aerosol inhalation system designed in-house. The system was composed of a whole-body rodent exposure chamber into which nicotine aerosol generated by a commercial ENDS device could be pumped, contained and then rapidly evacuated using a simple automated control system. The rectangular exposure chamber was constructed of 3/8” clear acrylic, measuring 24.4 × 17.5 × 25.5 cm, with a total volume of 10.89 L. The door of the chamber contained an 8 × 8 cm, 24 v DC muffin exhaust fan with a wire mesh covered 3D printed self-closing louvered grate. Also attached to the chamber were 3D printed holding attachments for the ENDS e-cigarette device (Smok MORPH) and an e-liquid atomizer tank. The atomizer tank (Innokin iSubV Vape Tank) was fitted with an iSub Innokin SS BVC 0.5-ohm stainless steel vaporizer coil. The ENDS atomizer tank was modified by filling the air vents with epoxy and drilling and tapping a hole in the metal base of the tank. A hose barb was screwed into the threaded hole. These modifications allowed the atomizer tank to be pressurized by air supplied from a 12 v DC diaphragm air pump. A clear length of 3/8” Tygon tubing attached to the mouthpiece of the tank captured aerosol emitted by the pressurized atomizer and directed it into the rodent exposure chamber. The function of the aerosol exposure system was controlled by an Arduino Uno single-board computer and three electromechanical relays which controlled the air pump, exhaust fan and ENDS firing switch. The air pump was calibrated by a precision flowmeter to deliver a flow rate of 1 L/min to the atomizer tank when activated. The exhaust fan was rated to produce a free-airflow rate of 1.48 cubic meters/min. The timing of the activation of the exhaust fan, ENDS and air pump relays were controlled by a custom-written C++ program uploaded to the Arduino from a laptop PC computer. Spent aerosol was exhausted directly into a laboratory fume hood.

Drug Discrimination Apparatus

Drug discrimination sessions were conducted in seven standard operant conditioning chambers (Med-Associates, St. Albans, VT). Each chamber was equipped with a lever on the right and left side of the front chamber wall. Above each lever was a yellow LED stimulus lamp. A food pellet dispenser located outside the chamber delivered 45 mg food pellets to a receptacle located between the two levers (F0021; Bioserv, Frenchtown, N.J., USA). A single 5-watt houselight was located at the top center of the chamber rear wall. The operant conditioning chambers were individually housed in sound-attenuating and ventilated cubicles. Drug discrimination schedule conditions and data recording were accomplished using a Med-associates interface and Med-PC version 4 control software running on a PC-compatible computer (Med-Associates, St. Albans, VT).

Discrimination Training

Rats were trained in 15-minute daily (M–F) sessions to respond on one lever following subcutaneous administration of 0.3 mg/kg nicotine (pretreatment time 10 minutes) and to respond on the alternate lever following subcutaneous saline administration. Designation of drug and vehicle levers were fixed for the individual animal but counterbalanced across subjects such that the drug lever was the right lever for four rats and the left lever for four rats. Responding on the correct lever in each daily session was reinforced with 45 mg sucrose pellets (Bioserve, Flemington, NJ). Initially, the fixed ratio value (FR) for each pellet delivery was FR1 until reliable responding occurred. The FR value was subsequently incremented over sessions to FR16. Responses on the incorrect lever reset the ratio requirement on the correct lever. Daily injections were administered on a double alternation sequence of training drug and vehicle (e.g., nicotine, nicotine, saline, saline). Training sessions were conducted until the rats met acquisition criteria consisting of: (1) the first completed FR16 was on the correct lever and (2) ≥80% of the total responding occurred on the correct lever for 8 of 10 consecutive training sessions. To maintain accurate stimulus control, as the study progressed the FR value for 1 rat was increased to FR24.

Substitution Test Procedure

Substitution test sessions were conducted twice weekly on Tuesday and Friday, providing that the rats continued to exhibit accurate stimulus control as shown by maintaining correct first FR lever selection and ≥80% percent overall correct-lever responding on the intervening Monday, Wednesday, and Thursday training sessions. Incorrect responding during a training session resulted in suspension of testing and additional daily training until the first FR was emitted on the correct lever and correct-lever responding was ≥80% percent on at least two consecutive days, which included both a vehicle and drug training day. Drug discrimination test sessions were identical to training sessions with the exception that completion of an FR on either lever resulted in food pellet delivery. Between substitution tests, the double alternation sequence of subcutaneously injected nicotine and saline training sessions was continued. Doses or concentrations of each compound were generally tested in ascending order. Completion of the subcutaneously injected nicotine dose effect curve (saline, 0.01, 0.03, 0.056, 0.1, 0.3, 0.56 mg/kg nicotine) was followed by concentration-response curves with aerosolized nicotine (0.01, 0.03, 0.1, 0.3,1, 1.7, 3 mg/ml nicotine e-liquid). For substitution tests with nicotine aerosol, the sessions were preceded by an aerosol puff exposure cycle using a single e-liquid concentration. A puff was defined as a single brief ENDS exposure cycle comprised of 10-second aerosol of aerosol generation, a 10-second hold during which aerosol was allowed to remain in the chamber, and a 10-second fan-forced evacuation of the nicotine aerosol from the chamber. All testing was done at an ENDS power setting of 36 W. A total of 10 consecutive puffs were administered in each aerosol exposure experiment with the exception of the experiment examining the effect of puff number. In that experiment, the total number of consecutive puffs of 3 mg/ml nicotine containing e-liquid was varied between 1–20. In all cases, following the completion of the last puff, the subject was immediately removed from the exposure chamber and placed into the operant chamber for substitution testing. For antagonism tests with mecamylamine, rats were subcutaneously injected with mecamylamine (0.1, 0.32, and 0.56 mg/kg) or saline, 15 minutes before receiving injected nicotine which was administered 10 minutes prior to the operant test session. In the case of aerosolized nicotine tests, rats were injected with mecamylamine 15 minutes prior to being exposed to the aerosolized nicotine puffing cycle. For the time course tests with nicotine aerosol, substitution test sessions were conducted after increasing post-exposure delays (1, 10-, 20-, 40- and 60-minute) following the completion of 10 puffs of aerosol generated from 3 mg/kg nicotine e-liquid. Prior to each concentration-effect or dose-effect curve, two control substitution test sessions were conducted, one with the training dose of injected nicotine (0.3 mg/kg) and a second with saline. For dose response curves that include nicotine aerosol, exposure to the aerosolization vehicle (propylene glycol:vegetable glycerin, PG:VG) was tested as an additional control. At least 7 rats were used to generate each dose- or concentration-effect curve.

Locomotor Activity Assay

Open field locomotor activity experiments were conducted in four Plexiglas open field arenas (43.5 cm × 43.5 cm × 30.5 cm) enclosed within sound-attenuating cubicles (Med-Associates Inc.). Each chamber was equipped with three 16-beam infrared arrays, which tracked distance traveled (cm). Data were collected via Activity Monitor software (version 7; Med-Associates). Rats were tested with nicotine twice weekly on Tuesday and Friday. On Mondays and Thursdays, rats underwent drug-free 1-hour habituation sessions. On test days, rats were administered a single nicotine tartrate (s.c.) dose in ascending order (vehicle, 0.1, 0.3, 0.56, 0.7, and 1 mg/kg) prior to each locomotor activity session. Ten minutes post-injection, the animals were placed into the locomotor chamber for 1 hour. On nicotine aerosol testing days, rats were exposed to the same ten 30-second puffs of nicotine aerosol (0.01, 0.1, 1, 10, and 30 mg/ml nicotine e-liquid concentration) or vehicle (50/50 PG:VG) and immediately placed into the locomotor activity chambers for 1 hour. As in the drug discrimination experiments, the ENDS power setting remained at a constant 36 W.

Data Analysis

For locomotor activity tests, distance traveled in cm for the entire 60-minute session was recorded and then compared with the vehicle control using two independent repeated-measures one-way ANOVAs followed by Sidak’s multiple comparisons post-hoc tests on significant main effects. For drug discrimination sessions, nicotine- and vehicle-lever responses, reinforcers earned and first fixed ratio values were recorded for each animal. Group means (±S.E.M.) were calculated for percentage nicotine-lever responding and response rate under each test condition. The percent nicotine-appropriate lever responding during the entire test session was used as measures of the ability of a test condition to substitute for the 0.3 mg/kg subcutaneous nicotine training dose. Any aerosol exposure condition or injected drug dose that suppressed response rates to the extent that the animal did not complete a fixed first ratio resulted in the exclusion of that rat’s data from the lever selection analyses, although that animal’s data were included in the response rate determination. The response rate for each test concentration or dose was expressed in responses/s. A criterion of 80% or greater nicotine-lever responding was selected to indicate full substitution for the nicotine training dose. Mean nicotine-lever responding between 20 and 79% was defined as partial substitution. Mean nicotine-lever responding of less than 20% was considered to be evidence of no substitution for the nicotine training condition. When possible, EC50 or ED50 values (and 95% confidence limits) for nicotine-lever selection were calculated based on the linear portion of each dose–effect curve. Calculations were performed using a Microsoft Excel spreadsheet on the basis of SAS Pharm/PCS version 4 (Tallarida and Murray, 1986). The ability of mecamylamine to reduce or block nicotine-like discriminative stimulus effects was determined by comparison of results following mecamylamine pretreatment with the nicotine control using repeated measures one-way ANOVAs and Sidak’s multiple comparison post-hoc tests.

Results

Drug Discrimination

A mean of 40.8 ± 7.1 total training sessions (26–49 session range) were required to reach acquisition criteria. The 4 male rats acquired injected nicotine (0.3 mg/kg subcutaneous) versus saline discrimination in a mean of 44 ± 1.4 training sessions (range 43–46 sessions). The 4 female rats acquired injected nicotine discrimination in a mean of 37.8 ± 9.4 training sessions (range 26–49 sessions). The upper panel of Fig. 1 shows the injected nicotine dose-response curve. Injected nicotine dose-dependently substituted for the nicotine training condition with an ED50 value of 0.04 mg/kg (CL: 0.03–0.055 mg/kg). The training dose of 0.3 mg/kg nicotine as well as a higher 0.56 mg/kg dose fully substituted for the 0.3 mg/kg subcutaneous nicotine training condition. Intermediate doses (0.03, 0.056, 0.1 mg/kg of injected nicotine) resulted in partial substitution for the nicotine training condition. The lowest dose of 0.01 mg/kg nicotine resulted in less than 20% nicotine-lever responding. A repeated measures (RM) one-way ANOVA showed that rates of operant responding were not altered by injected nicotine [F(7, 49) = 0.2828, P = 0.957, n = 8].

Fig. 1.
Fig. 1.

Dose-effect curve for injected nicotine in eight rats trained to discriminate 0.3 mg/kg of subcutaneous nicotine from saline. Points above SAL (open circle) and NIC (open square) represents the results of the subcutaneous saline and 0.3 mg/kg subcutaneous nicotine control sessions. Mean (±S.E.M.) percentage nicotine-lever selection across increasing doses of nicotine is shown in filled circles. Percent drug lever selection is shown in the upper panel. Mean (±S.E.M.) response rates in responses per second are shown in the lower panel.

The upper panel of Fig. 2 shows the concentration-effect curve for % nicotine-lever responding for nicotine aerosol generated from increasing nicotine e-liquid concentrations. Exposure to ten puffs of nicotine aerosol at an ENDS output setting of 36 W produced e-liquid nicotine concentration-dependent substitution for the injected nicotine training condition with an e-liquid nicotine EC50 of 0.08 mg/ml (CL: 0.044–0.15 mg/ml). Nicotine aerosol generated by e-liquid nicotine concentration of 1, 1.7, and 3 mg/ml produced greater than 80% nicotine-associated lever responding. Aerosol generated by nicotine e-liquid concentrations of 0.03, 0.1, and 0.3 mg/ml resulted in partial substitution for injected nicotine training condition. The partial substitution exhibited primarily from differing proportions of rats exhibiting responding predominantly on the nicotine or vehicle lever rather than from rats splitting total responding within a test between the nicotine and vehicle levers. Aerosol generated from the lowest nicotine e-liquid concentration; 0.01 mg/ml failed to substitute for injected nicotine. The bottom panel of Fig. 2 shows the effects of nicotine aerosol on rates of operant responding. A RM one-way ANOVA demonstrated that nicotine aerosol generated from e-liquid nicotine concentrations up to 3 mg/ml did not have significant effects on rates of operant responding [F (9,63) = 0.5739, P = 0.8134, n = 7].

Fig. 2.
Fig. 2.

Concentration-effect curve for nicotine-containing e-liquid in eight rats trained to discriminate 0.3 mg/kg of subcutaneous nicotine from saline. Point above SAL (open circle) NIC (open square) and PG:VG (open triangle) represents the results of subcutaneously injected saline, 0.3 mg/kg subcutaneously injected nicotine and vehicle vapor control sessions. Mean (±S.E.M.) percentage nicotine-lever selection following exposure to aerosol produced by increasing nicotine e-liquid concentrations is shown in filled circles. Percent drug lever selection is shown in the upper panel. Mean (±S.E.M.) response rates in responses per second is shown in the lower panel.

The upper panel of Fig. 3 illustrates the effect of pretreatment with the non-selective nicotinic receptor antagonist mecamylamine on the discriminative stimulus produced by the training dose of 0.3 mg/kg injected nicotine. A repeated measure one-way ANOVA revealed a statistically significant decrease in percent nicotine-associated lever responding produced by 0.3 mg/kg injected nicotine after mecamylamine pretreatment [F(3,21) = 7.327, P = 0.0015, n = 8]. Holm-Sidak’s post-hoc tests showed that doses of 0.32 and 0.56 mg/kg mecamylamine significantly attenuated nicotine-lever selection compared to nicotine + vehicle. The lower panel of Fig. 3 shows operant response rates after administration of saline + saline, saline + 0.3 mg/kg nicotine, 0.56 mg/kg mecamylamine + saline and 0.3 mg/kg nicotine combined with 0.1, 0.32 or 0.56 mg/kg mecamylamine. An RM one-way ANOVA showed that mecamylamine combined with nicotine did not significantly alter rates of operant responding compared with saline [F(3,21) = 0.4324, P = 0.730, n = 8]. Paired t tests demonstrated that saline + 0.56 mg/kg mecamylamine did not alter response rates compared with saline [t = 2.017, P = 0.0835], but saline + 0.3 mg/kg nicotine response rates were significantly higher than rates of responding following saline + saline administration [t = 2.940, P = 0.0217].

Fig. 3.
Fig. 3.

Dose-effect curve for 0.3 mg/kg subcutaneous nicotine + subcutaneous mecamylamine in 8 rats trained to discriminate 0.3 mg/kg subcutaneous nicotine from subcutaneous saline. Point above SAL+SAL (open circle), MEC+SAL (filled triangle) and SAL+NIC (open square) represents the results of saline+saline, 0.56 mg/kg mecamylamine+saline and saline+0.3 mg/kg nicotine control sessions. Mean (±S.E.M.) percentage nicotine-lever responding for 0.3 mg/kg injected nicotine combined with increasing doses of mecamylamine is shown in filled circles. Percent drug lever selection are shown in the upper panel. Mean (±S.E.M.) response rates in responses per second are shown in the lower panel. * Indicates statistically significant (P < 0.05) difference from saline+0.3 mg/kg nicotine control session. # Indicates a statistically significant difference (P < 0.05) from the saline+saline control condition.

The upper panel of Fig. 4 illustrates the effect of pretreatment with the non-selective nicotinic receptor antagonist mecamylamine on the discriminative stimulus produced by 10 puffs of nicotine aerosol generated from 3 mg/ml nicotine e-liquid. A repeated measure one-way ANOVA demonstrated that there was a statistically significant decrease in percent drug-lever responding produced by nicotine aerosol after mecamylamine pretreatment [F(3,18) = 4.447, = P = 0.0166, n = 8]. Holm-Sidak’s post-hoc tests revealed that 0.56 mg/kg mecamylamine significantly attenuated nicotine aerosol substitution compared to 3 mg/ml nicotine aerosol + vehicle. The lower panel of Fig. 4 shows operant response rates after administration of 0.56 mg/kg mecamylamine + saline, saline + 3 mg/ml nicotine aerosol and 3 mg/ml nicotine aerosol combined with 0.1, 0.32, or 0.56 mg/kg mecamylamine. An RM one-way ANOVA showed that mecamylamine combined with nicotine aerosol did not significantly alter rates of operant responding compared with nicotine aerosol + saline [F(3,18) = 0.2850, P = 0.8356, n = 8]. A paired t test showed that there was no significant difference in response rates between mecamylamine + saline and nicotine aerosol + saline [t = 0.3637, P = 0.7286, n = 8].

Fig. 4.
Fig. 4.

Dose-effect curve for 10 puffs of 3 mg/ml nicotine e-liquid + mecamylamine in eight rats trained to discriminate 0.3 mg/kg subcutaneous nicotine from saline. Point above MEC+SAL (filled triangle), NIC+SAL (open square) in the upper panel represents the results of 0.56 mg/kg subcutaneous mecamylamine + subcutaneous saline and 0.3 mg/kg subcutaneous nicotine+saline control sessions. Mean (±S.E.M.) percentage nicotine-lever responding after 10 puffs of aerosol generated from 3 mg/ml nicotine e-liquid combined with increasing doses of mecamylamine are shown in filled circles in the upper panel. Mean (±S.E.M.) response rates in responses per second are shown in the lower panel. * Indicates statistically significant (P < 0.05) difference from the saline+0.3 mg/kg nicotine control session.

Fig. 5 (upper panel) shows percent nicotine-lever responding produced by increasing numbers of puffs of nicotine aerosol generated from 3 mg/ml nicotine containing e-liquid. There was a puff-dependent increase in nicotine-lever selection. The 1- and 3-puff conditions produced 15% and 18% nicotine-appropriate responding, respectively. The 7-, 10- and 15-puff conditions produced partial substitution (43%, 61.2%, and 71.3%, respectively) and the greatest, 20-puff, condition engendered 96% nicotine-lever selection. An RM one-way ANOVA revealed that there was no significant difference in rates of responding following any of the nicotine puff exposures [F(6,24) = 1.040, P = 0.4136, n = 8] compared to the 50%VG/50%PG aerosol control condition (Fig. 5, lower panel).

Fig. 5.
Fig. 5.

Percentage nicotine lever-selection resulting from increasing number of puffs of nicotine aerosol produced by 3 mg/ml nicotine e-liquid. Point above SAL (open circle) NIC (open square) and PG:VG (open triangle) represent the 1 ml/kg subcutaneous saline, 0.3 mg/kg subcutaneous nicotine and aerosolized 50% PG:50%VG control sessions. Mean (±S.E.M.) percentage nicotine-lever responding after 1, 3, 7, 10, 15, or 20 puffs is shown in the upper panel (filled circles). Mean (±S.E.M.) response rates in responses per second for each test are shown in the lower panel.

Fig. 6 (upper panel) shows the effect of increasing the delay between exposure to 10 puffs of aerosol generated by 3 mg/ml nicotine e-liquid and the subsequent initiation of the discrimination test session on percent nicotine-lever responding. At a delay of 1 minute, nicotine aerosol produced 69% nicotine-lever responding. Nicotine-lever selection of 81.3% and 86.3% were produced at 10- and 20-minute delay, respectively. An RM one-way ANOVA demonstrated that there was a significant effect of puff delay on nicotine-associated lever selection [F(6,36) = 14.42, P < 0.0001, n = 7]. Holm-Sidak’s post-hoc tests revealed that the 3 mg/kg subcutaneous nicotine control as well as 1-, 10-, and 20-minute delays following 10 puffs of aerosol generated from e-liquid containing 3 mg/ml nicotine resulted in significantly greater nicotine-lever selection than the saline control condition. An RM one-way ANOVA showed that there were no differences in operant response rates (Fig. 6, lower panel) across the control conditions and the different delays [F(6,36) = 0.3588, P = 0.9000, n = 7].

Fig. 6.
Fig. 6.

Effect of increasing post-exposure delays on the discriminative stimulus effects resulting from 10 puffs of aerosol generated from 3 mg/ml nicotine e-liquid in eight rats trained to discriminate 0.3 mg/kg subcutaneous nicotine from saline. Point above SAL (open circle) and NIC (open square) represents the results of the 0.3 mg/kg subcutaneously injected nicotine and subcutaneous saline control sessions. Mean (±S.E.M.) percentages nicotine-lever responding at post-exposure discrimination session test delays of 1, 10, 20, 40, and 60 minutes are shown the upper panel (filled circles). Mean (±S.E.M.) response rates are shown in the lower panel. * Indicates statistically significant (P < 0.05) difference from the saline control session.

Locomotor Activity

The locomotor assay was used to provide a comparison of the acute behavioral effects of nicotine following administration via subcutaneous injection versus aerosol inhalation. The upper panel of Fig. 7 shows the effects of injected nicotine on distance traveled in each 60-minute test session. An RM one-way ANOVA revealed that there was a statistically significant increase in locomotor activity after S.C. nicotine injections [F(5, 35) = 11.9, P < 0.0001, n = 8]. Holm-Sidak’s post-hoc tests revealed that doses of 0.56, 0.7, and 1 mg/kg all produced significant increases in distance traveled compared with injected saline. The dose of 0.56 mg/kg S.C nicotine produced the maximum distance traveled of 161.5 m compared with 36.7 m following saline administration. The lower panel of Fig. 7 shows the concentration-effect curve for locomotor activity following exposure to aerosol produced from e-liquid containing increasing concentration of nicotine. An RM one-way ANOVA found that there was a statistically significant increase in locomotor activity after inhalation of nicotine aerosol [F(5,35) = 5.401, P = 0.0009, n = 8]. Holm-Sidak’s post-hoc tests revealed significant increases in locomotor activity following exposure to 10 puffs of nicotine aerosol generated by 0.1, 1, and 10 mg/ml nicotine-containing e-liquid relative to vehicle (PG:VG) exposure. The concentration of 1 mg/ml inhaled nicotine produced the maximum distance traveled, 212.1 m, comparing to 54.01 m after exposure to nicotine-free control aerosol.

Fig. 7.
Fig. 7.

Dose-effect curve for subcutaneous nicotine and concentration-effect curve for inhaled nicotine aerosol in eight rats tested for locomotor activity responses. Mean (±S.E.M.) meters traveled in the 60-minute test session for subcutaneous nicotine doses of 0.1, 0.3, 0.56, 0.7, and 1 mg/kg (filled circles), and 1 ml/kg subcutaneous saline pretreatment control session (open circle) are shown in upper panel. Mean (±S.E.M.) meters traveled following exposure to 10 puffs of aerosol produced by 0.01, 0.1, 1, 10, and 30 mg/ml nicotine e-liquid (filled squares) and PG:VG aerosol vehicle (open triangle) are shown in the lower panel. * Indicates statistically significant (P < 0.05) difference from saline or vehicle aerosol control.

Discussion

Prior studies have used a variety of nicotine inhalation systems to expose rodents to combustible cigarettes (Binns et al., 1976; Moir et al., 2008), nicotine vapor (George et al., 2010), and nicotine aerosol (Shao et al., 2017, 2013). Our e-cigarette aerosol generation and exposure system were specifically designed to build upon prior work by introducing and examining puffing conditions more closely modeling human e-cigarette use. To demonstrate that our delivery system could effectively deliver meaningful nicotine doses, we compared the acute behavioral effects of exposure to nicotine aerosol to injected nicotine using a locomotor activity assay. Subcutaneous doses of nicotine from 0.56 to 1 mg/kg as well as 10 puffs of aerosolized nicotine at e-liquid nicotine concentrations of 0.1 to 10 mg/ml produced statistically significant increases in locomotor activity. The maximum effects of aerosolized nicotine on locomotor activity were at least as great as maximum effects of injected nicotine, strongly supporting the conclusion that our system was an effective nicotine delivery device and that our initial exposure parameters were adequate to produce meaningful brain nicotine concentrations.

Exposure conditions in animal studies with nicotine aerosol have varied widely, and it is unclear how experimental parameters, such as exposure duration, nicotine concentrations in the aerosolized e-liquid, ENDS wattage setting and e-liquid vehicle composition, among others, impact abuse-relevant endpoints. Drug discrimination outcomes are CNS-mediated and determined by the similarity of the test condition to that of the training stimulus (Swedberg, 2016). As such, we hypothesized that drug discrimination would be an ideal means of differentiating vaping variables with abuse-relevant impact from those with less clinical relevance. Injected nicotine drug discrimination doses in rats have varied but usually fall within a range of 0.01 to 0.56 mg/kg. We chose to target a nicotine training dose on the lower end of this spectrum to increase the sensitivity of the assay (Stolerman et al., 2011). As expected from the extensive literature (Rosecrans, 1989; Stolerman et al., 1999; Wooters et al., 2009, Lefever et al., 2019), the discriminative stimulus effects of injected nicotine in the present study were orderly and dose dependent (Fig. 1).

The ability of inhaled drugs to fully substitute for an injected drug training stimulus has been repeatedly demonstrated for abused inhalant vapors, volatile anesthetic vapors and anesthetic gasses (for review, see Shelton, 2018). It is therefore interesting that a recent study (Lefever et al., 2019) reported that inhaled nicotine aerosol only engendered partial substitution (46–49%) for an injected nicotine training condition in mice. In contrast, in the present study, nicotine aerosol fully substituted for the 0.3 mg/kg injected nicotine training dose. In both our study and the prior experiment each ENDS activation was 10 s in duration but the exposure methods differed significantly in other respects. Specifically, Lefever et al. allowed the aerosol from each puff to remain in the chamber for 1 minute, removed the animal from the chamber for 2 minutes, and then repeated this sequence five times before the discrimination test session. In contrast, to the extent possible mimic the conditions present in human laboratory studies with e-cigarettes (Spindle et al., 2017) our puff sequences used a greater number of puffs, shorter interpuff interval and reduced puff exposure duration.

It is possible that the reduced latency between repeated aerosol exposures and subsequent testing could have produced the more robust aerosol substitution demonstrated in the present study as compared with the prior report. In our time course experiment (Fig. 6), we showed that full substitution of aerosolized nicotine for the injected training stimulus occurred for up to 20 minutes after the cessation of exposure, suggesting that timing alone may not be a sufficient explanation for our more robust stimulus effects. The stimulus effect of nicotine aerosol do, however, appear to be of shorter duration than that of injected nicotine based on data showing that doses of 0.2 and 0.4 mg/kg resulted in at least partial substitution for as long as 160 minutes after injection (Hirschhorn and Rosecrans, 1974). Although brief, the onset of the stimulus effects of aerosolized nicotine were quite rapid, with high levels of partial substitution occurring in as little as 1 minute post-exposure, supporting the conclusion that in rats, as in humans, there is rapid CNS penetration of aerosolized nicotine following inhalation. There are a number of additional differences between the present study and the previous report, including ENDS make and model, nicotine e-liquid concentration used to produce aerosol, species, and training dose. Any one or a combination of these factors may have played a role in the discrepant results. Regardless of the controlling variables involved, our results support that hypothesis that ENDS exposure parameters impact CNS-mediated behavioral outcomes.

Nicotine e-liquid concentration and number of puffs are considered two parameters that directly impact nicotine delivery in e-cigarette users (Lopez et al., 2016; St.Helen et al., 2016). In the present study, increasing the number of puffs increased the level of substitution of nicotine aerosol for the injected training dose. Likewise, e-liquid nicotine concentration also concentration-dependently increased nicotine-lever selection with concentrations of as low as 0.3 mg/ml producing partial substitution and 1–3 mg/ml nicotine e-liquid producing full substitution for the 0.3 mg/kg subcutaneous nicotine training dose. This would suggest that the efficacy of aerosolized nicotine as a discriminative stimulus is high given as few as 10 puffs of 1 mg/ml nicotine produces comparable interoceptive stimulus effects as that resulting from a moderate dose of injected nicotine. This finding is of particular importance given the nicotine concentrations present in human e-liquids sold to be used in devices similar to that employed in the present study can reach 20 mg/ml or greater (Raymond et al., 2018). Our results align with human studies that that suggest that simply mandating lower nicotine concentrations in e-liquids as was done by the European Union may not reduce their subjective effects sufficiently to impact usage (Kosmider et al., 2018). These data confirm that two variables identified as impacting the abuse-related effects of ENDS in human users can be systematically explored in rodents using the drug discrimination procedure. As such, the role of other variables identified to be important in humans ENDS users such as ENDS wattage, vehicle composition, additives and flavors can likewise be examined to determine if they also have a significant impact on the subjective effects of aerosolized nicotine.

Consistent with the literature, we demonstrated that the stimulus effects of 0.3 mg/kg injected nicotine are blocked by 5.6 mg/kg of the non-selective nicotinic acetylcholine receptor antagonist mecamylamine (Jutkiewicz et al., 2011; Gasior et al., 1999). Mecamylamine blockade of the stimulus effects of nicotine has been interpreted as evidence that the discriminative stimulus of injected nicotine is CNS-mediated. Similarly, 0.56 mg/kg of mecamylamine completely blocked the ability of 10 puffs of aerosolized e-liquid containing 3 mg/ml nicotine to substitute for injected nicotine. These data support the conclusion that inhaled nicotine substitution is also mediated by nicotinic acetylcholine receptors and that the injected nicotine training dose and aerosolized nicotine dose chosen for the antagonist study have at least roughly similar potencies for producing the stimulus cue.

Recently, the U.S. Food and Drug Administration expanded their regulatory scope to ENDS in a process known as “deeming.” Deeming emphasized data-driven conclusions focusing on the possible risks of ENDS use, spotlighting the need for additional preclinical research to explore the underlying mechanisms. Our new results demonstrate that our puff exposure methodology, designed to more closely model vaping behavior in humans, produces CNS-mediated discriminative stimulus effects comparable to those produced by injected nicotine in rodents and support the conclusion that drug discrimination can serve as a sensitive, translational measure for exploring variables which impact the abuse liability of ENDS. For example, drug discrimination may be valuable for investigating how flavors, e-liquid additives, nicotine concentrations, ENDS device type, and other factors impact the abuse-related effects of nicotine aerosol. Our data showing that the stimulus effects of nicotine aerosol are blocked by a nicotinic receptor antagonist also support a role for drug discrimination in evaluating existing pharmacotherapies for their utility in ENDS cessation therapy as well as developing new medications for this purpose.

In conclusion, the present study provides strong evidence that the discriminative properties of inhaled nicotine are equivalent to that of injected nicotine in rats. Several parameters such as number of puffs, concentration of nicotine solution used for aerosolization, and pretreatment time played key roles in modulating the discriminative stimulus of inhaled nicotine. These data support the hypothesis that this new e-cigarette aerosol exposure system coupled with the well validated drug discrimination paradigm may be a valuable rodent model for exploring the abuse-related effects of e-cigarettes. This strategy may be particularly useful for assessing the likely outcomes of regulatory strategies designed to retain the potential utility of e-cigarettes as smoking cessation aids and as harm reduction tools while minimizing the potential of e-cigarette abuse in non-smokers.

Acknowledgments

The authors would like to thank Kate Nicholson for her excellent technical support.

Authorship Contributions

Participated in research design: Alkhlaif, Shelton.

Conducted experiments: Alkhlaif.

Performed data analysis: Alkhlaif, Shelton.

Wrote or contributed to the writing of the manuscript: Alkhlaif, Shelton.

Footnotes

    • Received November 21, 2022.
    • Accepted February 24, 2023.

  • This work was supported by the National Institutes of Health National Institute of Drug Abuse [Grant R21-DA04842].

  • No author has an actual or perceived conflict of interest with the contents of this article.

  • dx.doi.org/10.1124/jpet.122.001520.

Abbreviations

CNS
central nervous system
ENDS
electronic nicotine delivery systems
FR
fixed ration
PG:VG
propylene glycol:vegetable glycerin
RM
repeated measures

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Research ArticleBehavioral Pharmacology

Stimulus Effects of Nicotine Aerosol

Yasmin Alkhlaif and Keith L. Shelton
Journal of Pharmacology and Experimental Therapeutics June 1, 2023, 385 (3) 171-179; DOI: https://doi.org/10.1124/jpet.122.001520
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