Rapid nicotine tolerance and cross-tolerance to varenicline in rhesus monkeys: drug discrimination

Abstract

Acute tolerance to effects of nicotine plays an important role in nicotine dependence, but the mechanism underlying these effects is unclear. Drug discrimination was used in the current study to examine the impact of nicotine pretreatment on sensitivity to the discriminative stimulus effects of nicotine and the FDA-approved smoking cessation pharmacotherapy varenicline. Rhesus monkeys (n=4) discriminated 0.032 mg/kg nicotine base i.v. from saline under an FR5 schedule of stimulus-shock termination. Both nicotine and varenicline increased drug-appropriate responding; ED₅₀ values (95% confidence limits) were 0.0087 (0.0025–0.030) and 0.028 (0.0096–0.082) mg/kg, respectively. Additional pretreatment injections of the training dose of nicotine (0.032 mg/kg, i.v.) produced tolerance to its discriminative stimulus effects and the magnitude of this effect was related to the number of pretreatment injections administered. Two pretreatment injections of the training dose of nicotine (0.032 mg/kg, i.v.) produced a 5.4-fold rightward shift in the nicotine dose-response function and a 7-fold rightward shift in the varenicline dose-response function. The duration of tolerance under these conditions was less than 60 min. These results demonstrate that tolerance to the discriminative stimulus effects of nicotine can be produced by acute nicotine exposure. Acute cross-tolerance from nicotine to varenicline is consistent with similar actions at nAChRs, and suggests that conditions resulting in acute nicotine tolerance could impact sensitivity to other nAChR agonists.

Introduction

Approximately 480,000 deaths in the United States are attributed to smoking-related causes annually (U. S. Department of Health and Human Services, 2014). Even with behavioral interventions and smoking cessation aids, less than 10% of smokers remain abstinent six months later (Babb, Malarcher, Schauer, Asman, & Jamal, 2017). Nicotine inhaled from tobacco smoke is typically dosed repeatedly at somewhat regular intervals, leading to cycles of receptor activation and desensitization. This is considered an important factor in the reinforcing effects of nicotine and the development of nicotine dependence (Picciotto, Addy, Mineur, & Brunzell, 2008), and it has been shown previously that satisfaction ratings in cigarette smokers are inversely related to the number of cigarettes smoked over the past six hours (Fant, Schuh, & Stitzer, 1995). This behavioral pattern is disrupted during episodes of sleep, leading to temporary abstinence and presumably withdrawal upon waking in the morning. The time to the first cigarette of the day is the single most valid predictor of smoking cessation outcomes (Baker et al., 2007). Thus acute tolerance to the nicotine in tobacco smoke plays an important role in nicotine dependence and an improved understanding could lead to insights for smoking cessation.

In humans, acute tolerance to the cardiovascular (Perkins, Stiller, & Jennings, 1991), discriminative stimulus (Perkins et al., 1996), and subjective effects (Perkins et al., 1993) of nicotine has been reported. Furthermore, acute tolerance to the subjective effects of nicotine is reported both in smokers and non-smokers (Perkins et al., 1993), although these two groups rate the subjective effects of nicotine differently. Rapid tolerance to various effects of nicotine has been also been demonstrated pre-clinically. For example, a sub-lethal dose of nicotine can protect mice from a lethal dose of nicotine (Barrass, Blackburn, Brimblecombe, & Rich, 1969), although the study attributed this tolerance to pharmacokinetics. Other studies examining rapid tolerance to the locomotor depressant (Stolerman, Bunker, & Jarvik, 1974) and discriminative stimulus effects of nicotine in rats (James, Villanueva, Johnson, Arezo, & Rosecrans, 1994; Robinson et al., 2006) were limited in their ability to demonstrate acute tolerance in all of the experimental subjects.

An i.v. nicotine discrimination assay in rhesus monkeys was developed and it has been reported previously that this discrimination is likely mediated by β2-containing nAChRs and has similar pharmacokinetics to smoking 1–2 cigarettes (Moerke, Zhu, Tyndale, Javors, & McMahon, 2017). During the development of the discrimination it was noted that cumulative dosing could not be adopted, as attempting to generate a dose-response curve for nicotine using this method failed to produce nicotine-lever responding up to doses well beyond the training dose. The apparent loss of sensitivity to nicotine during cumulative dosing appeared to reflect rapid, acute tolerance. Thus, a goal of the current study was to identify experimental conditions that reliably produce acute tolerance to the discriminative stimulus effects of nicotine. This was followed by a more systematic characterization of acute tolerance, including its relationship to nicotine pretreatment dose and time after nicotine pretreatment, followed by an additional goal of comparing the pharmacology of nicotine and varenicline.

Materials and Methods

Animals

Two male (PE and YA) and two female (BA and LA) adult rhesus monkeys (Macaca mulatta) discriminated 0.032 mg/kg nicotine i.v. One male was pharmacologically naïve; the other three monkeys received nicotinic and non-nicotinic drugs previously (Moerke et al., 2017). Monkeys weighed 7.5–10.8 kg and were fed primate chow (Harlan Teklad High Protein Monkey Diet; Madison, WI), fresh fruit and peanuts each day. They were housed individually in stainless steel cages in a room with controlled temperature and humidity on a 14/10-h light-dark cycle. There was continuous access to water in the home cages. These experiments were conducted in accordance with the 2011 Guide for Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 2011), and all experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at The University of Texas Health Science Center at San Antonio.

Surgery

Monkeys were anesthetized with ketamine (10 mg/kg, i.m.), followed by isoflurane (1.5–3.0%) inhaled via face mask. Catheters (heparin-coated polyurethane i.d.=1.02 mm, o.d.=1.68 mm; Instech Laboratories, Plymouth Meeting, PA) were inserted into a femoral, jugular or subclavian vessel. The catheter was secured to the vessel using suture silk (coated vicryl; Ethicon Inc., Somerville, NJ) and was connected to a vascular access port (Mida-cbas-c50; Instech Laboratories) placed subcutaneously at the midscapular region.

Drugs

Drugs were administered intravenously in a volume of 0.1–1 ml/kg. Nicotine hydrogen tartrate salt (Sigma-Aldrich, St. Louis, MO) and varenicline dihydrochloride (Research Technology Branch of the National Institute on Drug Abuse, Rockville, MD) were dissolved in physiological saline. For varenicline, drug doses are expressed as the weight, in mg/kg, of the salt; nicotine doses are expressed as the weight of the free base. For nicotine, the amount of sodium hydroxide (Sigma-Aldrich) required to bring the solution to a pH of 7 was calculated and accounted for in the volume when making the solution for a desired concentration. Drugs were studied from ineffective doses up to doses that produced greater than 80% drug-lever responding or that were judged to be the largest dose that could be safely studied. The order of drug dosing was non-systematic.

Apparatus

Monkeys were transferred from the home cage to commercially available chairs (Model R001; Primate Products, Miami, FL) that provided restraint. Feet were placed in shoes fitted with brass electrodes that were able to deliver a brief electric shock (3 mA, 250 ms) from an a/c generator (Coulbourn Instruments, Allentown, PA). Discrimination sessions were conducted in ventilated operant conditioning chambers in the presence of white noise. Each operant chamber contained a panel consisting of two red lights, one above each of two levers. Stimulus presentations were controlled and lever presses were recorded by a computer, a commercially available interface (MedAssociates, Inc., St. Albans, VT), and Med-PC software (MedAssociates, Inc.).

Discrimination training

Monkeys were trained once daily seven days per week to discriminate 0.032 mg/kg nicotine. For two monkeys (LA and PE), the left lever was assigned as the drug lever and the right lever was assigned as the saline lever; the lever assignments were reversed for the other two monkeys (BA and YA). On days the monkeys received an injection of the training dose of nicotine (0.032 mg/kg i.v.), responses on the drug lever were reinforced and on days the monkeys received an injection of saline, responses on the saline lever were reinforced. A training session began with an injection of the training dose of nicotine or saline, followed by 6 ml of heparinized saline, given via chronic indwelling catheter. This was followed by closing the door of the operant chamber and a 1-min pretreatment interval during which no lights in the operant chamber were illuminated and pressing the levers had no scheduled consequence. Following the conclusion of this interval, the red lights were illuminated, which signaled that the schedule of stimulus-shock termination (SST) was in effect. While the red lights were illuminated, a brief (250 ms) electric foot shock (3 mA) was scheduled to occur every 10 s; five consecutive responses on the correct lever (i.e., completion of the FR5) within 10 s extinguished the lights and postponed the shock schedule for 30 s. An incorrect response reset the FR5. During the 30-s timeout, no lights were illuminated and lever pressing had no scheduled consequence. Following the timeout, the lights were again illuminated red, the levers became active, and the shock schedule resumed. This continued for a duration of 5 min, at which point the lights were extinguished and the session ended (Fig. 1 top). The pattern of training with nicotine and saline was non-systematic and included schedules of single (i.e., nicotine, saline, nicotine) and double (nicotine, nicotine, saline, saline, nicotine) alternation, such that no training condition was repeated for more than two consecutive days. To be considered testable, monkeys were required to achieve seven consecutive training sessions for which greater than 80% of the total responses were made on the correct lever and less than five responses were made on the incorrect lever prior to delivery of the first reinforcer.

Fig. 1.

Timeline of discrimination training (top) or testing with one (middle) or two (bottom) pretreatment injections. For all tests, the appropriate control with saline given in place of nicotine pretreatment injections was performed.

Discrimination testing

Test sessions were scheduled so that approximately half followed saline training days and half followed nicotine training days. On test days, five consecutive responses on either lever resulted in reinforcement. Test days were always followed by training days, and monkeys were required to meet the discrimination training criteria, i.e., greater than 80% of the total responses on the correct lever and fewer than 5 responses on the incorrect lever prior to delivery of the first reinforcer, for three consecutive days before the next test, including one saline training day and one nicotine training day.

Previous experiments determined that the duration of action of the training dose of nicotine to produce discriminative stimulus effects was between 10 and 30 min (Moerke et al., 2017); i.e., no monkey responded on the nicotine lever 30 min after an injection of the training dose of nicotine. Thus, the final injection of all pretreatments to examine tolerance was followed by a 30 min interval prior to initiation of the test session, to eliminate any confound from the discriminative stimulus effects of a pretreatment overlapping into a test session. Pretreatments consisted of one, two or three injections of the training dose of nicotine (0.032 mg/kg, i.v.) administered at 10–15 min intervals. Then, a single dose of nicotine (or varenicline) was administered during each test session, thereby requiring multiple test sessions to generate dose-response functions. All nicotine pretreatments were accompanied by saline-matched controls in which the monkeys received one, two or three injections of saline, followed by a test session with a single dose of nicotine (or varenicline). To determine the duration of acute tolerance, intervals of 45 min and 60 min following the final pretreatment injection were tested in addition to the 30 min interval used for all other experiments.

Two pretreatment injections of the training dose of nicotine (given 45 and 30 min before a test session) was decided as the maximum safe cumulative nicotine pretreatment dose, and these conditions were used in determining the time course of the effect and to compare changes in sensitivity to nicotine and varenicline (Fig. 1 bottom). However, because there was no evidence for acute tolerance in monkey YA under these conditions, he was excluded from these experiments, as well as experiments with the smaller cumulative pretreatment dose of nicotine (Fig. 1 middle). In monkey YA, three injections of the training dose were required to demonstrate tolerance, but this pretreatment was not repeated due to concerns of toxicity.

Data analyses

Two primary measures were recorded: 1) the total number of responses on the drug lever divided by the total number of responses on both levers and 2) the average rate of responding in responses/sec. Responses made during timeouts were excluded. The mean of the most recent five saline training sessions for which the subject met the training criteria were considered to be the control response rate for that subject. Response rate during tests was then calculated as a percentage of the individual control response rate. Data were plotted for each animal individually or averaged across subjects, expressed as a mean ± 1 standard error of the mean (SEM), as a function of dose or time. ED₅₀ values were calculated with linear regression of data from each subject using GraphPad Prism (v. 5.0; GraphPad Scientific, San Diego, CA, USA). For group data analysis, the common best-fitting slope was used to calculate ED₅₀ values (Kenakin, 1997). The ED₅₀ values were compared by calculating potency ratios and 95% confidence limits using parallel line analyses (Tallarida & Raffa, 1992). The ED₅₀ values were considered significantly different when the 95% confidence limits of the potency ratio did not include 1.

Results

Pretreatment with one injection of saline or nicotine

Nicotine dose-dependently increased drug-lever responding (Supplementary Fig. 1 top panels) but did not alter response rate (Supplementary Fig. 1 bottom panels). When calculated as an average from 10 saline training sessions, excluding drug training sessions, rate of responding for each monkey was 3.47, 2.24, 2.08, and 1.54 responses per s. For two of three monkeys, one pretreatment injection of the training dose of nicotine (0.032 mg/kg, i.v.) produced rightward shifts in the nicotine dose-response function. For monkeys BA and LA, the ED₅₀ values for nicotine after saline pretreatment were 0.0042 and 0.0071 mg/kg, respectively. Pretreatment with 0.032 mg/kg nicotine increased the respective ED₅₀ values to 0.013 and 0.023 mg/kg (i.e., 3.1- and 3.2-fold). The nicotine ED₅₀ value after saline pretreatment was 0.0075 mg/kg in monkey PE; 0.032 mg/kg nicotine pretreatment did not modify the potency of nicotine in monkey PE.

Pretreatment with two injections of saline or nicotine

Nicotine dose-dependently increased responding on the drug lever (Supplementary Fig. 2 top panels), and decreased response rate in one monkey at the maximum dose tested (Supplementary Fig. 2 bottom panels). For three of four monkeys, two nicotine pretreatment injections (each 0.032 mg/kg, i.v.) shifted the nicotine dose-response functions rightward. For monkeys BA, LA, and PE, the control nicotine ED₅₀ values were 0.0042, 0.0086, and 0.024 mg/kg, respectively. Two pretreatment injections of 0.032 mg/kg nicotine increased the nicotine ED₅₀ values to 0.056, 0.042, and 0.042 mg/kg, respectively, corresponding to rightward shifts of 13.3-, 4.9-, and 1.8-fold. For monkey YA, the control nicotine ED₅₀ value (0.0074 mg/kg) was not modified by pretreatment with two injections of 0.032 mg/kg nicotine.

Relationship between pretreatment dose and acute nicotine tolerance

As the number of pretreatment injections of 0.032 mg/kg nicotine i.v. increased, there was a corresponding attenuation of the discriminative stimulus effects of the nicotine training dose (Fig. 2 top left), but no change in response rate (Fig. 2 bottom left). After saline pretreatment, all four monkeys responded predominantly on the drug lever at the training dose. Although a single nicotine pretreatment injection resulted in rightward shifts of the nicotine dose-response function in two monkeys (BA and LA), all four monkeys maintained 100% nicotine-lever responding at the nicotine training dose. Two pretreatment injections of nicotine produced rightward shifts in the nicotine dose-response functions including marked reductions in the effects of the training dose in monkeys BA, LA, and PE. In contrast, monkey YA maintained 100% nicotine-lever responding at the training dose, resulting in a mean of 27.78% nicotine-lever responding. Increasing the number of nicotine pretreatment injections to three decreased responding on the nicotine lever at the training dose to 0%. Concerns about toxicity precluded further testing with three pretreatment injections of 0.032 mg/kg nicotine i.v.

Fig. 2. Discriminative stimulus and rate effects of the training dose of nicotine (0.032 mg/kg i.v.) after 0–3 nicotine pretreatment injections (n=4) (left panels) and at various times following nicotine pretreatment (0.064 mg/kg, i.v.) (n=3) (right panels).

Abscissa: cumulative pretreatment dose of nicotine in mg/kg, i.v. (left panels) or time in minutes after last pretreatment injection (right panels). Ordinate: mean (±S.E.M.) percentage of responding on the nicotine lever (top panels) and mean (±S.E.M.) rate of responding expressed as a percentage of control rate (bottom panels). All monkeys contributed to all data points.

Time course of acute nicotine tolerance

Tolerance to the discriminative stimulus effects of the nicotine training dose varied significantly as a function of time following two pretreatment injections of nicotine (F_2,6=75.0, p<0.05). This pretreatment decreased mean nicotine-lever responding to 0% at the training dose of nicotine (0.32 mg/kg, i.v.) at 30 min in the three monkeys for which acute tolerance could be observed under these conditions (Fig. 2 top right). Significant attenuation of nicotine-lever responding was also observed 45 min following pretreatment, but not at 60 min, at which point responding on the nicotine lever at the training dose returned to 100%. Response rate was not significantly modified at any time after nicotine pretreatment (Fig. 2 bottom right).

Pretreatment with two injections of saline or nicotine: cross-tolerance to varenicline

Varenicline dose-dependently increased responding on the drug lever after two pretreatment injections of saline or nicotine, except in one monkey for which higher doses of varenicline could not be tested (Supplementary Fig. 3 top panels) and did not alter response rates (Supplementary Fig. 3 bottom panels). For monkeys BA and LA, the varenicline ED₅₀ values after saline pretreatment were 0.024 and 0.041 mg/kg, respectively. Two pretreatment injections of 0.032 mg/kg nicotine increased the varenicline ED₅₀ values to 0.13 and 0.17 mg/kg, respectively (i.e., 5.4- and 4.1-fold, respectively). For monkey PE, the ED₅₀ value for varenicline after saline pretreatment was 0.024 mg/kg. After nicotine pretreatment in monkey PE, varenicline produced a maximum of 4% responding on the drug-appropriate lever up to the largest dose that could be safely tested. The minimum rightward shift in the varenicline dose-response function for this monkey would be 18-fold, if the next larger dose of varenicline (0.56 mg/kg) was assumed to produce 100% drug-lever responding.

Comparison of tolerance to nicotine and cross-tolerance to varenicline

Nicotine and varenicline dose-dependently increased responding on the nicotine-appropriate lever (Fig. 3 top panels), whereas neither drug significantly modified response rate (Fig. 3 bottom panels). For discriminative stimulus effects, the slopes of the nicotine and varenicline dose-response functions determined after saline pretreatment were not significantly different from each other (F_1,9=1.29, p=0.29). The ED₅₀ values (95% confidence limits) of nicotine and varenicline after saline pretreatment were 0.0087 (0.0025–0.030) and 0.028 (0.0096–0.082) mg/kg, respectively, and were not significantly different. When the dose-response functions for nicotine and varenicline were re-determined after two pretreatment injections of 0.032 mg/kg nicotine i.v., the ED₅₀ values (95% confidence limits) of nicotine and varenicline were 0.047 (0.027–0.082) and 0.20 (0.075–0.52) mg/kg, respectively, reflecting significant rightward shifts of 5.4- and 7.1-fold, respectively.

Fig. 3. Discriminative stimulus and rate effects of nicotine (circles) and varenicline (squares) with (closed symbols) and without (open symbols) nicotine pretreatment (0.064 mg/kg, i.v.) (n=3).

Abscissae: dose of nicotine (left panels) or varenicline (right panels) in mg/kg. Ordinates: mean (±S.E.M.) percentage of responding on the nicotine lever (top panels) and mean (±S.E.M.) rate of responding expressed as a percentage of control rate (bottom panels). All monkeys contributed to all data points.

Discussion

In monkeys discriminating 0.032 mg/kg nicotine i.v., tolerance to the discriminative stimulus effects of nicotine was evident 30 minutes but not 60 minutes following nicotine pretreatment. Whereas drug-appropriate responding at the training dose of nicotine was near 100% under control conditions, responding at the training dose was reduced to nearly 0% with increases in the pretreatment dose of nicotine (0.032 mg/kg per injection, 1–3 injections). Tolerance was surmountable, resulting in a parallel rightward shift of the nicotine dose-response function. Nicotine pretreatment also resulted in cross-tolerance to the nicotine-like effects of varenicline. Cross-tolerance to varenicline was not greater than tolerance to nicotine, indicative of similar pharmacological mechanisms between drugs.

The literature examining acute tolerance to the discriminative stimulus effects of nicotine reveals significant individual variability in tolerance magnitude. For example, in humans, acute tolerance to the discriminative stimulus effects of nicotine was significantly greater in females than it was in males (Perkins, Fonte, Meeker, White, & Wilson, 2001). Although the sample size of the current study was too small to draw firm conclusions about sex differences, it is worth noting that, of the four monkeys included in this study, the two most sensitive to tolerance with nicotine pretreatment were female (LA and BA) and the two least sensitive were male (PE and YA). As females have poorer outcomes for smoking cessation as compared to males (Wetter et al., 1999), increasing the number of males and females in the experiment to properly examine sex differences in acute tolerance would be of interest, as one potential underlying factor in poorer outcomes for females could be sensitivity to acute tolerance.

Although the mechanism by which nicotine confers acute tolerance is not known, acute tolerance to the discriminative stimulus effects of nicotine in rats has been linked to nAChR desensitization by a strong correlation between biomarkers of receptor desensitization and behavior (Robinson et al., 2006; Zhang et al., 2000). The average duration of tolerance to the discriminative stimulus effects of nicotine in rodents is 42 minutes (James et al., 1994), which is comparable to the time frame of acute tolerance reported here. In humans, electrophysiological approaches have been used to suggest that plasma nicotine levels from cigarette smoking desensitize midbrain nAChRs with a time course of recovery of minutes (Pidoplichko, DeBiasi, Williams, & Dani, 1997; Wooltorton, Pidoplichko, Broide, & Dani, 2003). This is consistent with the time course for acute tolerance to the subjective effects of nicotine (i.e., “arousal”), which were present up to 60 minutes following nicotine pretreatment, but not the time course for acute tolerance to nicotine-induced tachycardia, which lasted at least 2 hours (Perkins et al., 1995). It has been shown that different nAChR subtypes have different rates of desensitization, and that β2-containing subtypes desensitize faster than β4-containing subtypes (Quick & Lester, 2002). Thus, the different time courses for acute tolerance to nicotine based on the endpoint measured would be consistent with a role for desensitization in this process, as β2-containing subtypes mediate the abuse-related (e.g., subjective) effects of nicotine (Gotti et al., 2010), but appear not to play a significant role in the cardiovascular (e.g., tachycardia) effects of nicotine (Jutkiewicz, Rice, Carroll, & Woods, 2013). Given that we have reported previously the importance of β2-containing nAChRs in mediating the discriminative stimulus effects of nicotine in the current assay (Moerke et al., 2017), it is plausible that desensitization of these receptors may be involved in the acute tolerance that was observed.

Varenicline, a partial α4β2 nAChR agonist, is hypothesized to function as an effective pharmacotherapy for smoking cessation by producing partial nicotine-like effects itself while antagonizing effects of nicotine (i.e., functioning as a low efficacy agonist), which is supported by evidence in vitro. For example, varenicline has lower efficacy than nicotine to stimulate dopamine release from rat brain slices (Rollema et al., 2007) and to evoke current in cells transfected with hα4β2 nAChRs (Coe et al., 2005). Furthermore, given in combination with nicotine it antagonizes nicotine’s effects in these assays (Coe et al., 2005; Rollema et al., 2007). However, the effects of varenicline in vivo are less consistent with activity as a low efficacy agonist. In mice discriminating nicotine, although no dose of varenicline fully substitutes for nicotine, percentage of nicotine-appropriate responding increases up to a dose of varenicline that eliminates responding, even when multiple training doses of nicotine are studied (Cunningham & McMahon, 2013; de Moura & McMahon, 2017; Rodriguez et al., 2014). Furthermore, it has been shown in both monkeys (Cunningham, Javors, & McMahon, 2012; Moerke et al., 2017) and rats (Jutkiewicz, Brooks, Kynaston, Rice, & Woods, 2011; Rollema et al., 2007) that varenicline fully substitutes for the nicotine discriminative stimulus, even when both small and large nicotine doses are trained (Jutkiewicz et al., 2011). In monkeys, rather than antagonizing the discriminative stimulus effect of nicotine, the effects of varenicline and nicotine were synergistic (Cunningham et al., 2012), and there was no evidence of antagonism by varenicline in rats trained to discriminate a relatively large dose of nicotine, and only limited antagonism in rats discriminating a small dose of nicotine (Jutkiewicz et al., 2011). However, results from these nicotine discriminations do not rule out the possibility that varenicline has lower efficacy than nicotine in vivo. Instead, the amount of efficacy required under the parameters that have been studied (i.e., in different species with different training doses of nicotine) may not have been high enough to reveal differences in efficacy between nicotine and varenicline. Presumably, another way to increase the efficacy demand of the assay is to produce tolerance; according to receptor theory, loss of sensitivity to a low efficacy agonist (i.e., varenicline) is expected to be greater than loss of sensitivity to a higher efficacy agonist (i.e., nicotine) (Kenakin, 1997). Although chronic tolerance readily develops to some effects of nicotine (e.g., decreases in body temperature), previous efforts examining the discriminative stimulus effects of nicotine have failed to show the development of chronic tolerance (Shoaib, Thorndike, Schindler, & Goldberg, 1997). Furthermore, in the current study, examining acute tolerance to the discriminative stimulus effects of nicotine, tolerance to nicotine did not significantly differ from cross-tolerance to varenicline. Thus, even under conditions that might be expected to reveal differences in efficacy, varenicline and nicotine exhibit strikingly similar effects.

In summary, there was a rapid, transient loss of sensitivity to the discriminative stimulus effects of nicotine that depended upon nicotine pretreatment dose. Previous results suggesting that the current discrimination is mediated by α4β2* nAChRs (Moerke et al., 2017) suggest that α4β2* receptors are also mediating acute tolerance, possibly by desensitization of these receptors. Regardless of the underlying mechanism, sensitivity to varenicline is impacted similarly, suggesting that other nAChR agonists producing nicotine-like effects through the same pharmacological mechanism as nicotine and varenicline will be similarly impacted.

Supplementary Material

1

Supplementary Fig. 1 Discriminative stimulus and rate effects of nicotine with (filled symbols) and without (open symbols) nicotine pretreatment (0.032 mg/kg, i.v.) in individual monkeys. Abscissae: nicotine dose in mg/kg. Ordinates: percentage of responding on the nicotine lever (top panels) and rate of responding expressed as a percentage of control rate (bottom panels). All points represent data from a single test session, except where numbers in parentheses indicate an average of two test sessions.

Supplementary Fig. 2 Discriminative stimulus and rate effects of nicotine with (filled symbols) and without (open symbols) nicotine pretreatment (0.064 mg/kg, i.v.) in individual monkeys. Abscissae: nicotine dose in mg/kg. Ordinates: percentage of responding on the nicotine lever (top panels) and rate of responding expressed as a percentage of control rate (bottom panels). All points represent data from a single test session, except where numbers in parentheses indicate an average of two test sessions.

Supplementary Fig. 3 Discriminative stimulus and rate effects of varenicline with (filled symbols) and without (open symbols) nicotine pretreatment (0.064 mg/kg, i.v.) in individual monkeys. Abscissae: varenicline dose in mg/kg. Ordinates: percentage of responding on the nicotine lever (top panels) and rate of responding expressed as a percentage of control rate (bottom panels). All points represent data obtained from a single test session.

Public Significance Statement.

This study provides evidence for a rapid loss of sensitivity to nicotine and varenicline following acute nicotine pretreatment in a non-human primate model of subjective effects. Cross-tolerance to varenicline was not significantly different from tolerance to nicotine, underscoring similarities in the receptor mechanism of action of these nAChR agonist-based therapies.