Rationale The extent to which non-α4β2 versus α4β2* nAChRs contribute to the behavioral effects of varenicline and other nAChR agonists is unclear. Objectives The purpose of this study was to characterize the discriminative stimulus effects of varenicline and nicotine using various nAChR agonists and antagonists to elucidate possible non-α4β2 nAChR mechanisms. Methods Separate groups of male C57BL/6J mice were trained to discriminate varenicline (3.2 mg/kg) or nicotine (1 mg/kg). Test drugs included mecamylamine, the nAChR agonists epibatidine, nicotine, cytisine, varenicline, RTI-102, the β2-containing nAChR antagonist dihydro-β-erythroidine (DHβE), the α7 nAChR agonist PNU-282987, the α7 antagonist methyllycaconitine (MLA), the α3β4 antagonist 18-methoxycoronaridine (18-MC), and the non-nAChR drugs midazolam and cocaine. Results In nicotine-trained mice, maximum nicotine-appropriate responding was: 95% nicotine, 94% epibatidine; 63% varenicline; 58% cytisine; and less than 50% for RTI-102, PNU-282987, midazolam, and cocaine. In varenicline-trained mice, maximum varenicline-appropriate responding was: 90% varenicline, 86% epibatidine; 74% cytisine; 80% RTI-102; 50% cocaine; and 50% or less for nicotine, PNU-282987, and midazolam. Drugs were studied to doses that abolished operant responding. Mecamylamine antagonized the discriminative stimulus effects, but not the rate-decreasing effects, of nicotine and varenicline. DHβE antagonized the discriminative stimulus and rate-decreasing effects of nicotine, but not varenicline in either the nicotine or varenicline discrimination assays. The discriminative stimulus, but not rate-decreasing, effects of epibatidine were antagonized by DHβE regardless of the training drug. Conclusions These results suggest that α4β2* nAChRs differentially mediate the discriminative stimulus effects of nicotine and varenicline, and suggest that varenicline has substantial non-α4β2 nAChR activity.

The tobacco dependence pharmacotherapies varenicline and cytisine act as partial α4β2 nAChR agonists. However, the extent to which α4β2 nAChRs mediate their in vivo effects remains unclear. Nicotine, varenicline, cytisine, and epibatidine were studied in male C57BL/6J mice for their effects on rates of fixed ratio responding and rectal temperature, alone and in combination with the nonselective nAChR antagonist mecamylamine and the α4β2 nAChR antagonist DHβE. The effects of nicotine, varenicline, cytisine, epibatidine, and cocaine were assessed before and during chronic nicotine treatment. The rate-decreasing and hypothermic effects of nicotine, varenicline, cytisine, and epibatidine were antagonized by mecamylamine (1 mg/kg), but only the effects of nicotine and epibatidine were antagonized by DHβE (3.2 mg/kg). Chronic nicotine produced 4.7 and 5.1 fold rightward shifts in the nicotine dose-effect functions to decrease response rate and rectal temperature, respectively. Nicotine treatment decreased the potency of epibatidine to decrease response rate and rectal temperature 2.2 and 2.9 fold, respectively, and shifted the varenicline dose-effect functions 2.0 and 1.7 fold rightward, respectively. Cross-tolerance did not develop from nicotine to cytisine. These results suggest that the in vivo pharmacology of tobacco cessation aids cannot be attributed to a single nAChR subtype; instead, multiple receptor subtypes differentially mediate their effects.

Rationale Receptor mechanisms underlying the in vivo effects of nicotinic acetylcholine receptor (nAChR) drugs need to be determined to better understand possible differences in therapeutic potential. Objective This study compared the effects of agonists that are reported either to differ in intrinsic activity (i.e., efficacy) at α4β2 nAChR in vitro or to have in vivo effects consistent with differences in efficacy. The drugs included nicotine, varenicline, cytisine, epibatidine, and three novel epibatidine derivatives 2′-fluoro-(4-nitrophenyl) deschloro-epibatidine (RTI-7527-102), 2′-fluorodeschloroepibatidine (RTI-7527-36), and 3′-(3″-dimethylaminophenyl)-epibatidine (RTI-7527-76). Methods Mice discriminated nicotine base (1 mg/kg base) from saline; other mice were used to measure rectal temperature. Results In the nicotine discrimination assay, the maximum percentage of nicotine-appropriate responding varied: 92% for nicotine, 84% for epibatidine, 77% for RTI-7527-36, 71% for varenicline, and significantly less for RTI-7527-76 (58%), RTI-7527-102 (46%), and cytisine (33%). Each drug markedly decreased rectal temperature by as much as 12 °C. The rank order potency in the discrimination and hypothermia assays was epibatidine > RTI-7527-36 > nicotine > RTI-7527-102 > varenicline = cytisine = RTI-7527-76. The nAChR antagonist mecamylamine (3.2 mg/kg) antagonized the discriminative stimulus effects of epibatidine and RTI-7527-102, as well as the hypothermic effects of every drug except cytisine. The β2-subunit selective nAChR antagonist dihydro-β-erythroidine (DHβE; up to 10 mg/kg) antagonized hypothermic effects, but less effectively so than mecamylamine. Conclusions The marked hypothermic effects of all drugs except cytisine are due in part to agonism at nAChR containing β2 subunits. Differential substitution for the nicotine discriminative stimulus is consistent with differences in α4β2 nAChR efficacy; however, these results suggest that multiple nAChR receptor subtypes mediate the effects of the agonists.

Some drugs that are positive allosteric nAChR modulators in vitro, desformylflustrabromine (dFBr), PNU-120596 and LY 2087101, have not been fully characterized in vivo. These drugs were examined for their capacity to share or modify the hypothermic and discriminative stimulus effects of nicotine (1 mg/kg s.c.) in male C57Bl/6J mice. Nicotine, dFBr, and PNU-120596 produced significant hypothermia, whereas LY 2087101 (up to 100 mg/kg) did not. Nicotine dose-dependently increased nicotine-appropriate responding and decreased response rate; the respective ED50 values were 0.56 mg/kg and 0.91 mg/kg. The modulators produced no more than 38% nicotine-appropriate responding up to doses that disrupted operant responding. Rank order potency was the same for hypothermia and rate-decreasing effects: nicotine>dFBr>PNU-120596=LY 2087101. Mecamylamine and the α4β2 nAChR antagonist dihydro-β-erythroidine, but not the α7 antagonist methyllycaconitine, antagonized the hypothermic effects of nicotine. In contrast, mecamylamine did not antagonize the hypothermic effects of the modulators. The combined discriminative stimulus effects of DFBr and nicotine were synergistic, whereas the combined hypothermic effects of nicotine with either dFBr or PNU-120596 were infra-additive. PNU-120596 did not modify the nicotine discriminative stimulus, and LY 2087101 did not significantly modify either effect of nicotine. Positive modulation of nicotine at nAChRs by PNU-120596 and LY 2087101 in vitro does not appear to confer enhancement of the nAChR-mediated hypothermic or discriminative stimulus effects of nicotine. However, dFBr appears to be a positive allosteric modulator of some behavioral effects of nicotine at doses of dFBr smaller than the doses producing unwanted effects (e.g. hypothermia) through non-nAChR mechanisms.

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