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. 2015 Jun;172(11):2864-77.
doi: 10.1111/bph.13090. Epub 2015 Mar 24.

Anxiolytic-like and anxiogenic-like effects of nicotine are regulated via diverse action at β2*nicotinic acetylcholine receptors

S M Anderson  1 , D H Brunzell  1
Affiliations

Anxiolytic-like and anxiogenic-like effects of nicotine are regulated via diverse action at β2*nicotinic acetylcholine receptors

S M Anderson et al. Br J Pharmacol. 2015 Jun.

Abstract

Background and purpose: Nicotine dose-dependently activates or preferentially desensitizes β2 subunit containing nicotinic ACh receptors (β2*nAChRs). Genetic and pharmacological manipulations assessed effects of stimulation versus inhibition of β2*nAChRs on nicotine-associated anxiety-like phenotype.

Experimental approach: Using a range of doses of nicotine in β2*nAChR subunit null mutant mice (β2KO; backcrossed to C57BL/6J) and their wild-type (WT) littermates, administration of the selective β2*nAChR agonist, 5I-A85380, and the selective β2*nAChR antagonist dihydro-β-erythroidine (DHβE), we determined the behavioural effects of stimulation and inhibition of β2*nAChRs in the light-dark and elevated plus maze (EPM) assays.

Key results: Low-dose i.p. nicotine (0.05 mg·kg(-) 1) supported anxiolysis-like behaviour independent of genotype whereas the highest dose (0.5 mg·kg(-1) ) promoted anxiogenic-like phenotype in WT mice, but was blunted in β2KO mice for the measure of latency. Administration of 5I-A85380 had similar dose-dependent effects in C57BL/6J WT mice; 0.001 mg·kg(-1) 5I-A85380 reduced anxiety on an EPM, whereas 0.032 mg·kg(-1) 5I-A85380 promoted anxiogenic-like behaviour in both the light-dark and EPM assays. DHβE pretreatment blocked anxiogenic-like effects of 0.5 mg·kg(-1) nicotine. Similarly to DHβE, pretreatment with low-dose 0.05 mg·kg(-1) nicotine did not accumulate with 0.5 mg·kg(-1) nicotine, but rather blocked anxiogenic-like effects of high-dose nicotine in the light-dark and EPM assays.

Conclusions and implications: These studies provide direct evidence that low-dose nicotine inhibits nAChRs and demonstrate that inhibition or stimulation of β2*nAChRs supports the corresponding anxiolytic-like or anxiogenic-like effects of nicotine. Inhibition of β2*nAChRs may relieve anxiety in smokers and non-smokers alike.

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Figures

Figure 1
Figure 1
Genetic evidence that β2*nAChRs contribute to anxiogenic-like effects of nicotine. Results shown are from WT and β2KO mice. (A) As predicted, nicotine treatment had a dichotomous effect on anxiety-like behaviour. A low dose of nicotine (0.05 mg·kg−1 i.p.) increased time spent in the light chamber, suggestive of a an anxiolytic-like phenotype, and a high dose (0.5 mg·kg−1 i.p.) decreased time in the light chamber, suggestive of an anxiogenic-like phenotype. (B) For latency, mice, receiving high-dose nicotine required more time to enter the light chamber than their respective saline-treated controls, an effect that was blunted in β2KO mice, demonstrating that β2*nAChRs contribute to this measure. (C) Compared with VEH controls, mice treated with 0.5 mg·kg−1 i.p. nicotine showed reduced movement counts independent of genotype, suggesting that nicotinic receptor subtypes other than β2*nAChRs contributed to reduced horizontal activity in this task. Data are represented as means ± SEM. *P < 0.05 compared with saline-treated mice of same genotype, **P < 0.05 compared with WT mice receiving the same dose.
Figure 2
Figure 2
Administration of the selective β2*nAChR agonist 5I-A85380 results in anxiogenic-like behaviour in the light–dark assay. (A) Mice administered 0.032 mg·kg−1 i.p. of the selective β2*nAChR agonist 5-Iodo-A85380 (5I-A85380) spent less time in the light chamber, (B) showed longer latencies to enter the light chamber and (C) showed less horizontal activity than mice receiving saline VEH. Data are reported as means ± SEM; *P < 0.05 compared with saline VEH.
Figure 3
Figure 3
Administration of selective β2*nAChR agonist 5I-A85380 has a bimodal effect on anxiety-like behaviour in EPM assay. (A) Mice receiving a low dose (0.001 mg·kg−1 i.p.) of 5I-A85380 spent more time in the open arms of an EPM, and (C) made more entries into the open arms than saline-injected mice, indicative of anxiolytic-like behaviour, (B) whereas mice injected with a high dose (0.032 mg·kg−1 i.p.) of 5I-A85380 spent more time in the closed arms than VEH-injected controls, indicative of anxiogenic-like behaviour. (D) There was no effect of 5I-A85380 on closed-arm entries to suggest non-specific effects of 5I-A85380 on locomotor behaviour. Data are reported as means ± SEM; *P < 0.05 compared with saline VEH.
Figure 4
Figure 4
Selective antagonism of β2*nAChRs via pretreatment with DHβE (PRE DHβE) blocks the anxiogenic-like effects of nicotine (NIC) in the light–dark assay. (A) Mice receiving saline pre-injection prior to high-dose nicotine (VEH/0.5 NIC) spent less time in the light chamber than mice given saline VEH only (VEH/VEH). A pre-injection of 2 mg·kg−1 i.p. DHβE effectively blocked this anxiogenic-like effect of 0.5 mg·kg−1 i.p. nicotine treatment (DHβE/0.5 NIC); DHβE/0.5 NIC mice spent significantly more time in the light chamber and (B) showed shorter latencies to enter the light chamber than VEH/0.5 NIC mice. (C) Pre-injections of DHβE also blocked high-dose nicotine-associated reductions in movement counts. DHβE/0.5 NIC mice showed significantly greater horizontal activity than VEH/0.5 NIC mice. (D) VEH/0.05 mg·kg−1 i.p. mice showed elevated time spent in the light chamber compared with VEH/VEH mice. This anxiolytic-like effect was not impacted by pre-injection of 2 mg·kg−1 i.p. DHβE, suggesting that the anxiolytic-like effects of low-dose nicotine do not require activation of β2*nAChRs. (E) Neither low-dose nicotine nor DHβE affected latency to enter the light chamber, (F) but both VEH/0.05 NIC and DHβE/VEH mice showed increased movement counts compared with controls. Data are reported as means ± SEM; *P < 0.05 compared with VEH/VEH; **P < 0.05 compared with VEH/0.5 NIC.
Figure 5
Figure 5
Pretreatment with an anxiolytic-like low dose of nicotine (PRE NIC) blocks rather than accumulates with an anxiogenic-like high dose of nicotine (NIC) in the light–dark assay. (A) Pretreatment with an anxiolytic-like dose of 0.05 mg·kg−1 i.p. nicotine blocked the anxiogenic-like effects of high-dose nicotine (0.05 NIC/0.5 NIC) as measured by more time spent in the light chamber (B) and less time required to enter the light chamber than mice pre-injected with saline VEH prior to an anxiogenic dose of 0.5 mg·kg−1 nicotine (VEH/0.5 NIC). (C) Pre-injections of 0.05 mg·kg−1 nicotine also blocked nicotine-induced reductions in horizontal activity. (D) Nicotine pretreatment did not significantly affect the percentage of time spent in the light chamber, (E) latencies to enter the light chamber or (F) horizontal activity in the light–dark assay. Data are reported as means ± SEM; *P < 0.05 compared with VEH/VEH; **P < 0.05 compared with VEH/0.5 NIC.
Figure 6
Figure 6
Pre-injection of low-dose nicotine reverses anxiogenic-like effects of high-dose nicotine during EPM assay. (A–C) Mice injected with 0.5 mg·kg−1 i.p. nicotine (VEH/0.5 NIC) spent less time in the open arms, more time in the closed arms and made fewer entries into the open arms of an EPM than saline-injected controls (VEH/VEH), demonstrating an anxiogenic-like effect of high-dose nicotine in this task. Pre-injections of 0.01 mg·kg−1 i.p. nicotine blocked this effect, as 0.01 NIC/0.5 NIC mice (A) spent significantly more time in the open arms and (B) less time in the closed arms and made more open-arm entries than VEH/0.5 NIC mice. These data suggest that pre-injections of low-dose nicotine effectively block the anxiogenic-like effects of 0.5 mg·kg−1 i.p. nicotine in an EPM assay. (D) Nicotine pretreatment did not block reductions in closed-arm entries observed in VEH/0.5 mice. Data are reported as means ± SEM. *P < 0.05 compared with VEH/VEH; **P < 0.05 compared with VEH/0.5 NIC.
Figure 7
Figure 7
High doses of nicotine and 5I-A85830 did not affect mouse rotorod performance or open-field horizontal activity under dim lighting. Mice tested at 0 and 10 min post-injection did not show any impairment in rotorod performance following i.p. injection of anxiogenic-like doses of 0.5 mg·kg−1 nicotine or 0.0032 mg·kg−1 of the β2*nAChR-selective agonist, 5I-A85380. Mice habituated to an open field also failed to show any effect of drug exposure on distance travelled under low-anxiety conditions. Data are reported as means ± SEM.
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