Time course of nicotine plasma concentrations in mice pretreated with menthol.

<p>Nicotine was administered (2.5 mg/kg s.c.), 30 min after pretreatment with vehicle or menthol (100 mg/kg i.p.). Each time point represents the mean ± SEM of 7 to 10 animals. For the vehicle pretreatment, values for nicotine plasma levels at 2 h were below the limits of detection. Each point represents the mean ± SE of 8–12 mice. Nic = nicotine.</p

Effects of menthol pretreatment on nicotine-induced antinociception and hypothermia dose-response curves in mice.

<p>Vehicle or menthol (100 mg/kg, ip) was administered 30 min before various doses of nicotine (0.5, 1.5, 2, and 2.5 mg/kg s.c.) and mice were tested in <b>(A)</b> the tail-flick test, <b>(B)</b> the hot-plate test, and <b>(C)</b> hypothermia. Each point represents the mean ± SE of 8–12 mice.</p

Effects of menthol on nicotine withdrawal signs. (A) Elevated plus-maze test, (B) Somatic signs and in (C) Hyperalgesia.

<p>Withdrawal from nicotine induced a: A) no significant change in anxiety- related behavior, B) significant increase in somatic signs, and C) a significant decrease in hot plate latency. Compared to vehicle, pretreatment with menthol (100 mg/kg, i.p. for 7 days) A) significantly increased expression of anxiety-related behavior; B) a further increase in somatic signs, and C) a decrease in hot plate latency in mice. Each point represents the mean ± S.E.M. of 6–8 mice per group. *<i>P</i> < 0.05 compared with control MP/Saline/Vehicle group. #<i>P</i> < 0.05 compared with MP/Nicotine/ Vehicle group. MP = minipump; Nic = nicotine; Sal = saline; Veh = vehicle.</p

Summary of the potency of nicotine in male and female mice in different behavioral tests.

<p>ED₅₀ values (±CL) were calculated from the dose-response curve of the different groups and expressed as mg/kg.</p><p>Animals were pretreated with either vehicle or menthol (100 mg/kg, i.p.) followed by nicotine (2.5 mg/kg s.c.) at different doses and then tested 5 min (tail-flick and hot-plate tests) or 20 min later (hypothermia). Each point represents the mean ± SE of 6 to 8 mice.</p

Nicotine plasma levels in adult male ICR mouse after pre-treatment with vehicle or menthol in the withdrawal testing.

<p>*P<0.05 compared to vehicle/nicotine group.</p><p>Mice received nicotine (12 mg/kg/day) for 7 days. On day 8, mice were sacrificed and blood samples were drawn. Values are shown as mean ± S.E.M. (n = 6-8/group)</p

Impact of menthol treatment on nicotine pharmacokinetic parameters.

<p>*p < 0.05 compared to Menthol.</p><p>Pharmacokinetic parameters of plasma nicotine in mice pre-treated i.p. with menthol (100 mg/kg) or vehicle 30 minutes prior to nicotine (2.5 mg/kg, s.c.). Results were derived using data (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137070#pone.0137070.g001" target="_blank">Fig 1</a> [time-concentration graph]).</p

Menthol mediated changes in nAChR expression in the brain.

<p>Western Blot detection of <b>α</b>4 and <b>β</b>2 nAChR subunits in membrane fractions of treated mice. Representative immunoblots showing the expression of <b>α</b>4 and <b>β</b>2 nAChR subunit bands in <b>(A)</b> Hippocampus, <b>(B)</b> Striatum, <b>(C)</b> Prefrontal Cortex, and <b>(D)</b> all three regions when expressed relative to nicotine treatment alone. GAPDH was used as a loading control and a normalizing factor for protein loading across lanes. Histograms show average values of optical density (OD) measurements of the immunoreactive bands relative to the vehicle lane (n = 6 mice per group; and all samples were run in triplicates). Student’s test *p < 0.05, **p < 0.01.</p

Effects of menthol on the time course of nicotine’s effects in (A) the tail-flick test, (B) the hot-plate assay, and (C) body temperature in mice.

<p>Animals were pretreated with either menthol (100 mg/kg i.p.) or vehicle and 30 min later received nicotine (2.5 mg/kg, s.c.). A control group (vehicle/vehicle) is also represented in all three tests. Mice were tested at different time points after injection. Each point represents the mean ± SE of 8–12 mice. *p < 0.05 compared with vehicle/nicotine group.</p

Effect of (A) pretreatment time and (B) dose of menthol’s effect in the tail-flick.

<p><b>(A)</b> Mice pretreated with vehicle or menthol (100 mg/kg, i.p.) and various time points (5, 30, 60 and 120 min) later, they were given nicotine (2.5 mg/kg, s.c.). Mice were then evaluated for antinociception 45 min after nicotine administration. <b>(B)</b> Mice pretreated with vehicle or different doses of menthol (10, 50, 100 or 200 mg/kg, i.p.) and 30 min later, they were given nicotine (2.5 mg/kg, s.c.). Mice were then evaluated for antinociception 45 min after nicotine administration. Each point represents the mean ± SE of 8–12 mice. *p < 0.05 compared with control.</p