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.2016 Mar 9;36(10):2957-74.
doi: 10.1523/JNEUROSCI.4194-15.2016.

Menthol Alone Upregulates Midbrain nAChRs, Alters nAChR Subtype Stoichiometry, Alters Dopamine Neuron Firing Frequency, and Prevents Nicotine Reward

Affiliations

Menthol Alone Upregulates Midbrain nAChRs, Alters nAChR Subtype Stoichiometry, Alters Dopamine Neuron Firing Frequency, and Prevents Nicotine Reward

Brandon J Henderson et al. J Neurosci..

Abstract

Upregulation of β2 subunit-containing (β2*) nicotinic acetylcholine receptors (nAChRs) is implicated in several aspects of nicotine addiction, and menthol cigarette smokers tend to upregulate β2* nAChRs more than nonmenthol cigarette smokers. We investigated the effect of long-term menthol alone on midbrain neurons containing nAChRs. In midbrain dopaminergic (DA) neurons from mice containing fluorescent nAChR subunits, menthol alone increased the number of α4 and α6 nAChR subunits, but this upregulation did not occur in midbrain GABAergic neurons. Thus, chronic menthol produces a cell-type-selective upregulation of α4* nAChRs, complementing that of chronic nicotine alone, which upregulates α4 subunit-containing (α4*) nAChRs in GABAergic but not DA neurons. In mouse brain slices and cultured midbrain neurons, menthol reduced DA neuron firing frequency and altered DA neuron excitability following nAChR activation. Furthermore, menthol exposure before nicotine abolished nicotine reward-related behavior in mice. In neuroblastoma cells transfected with fluorescent nAChR subunits, exposure to 500 nm menthol alone also increased nAChR number and favored the formation of (α4)3(β2)2 nAChRs; this contrasts with the action of nicotine itself, which favors (α4)2(β2)3 nAChRs. Menthol alone also increases the number of α6β2 receptors that exclude the β3 subunit. Thus, menthol stabilizes lower-sensitivity α4* and α6 subunit-containing nAChRs, possibly by acting as a chemical chaperone. The abolition of nicotine reward-related behavior may be mediated through menthol's ability to stabilize lower-sensitivity nAChRs and alter DA neuron excitability. We conclude that menthol is more than a tobacco flavorant: administered alone chronically, it alters midbrain DA neurons of the nicotine reward-related pathway.

Keywords: dopamine neuron; electrophysiology; menthol; nicotine; nicotinic receptor; reward.

Copyright © 2016 the authors 0270-6474/16/362957-18$15.00/0.

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Figures

Figure 1.
Figure 1.
Chronic menthol alone upregulates α4* and α6* nAChRsin vivo.A, Montage of 60× images from a vehicle-treated α4-mCherry mouse. Scale bar, 100 μm.B, 60× images of α4-mCherry direct fluorescence in the VTA, SNc, and SNr of mice treated with vehicle or menthol (2 mg/kg/h, 10 d). Scale bars, 10 μm.D, Montage of 60× images from a vehicle-treated α6-GFP mouse. Scale bar, 100 μm.E, 60× images of α6-GFP direct fluorescence in the VTA and SNc of mice treated with vehicle or menthol. Scale bars, 10 μm.B,E, Display lookup table “fire” was used in ImageJ.C,F, Quantification of α4-mCherry and α6-GFP intensities; transparent circles are mean intensities of individual mice.G, Representative TIRFM images of Neuro-2a cells transfected with α4-SEP and β2 nAChR subunits or with α6-SEP, β2, and β3 nAChR subunits. Menthol (500 nm) was added 24 h before imaging. Scale bars, 10 μm. In each panel, the left image is at pH 7.4 and the right image is the same cell at pH 5.4.H, PMID was quantified for SEP nAChRs.I, RNA transcript levels of α4 and β2 nAChR subunits in Neuro-2a cells, detected by RNA sequencing, are unaffected by 500 nm menthol treatment (24 h). For all panels, data are mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.005 (unpairedt test). For all panels, number in parenthesis indicatesn value.
Figure 2.
Figure 2.
In midbrain DA neurons, chronic menthol does not significantly alter ACh-induced response amplitudes but accelerates their decay and decreases the baseline firing rate.A–D, Midbrain slices; VTA DA neurons, bregma ∼−3.1 mm.B1,B2, Fluorescent neurons in slices from α6-GFP mice.C1, Currents induced by ACh puffs.C2, Baseline current-clamp traces for no drug-treated and for menthol-treated mice.E–H, Midbrain cultures; putative DA neurons.E1–E4, Fluorescent neurons from β3-GFP or TH-GFP mice.F1, Currents induced by ACh puffs.F2, Baseline current-clamp traces for no drug-treated and for chronic menthol-treated neurons.D1,D2,G1,G2, Summary of ACh response amplitude and decay time constant.D3, Histograms for DA neuron firing frequency for vehicle-treated and menthol-treated mouse brain slices (n = 16 and 11 for vehicle treated and menthol treated, respectively).G3, Histograms for DA neuron firing frequency for no drug-treated and menthol-treated cultured neurons (n = 23 and 31 for no drug treated and menthol treated, respectively).E–H, Chronic menthol treatment with cultured midbrain DA neurons was 500 nm, 7–10 d.H, Chronic menthol alters action potential properties of midbrain DA neurons.H1, Mean action potential spike waveform for a no drug-treated and menthol-treated midbrain DA neuron.H2,H3, Quantification of antipeak amplitude and action potential half-width.H2,n = 12 for both no drug-treated and menthol-treated cultured DA neurons.H3,n = 19 and 26 for no drug-treated and menthol-treated cultured DA neurons, respectively. In all cases (C1,F1), arrows indicate 100 ms puff of 300 μm ACh.D1,D2,G1,G2, For all bar graphs, number in parenthesis indicatesn value. Data are mean ± SEM; *p < 0.05; **p < 0.005 (unpairedt test).
Figure 3.
Figure 3.
Long-term menthol treatment prevents nAChR-induced acceleration of DA neuron firing frequency.A, Current-clamp recordings from cultured midbrain DA neurons treated with no drug (A1) or treated with 500 nm menthol over 10 d (A2) before and after an ACh puff.B1, Time course of firing frequency before and after a 100 ms ACh puff. Dashed lines are mean firing frequency for no drug-treated and menthol-treated neurons before the ACh puff.B2, Quantification of firing frequency in no drug-treated and menthol-treated conditions before and after ACh puff. Mean for pre-ACh (−) and post-ACh (+) puff are the 3 s before and after the ACh puff. Data are mean ± SEM; *p < 0.05; **p < 0.001 (unpairedt test). In all cases, arrows indicate 100 ms puff of 300 μm ACh.C, Current-clamp recordings from cultured midbrain DA neurons treated with 500 nm menthol (10 d) without sulpiride (C1) or with acute exposure to 1 μm sulpiride (C2). In all traces, arrows show a 100 ms puff of 300 μm ACh. Number in parenthesis indicatesn value.
Figure 4.
Figure 4.
Chronic menthol before nicotine exposure abolishes nicotine reward-related behavior.A, Schematic of CPP assay. Mice were implanted with osmotic minipumps for 20 d to continuously deliver 2 mg/kg/h menthol or vehicle 10 d before and during CPP training.B, Saline control mice did not exhibit a change in baseline preference. Mice implanted with osmotic minipumps delivering vehicle exhibited reward-related behavior to 0.5 mg/kg nicotine, but those receiving menthol exhibited no reward-related behavior to 0.5 mg/kg nicotine. Data are mean ± SEM; one-way ANOVA,F = 8.28; *p < 0.05,post hoc Tukey's test. Number in parenthesis indicatesn value.C, Mice exhibited no significant preference for the white or black chambers during the initial test phase.
Figure 5.
Figure 5.
Chronic menthol alters nAChR stoichiometry and favors low-sensitivity nAChRs.A, Concentration–response curves of Neuro-2a cells transfected with α4-eGFPβ2 nAChRs treated with no drug (black) or with menthol (red; 24 h, 500 nm).C, Concentration–response curves of Neuro-2a cells transfected with α6-eGFP, β2, and β3 nAChR subunits treated with no drug (black), with menthol (red), or with α6-eGFPβ2DM nAChRs with no drug (blue).B1,D1, Linear plot of donor dequenching versus acceptor (mCherry) photodestruction for no drug and for menthol (24 h, 500 nm) treatments.B2,D2, Mean FRET efficiency. FRET efficiency was calculated from the linear plot shown inB1 andD1 as described previously (Drenan et al., 2008a; Nichols et al., 2014).B2,D2, Data are mean ± SEM; **p < 0.01; ***p < 0.001 (unpairedt test).E,F, Summary of menthol-induced changes in nAChR stoichiometry.E, α4β2 nAChRs assemble in two stoichiometries: (α4)2(β2)3 and (α4)3(β2)2. We found that under no drug treatment the α4β2 nAChR pool is mixed. Following 24 h menthol treatment, α4β2 nAChRs assemble almost entirely in the low-sensitivity (α4)3(β2)2 stoichiometry.F, α6β2β3 nAChRs assemble efficiently under no drug treatment, but following 24 h menthol treatment the receptor pool is changed to a mixture of α6β2β3 and α6β2 nAChRs. Number in parenthesis indicatesn value.
Figure 6.
Figure 6.
Long-term menthol decreases α6β2β3 nAChR peak current amplitude and alters α4* and α6* nAChR desensitization.A1,B1,C1,D1, Mean waveforms recorded from Neuro-2a cells expressing α6-eGFPβ2β3 or α4-eGFPβ2 nAChRs in the absence or presence of menthol (24 h, 500 nm).A1,C1, 300 ms puff of ACh.B1,D1, 10 s puff of ACh.B1, 10 s puffs reveal a sustained rebound current that is likely caused by recovery from agonist-mediated channel block.A2,C2, Mean of peak amplitude of 300 ms puffs.B2,D2, Mean decay time constants for 10 s puffs. Numbers in parenthesis indicaten value.E,F, α4-GFPβ2 and α6-GFPβ2β3 nAChRs treated with no drug (black) or with menthol (green). nAChRs were desensitized with 1 μm nicotine (10 min) and then allowed to recover. In all panels, data are mean ± SEM; *p < 0.05; ***p < 0.005 (unpairedt test). In all cases, menthol treatment was 24 h, 500 nm. In all cases, menthol-containing media was removed at the start of assays. Number in parenthesis indicatesn value.
Figure 7.
Figure 7.
Menthol upregulates α6β2(non-β3) nAChRs. Neuro-2a cells were transfected with α6-eGFP and β2 nAChR subunits. Where indicated, menthol (500 nm) was present for 24 h before imaging.A, Representative traces showing 300 μm ACh-induced currents for cells treated with no drug or with menthol (24 h, 500 nm).B1,B2, Summary of peak current and percentage of cells exhibiting function.C1, Representative TIRF images. Scale bars, 10 μm. In each panel, left image is at pH 7.4 and right is the same cell imaged at pH 5.4.C2, Mean PMID (mean ± SEM).B1,C2, *p < 0.05, unpairedt test. Number in parenthesis indicatesn value.
Figure 8.
Figure 8.
Long-term menthol upregulates nAChRs via endoplasmic reticulum–Golgi trafficking.A1,A2, Representative TIRFM images of Neuro-2a cells transfected with α4-SEP plus β2 or with α6-SEP plus β2 plus β3 nAChR subunits. Nicotine (50 or 100 nm), menthol (500 nm), or CI-976 (20 μm) were present 24 h before imaging. Scale bars, 10 μm. In each panel, the left image is at pH 7.4 and right image is the same cell at pH 5.4.B1,B2, PMID was quantified for SEP nAChRs.C1–C4, Representative confocal images of a Neuro-2a cell transfected with α6-mCherry, β2, and β3 subunits plus Sec24D-eGFP. Scale bars, 5 μm.C1, Sec24D-eGFP fluorescence.C2, α6 Fluorescence.C3, MergedC1 andC2 image.C4, ERES demarcated for quantification.D, Quantification of the α4-mCherry or α6-mCherry fluorescence in ERES. Data are mean ± SEM. *p < 0.05; **p < 0.005 (ANOVA,post hoc Tukey's test). Number in parenthesis indicatesn value.
Figure 9.
Figure 9.
TrpM8 agonist icilin does not alter PM nAChR number.A1,B1, Representative TIRFM images of Neuro-2a cells transfected with α4-SEPβ2 and α6-SEPβ2β3 nAChRs. Icilin, at listed concentrations, was present 24 h before imaging. Scale bars, 10 μm. In each panel, the left image is at pH 7.4 and the right image is the same cell at pH 5.4.A2,B2, PMID was quantified for SEP nAChRs. Number in parenthesis indicatesn value.
Figure 10.
Figure 10.
Chronic menthol and chronic nicotine differentially alter midbrain DA neurons. Schematic of nAChRs residing in midbrain GABA (SNr) and DA (VTA and SNc) neurons. Nicotine robustly increases higher-sensitivity α4* nAChR number in SNr GABAergic neurons, but does not change α4* nAChR number in SNc or VTA DA neurons (Nashmi et al., 2007; Xiao et al., 2009). Long-term nicotine exposure decreases firing frequency of DA neurons, but this is transiently accelerated upon exposure to nAChR agonists (i.e., nicotine; Nashmi et al., 2007; Xiao et al., 2009). Long-term menthol exposure increases lower-sensitivity α4* and α6* nAChR number in SNC and VTA DA neurons, but does not alter SNr GABAergic α4* nAChR number. DA neuron firing frequency, which decreases following long-term menthol exposure, does not accelerate following exposure to nAChR agonists. The opposing effects of nicotine and menthol on midbrain neurons may explain how long-term menthol treatment before nicotine exposure abrogates nicotine reward-related behavior.
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