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

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.

Henderson BJ ，Wall TR ，Henley BM ，Kim CH ，Nichols WA ，Moaddel R ，Xiao C ，Lester HA ... -  《-》

Menthol Enhances Nicotine Reward-Related Behavior by Potentiating Nicotine-Induced Changes in nAChR Function, nAChR Upregulation, and DA Neuron Excitability.

Henderson BJ ，Wall TR ，Henley BM ，Kim CH ，McKinney S ，Lester HA ... -  《-》

Functional Upregulation of α4* Nicotinic Acetylcholine Receptors in VTA GABAergic Neurons Increases Sensitivity to Nicotine Reward.

Chronic nicotine exposure increases sensitivity to nicotine reward during a withdrawal period, which may facilitate relapse in abstinent smokers, yet the molecular neuroadaptation(s) that contribute to this phenomenon are unknown. Interestingly, chronic nicotine use induces functional upregulation of nicotinic acetylcholine receptors (nAChRs) in the mesocorticolimbic reward pathway potentially linking upregulation to increased drug sensitivity. In the ventral tegmental area (VTA), functional upregulation of nAChRs containing the α4 subunit (α4* nAChRs) is restricted to GABAergic neurons. To test the hypothesis that increased functional expression of α4* nAChRs in these neurons modulates nicotine reward behaviors, we engineered a Cre recombinase-dependent gene expression system to selectively express α4 nAChR subunits harboring a "gain-of-function" mutation [a leucine mutated to a serine residue at the 9' position (Leu9'Ser)] in VTA GABAergic neurons of adult mice. In mice expressing Leu9'Ser α4 nAChR subunits in VTA GABAergic neurons (Gad2(VTA):Leu9'Ser mice), subreward threshold doses of nicotine were sufficient to selectively activate VTA GABAergic neurons and elicit acute hypolocomotion, with subsequent nicotine exposures eliciting tolerance to this effect, compared to control animals. In the conditioned place preference procedure, nicotine was sufficient to condition a significant place preference in Gad2(VTA):Leu9'Ser mice at low nicotine doses that failed to condition control animals. Together, these data indicate that functional upregulation of α4* nAChRs in VTA GABAergic neurons confers increased sensitivity to nicotine reward and points to nAChR subtypes specifically expressed in GABAergic VTA neurons as molecular targets for smoking cessation therapeutics.

Ngolab J ，Liu L ，Zhao-Shea R ，Gao G ，Gardner PD ，Tapper AR ... -  《-》

Menthol Stereoisomers Exhibit Different Effects on α4β2 nAChR Upregulation and Dopamine Neuron Spontaneous Firing.

Henderson BJ ，Grant S ，Chu BW ，Shahoei R ，Huard SM ，Saladi SSM ，Tajkhorshid E ，Dougherty DA ，Lester HA ... -  《eNeuro》

Why flavored vape products may be attractive: Green apple tobacco flavor elicits reward-related behavior, upregulates nAChRs on VTA dopamine neurons, and alters midbrain dopamine and GABA neuron function.

While nicotine is the primary addictive component in tobacco products, additional flavors have become a concern with the growing popularity of electronic nicotine delivery systems (ENDS). For this reason, we have begun to investigate popular tobacco and ENDS flavors. Here, we examined farnesol, a chemical flavorant used in green apple and fruit flavors in ENDS e-liquids, for its ability to produce reward-related behavior. Using male and female 3-6 month old C57BL/6 J mice and farnesol doses of 0.1, 1, and 10 mg/kg we identified a sex-dependent effect in a conditioned place preference assay: farnesol-alone produces reward-related behavior in only male mice. Despite this sex-dependent effect, 1.0 mg/kg farnesol enhances locomotor activity in both male and female mice. To understand farnesol's effect on reward-related behavior, we used whole-cell patch-clamp electrophysiology and confocal microscopy to investigate changes in putative dopamine and GABA neurons. For these approaches, we utilized genetically modified mice that contain fluorescent nicotinic acetylcholine receptors (nAChRs). Our electrophysiological assays with male mice revealed that farnesol treatment increases ventral tegmental area (VTA) dopamine neuron firing frequency and this may be due to a decrease in inhibitory tone from GABA neurons. Our microscopy assays revealed that farnesol treatment produces a significant upregulation of α6* nAChRs in male mice but not female mice. This was supported by an observed increase in α6* nAChR function in additional electrophysiology assays. These data provide evidence that popular tobacco flavorants may alter smoking-related behavior and promote the need to examine additional ENDS flavors.

Avelar AJ ，Akers AT ，Baumgard ZJ ，Cooper SY ，Casinelli GP ，Henderson BJ ... -  《-》