Linking Nicotine, Menthol, and Brain Changes

C H A P T E R

12 Linking Nicotine, Menthol, and Brain Changes Brandon J. Henderson Department of Biomedical Sciences, Marshall University, Joan C. Edwards School of Medicine, Huntington, WV, United States

Abbreviations GABA nAChRs SNc SNr VTA

12.2 BRIEF HISTORY OF MENTHOL CIGARETTES

γ-aminobutyric acid nicotinic acetylcholine receptors substantia nigra pars compacta substantia nigra pars reticulata ventral tegmental area

The first menthol cigarette was likely the “Spud,” which was marketed by Axton-Fisher in 1926 (Proctor, 2012). Soon after (1933), the tobacco company Brown and Williamson launched their Kool brand of menthol cigarettes. Since the 1950s, there have been several arguments whether menthol cigarettes were disproportionately marketed to African Americans. Discussing this would take away from the focus of this chapter, but a thorough review can be found elsewhere (Lochlann Jain, 2003). The most notable public health concerns involve menthol’s role in nicotine addiction. When we examine smokers of all ages, smokers of menthol cigarettes quit at a significantly reduced rate when compared to smokers of nonmenthol cigarettes (Delnevo, Gundersen, Hrywna, Echeverria, & Steinberg, 2011; D’Silva et al., 2012). This of course increases the risk of smoking-related negative outcomes: cancers of the lung, esophagus, oral cavity, and larynx and proposed higher incidence in heart disease and hypertension. The concern has become so great that the U.S. Food and Drug Administration (FDA) and World Health Organization (WHO) conducted independent investigations and concluded that menthol does increase smoking initiation and causes more intense addiction (FDA, 2012; WHO, 2016). Young smokers of menthol cigarettes are twice as likely to transition to lifelong smoking compared to youths smoking nonmenthol cigarettes (D’Silva et al., 2012). As e-cigarettes grow in popularity, smoking rates of flavored products (including menthol) are increasing, especially among youth smokers (CDC, 2016; Villanti et al., 2016). This troubles many public health experts, as they fear this may contribute to a new generation of nicotine addicts.

12.1 INTRODUCTION Menthol is the most popular tobacco flavorant worldwide. In America and European countries, the smoking rate of menthol cigarettes is 30% of the smoking population (Caraballo & Asman, 2011). Elsewhere, the rates of smoking menthol cigarettes vary with the highest being the Philippines (60%) (WHO, 2016). African Americans exhibit an incredibly high rate of smoking menthol cigarettes: 75% of adults and 90% of youth smokers (D’Silva, Boyle, Lien, Rode, & Okuyemi, 2012). The general youth smoking population in America also prefers menthol cigarettes over nonflavored cigarettes (Villanti et al., 2016). Menthol not only is found in high doses (3–20 mg/cigarette) in menthol-labeled cigarettes but also is present in 98% of nonmenthol cigarettes at low doses (2–70 μg/ cigarette) (Ai et al., 2015). Menthol is a sweet-scented monoterpenoid derived from peppermint oil. It produces cooling sensations topically through its agonist activity on transient receptor potential melastatin-8 (TrpM8) ( Journigan & Zaveri, 2013). Research into menthol’s actions is currently expanding due to increased public health concerns about its role in reduced smoking cessation rates, cardiovascular effects, and incidents of smoking-related cancer (discussed further below). Here, we discuss menthol, nicotine, and the changes in the brain that result from their combined actions.

Neuroscience of Nicotine https://doi.org/10.1016/B978-0-12-813035-3.00012-5

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Copyright © 2019 Elsevier Inc. All rights reserved.

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12. LINKING NICOTINE, MENTHOL, AND BRAIN CHANGES

12.3 MENTHOL AND NICOTINE: EARLY CLINICAL FINDINGS

12.4 MENTHOL’S ACTIONS ON CYS-LOOP RECEPTORS

How could menthol reduce the quit rates of smokers? Some studies suggest that menthol decreases airway irritation and allows smokers to inhale more nicotine. Clinical reports show that the puff duration, puff volume, and number of puffs are significantly increased with menthol cigarettes (Ahijevych & Parsley, 1999). There is also evidence that menthol decreases the metabolism of nicotine, allowing elevated concentrations of nicotine in the plasma (Alsharari et al., 2015; Benowitz, Herrera, & Jacob 3rd, 2004). Despite this, there are counterarguments as other reports show that plasma levels of nicotine fail to increase significantly with menthol (Ashley, Dixon, Sisodiya, & Prasad, 2012). The most dramatic finding regarding the smoking of menthol and nonmenthol cigarettes is the effect on nicotinic acetylcholine receptor (nAChR) upregulation. The upregulation of β2-containing (β2*) nAChRs is a wellcharacterized feature of chronic nicotine exposure. This occurs in human smokers ( Jasinska, Zorick, Brody, & Stein, 2013), rodents (Henderson et al., 2017), and cell lines (Srinivasan et al., 2011). This is believed to be a biomarker for nicotine addiction as this event plays an important role in altering the dopamine reward pathway (Faure, Tolu, Valverde, & Naude, 2014; Nashmi et al., 2007). In the context of humans that smoke menthol or nonmenthol cigarettes, Brody et al. (2013) found that smokers of menthol cigarettes exhibit more β2* nAChR upregulation in several brain regions compared to nonmenthol smokers. These brain areas included the prefrontal cortex, corpus callosum, hypothalamus, cerebellum, and brain stem. Many of these regions play an important role in the dopamine reward pathway (Fig. 12.1), and this finding was a key step toward understanding why smokers of menthol cigarettes quit at lower rates.

The molecular actions of menthol have been studied on several classes of ion channels (for a complete review, see Oz, El Nebrisi, Yang, Howarth, and Al Kury (2017)) including the Cys-loop family of receptors. Many of these studies use fairly high doses (>50 μM) of menthol (Table 12.1), while a few use lower doses (<1 μM) that are believed to be smoking-relevant (discussed below). The Cys-loop ligand-gated ion channel family includes nAChRs, γ-aminobutyric acid (GABA), glycine,

TABLE 12.1 Acute Actions of Menthol on Cys-loop Receptors IC50 or EC50

Stereoselective?

GABAA

Positive allosteric modulatora,b

60 μM, (+)menthol 160 μM, ( )menthol

Yes

Glycine

Positive allosteric modulatora

75 μM

No

5-HT3

Noncompetitive antagonistc

163 μM

No

α3β4 nAChR

Noncompetitive antagonistd

69–100 μM

Unknown

α7 nAChR

Noncompetitive antagoniste

33 μM

No

α4β2 nAChR

Negative allosteric modulatorf

111 μM

Unknown

a b c d e f

Hall et al. (2004). Corvalan, Zygadlo, and Garcia (2009). Ashoor et al. (2013a). Ton et al. (2015). Ashoor et al. (2013b). Hans, Wilhelm, and Swandulla (2012).

FIG. 12.1 Human smokers exhibit an upregulation of brain β2* nAChRs in several regions that play a role in addictive behavior. (A) Schematic of dopamine neuron projections (red) that originate from the substantia nigra and ventral tegmental area. (B) Schematic of brain regions that exhibit β2* nAChR upregulation in the brains of smokers (see Jasinska et al., 2013).

12.5 MENTHOL ENHANCES NICOTINE REWARD BY ALTERING MIDBRAIN DOPAMINE NEURONS

5-hydroxytryptophan 3 (5-HT3), and zinc-activated ion channel receptors. Each of these receptors is composed of a pentameric protein assembly and have a wellcharacterized loop formed by highly conserved amino acids between two cysteine (Cys) residues that form a disulfide bond (Sine & Engel, 2006). Menthol was discovered to be a negative allosteric modulator of α4β2 nAChRs and a noncompetitive antagonist of α7 and α3β4 nAChRs, with IC50 values of 111, 35, and 70 μM, respectively (Ashoor et al., 2013b; Hans et al., 2012; Ton et al., 2015) (Table 12.1). Additionally, menthol has been shown to be a noncompetitive antagonist of 5-HT3 receptors (IC50, 163 μM), a positive allosteric modulator of GABAA receptors (EC50, 60–160 μM), and a positive allosteric modulator of glycine receptors (EC50, 50 μM) (Ashoor et al., 2013a; Hall et al., 2004; Lau, Karim, Goodchild, Vaughan, & Drew, 2014). While many investigations examined ( )-menthol only, one identified a significant difference in the pharmacological actions of (+)-menthol and ( )-menthol (Hall et al., 2004). Here, both (+)-menthol and ( )-menthol acted as potentiators of GABAA currents, but (+)-menthol was significantly more potent than ( )-menthol. Thus, there may be more stereospecific interactions of menthol on Cys-loop receptors that are yet to be discovered. The concentrations used in these investigations probably exceed those found in vivo as the smoking-relevant concentration of menthol is likely <8 μM (Benowitz et al., 2004). These findings using high μM concentrations of menthol may not explain the observations of enhanced β2* nAChR upregulation in menthol smokers (Brody et al., 2013), but several implications can be made from these observations. GABA neurons play an important role in the nicotine reward pathway; thus, menthol’s ability to potentiate GABA signaling could exert significant changes in the brain that contribute to nicotine addiction (discussed further below). Given menthol’s ability to pharmacologically manipulate Cys-loop receptors, there has been great interest in identifying its binding site. In studies that compared menthol to the actions of benzodiazepines and the anesthetic propofol, it was revealed that menthol and propofol may share the same binding site on GABAA receptors (Watt et al., 2008). Given the conserved chemical features between menthol and propofol, it is not unbelievable that menthol binds GABAA receptors in a manner similar to propofol. Computational methods have been employed to identify an allosteric binding site on nAChRs (Ashoor et al., 2013b). Here, the muscle-type nAChR was used as a model due to its similarity to α7 nAChRs and because, at the time, it was the only nAChR that had a solved, fulllength crystal structure. Here, the binding of menthol was found to be in the transmembrane region, similar to the findings of Watt et al. (2008). Despite this, the field is still far from identifying a menthol-binding site on nAChRs.

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12.5 MENTHOL ENHANCES NICOTINE REWARD BY ALTERING MIDBRAIN DOPAMINE NEURONS To understand how menthol acts with nicotine to enhance nAChR upregulation (as observed by Brody et al.) and possibly reduce smoking quit rates, several investigations were completed to examine menthol’s ability to alter nicotine’s actions in the brain. From rodent models, we know that nAChR subunits are important for nicotine reward and reinforcement: Individual deletions of α4, α6, or β2 nAChR subunits prevent selfadministration of nicotine in mice, and selective reexpression of these deleted subunits in the VTA (not SNc or SNr) reinstates self-administration behavior (Pons et al., 2008) (see Fig. 12.2 for a schematic of neuronal inputs to VTA dopamine neurons). Selective activation of α6β2* or α4β2* nAChRs by smoking-relevant concentrations of nicotine stimulates depolarization and elevates firing frequency of VTA DA neurons (Engle, Shih, McIntosh, & Drenan, 2013; Liu, Zhao-Shea, McIntosh, Gardner, & Tapper, 2012). Together, these results clearly show that VTA α4β2*, α6β2*, and α4α6β2* nAChRs are the primary targets of nicotine and mediate its rewarding and reinforcing properties.

12.5.1 Menthol Enhances Nicotine-Induced Upregulation of nAChRs Nicotine’s effect on the brain has been well characterized. Nicotine upregulates nAChRs, alters the firing frequency and excitability of dopamine and GABA neurons, and enhances dopamine release in the nucleus accumbens (see Faure et al. (2014) for a complete review). Recently, an investigation into menthol’s effect on these well-characterized nicotine-induced changes in the brain was completed (Henderson et al., 2017). Experiments using mice revealed that menthol plus nicotine upregulates α4β2* and α4α6β2* nAChRs (not α6β2* nAChRs) in the VTA and SNr significantly more than nicotine alone (Henderson et al., 2017) (see Fig. 12.3). Another study using mice also reported that menthol enhances nicotine’s upregulation of β2* nAChR subunits in the prefrontal cortex (Alsharari et al., 2015). These studies provided a nAChR subtype-specific, region-specific, and cell-specific correlate to the assays using human brain imaging (Brody et al., 2013). Menthol Enhances Nicotine Reward and Reinforcement Conditioned place preference assays have been used to examine how menthol alters nicotine reward-related behavior (Henderson et al., 2017). Conditioned place preference is an experimental method based on Pavlovian training, used to study drug reward-related behavior.

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FIG. 12.2 (A) Schematic of dopamine reward pathway with the midbrain (VTA and SNr) highlighted for panel B1. (B1) Schematic of neurons contributing to nicotine reward in the midbrain and the nAChR subtypes involved. (B2) Key for nAChR subtypes in B1. Additional abbreviations: Amyg, amygdala; LDT, lateral dorsal tegmentum; LHB, lateral habenula; PPTG, pedunculopontine tegmental nucleus; STN, subthalamic nucleus.

FIG. 12.3 α4β2*, α6β2*, and α4α6β2* nAChRs on VTA dopamine neurons are upregulated by nicotine and nicotine plus menthol. (A1-2) Schematic of a VTA dopamine neuron projecting from the midbrain to anterior regions of the brain (the nucleus accumbens, prefrontal cortex, and striatum). (B) Schematic of nAChRs on the cell surface that are upregulated by nicotine or nicotine plus menthol. Sizes of arrows indicate the degree of upregulation.

This method has frequently been applied to the study of nicotine reward-related behavior (Henderson et al., 2017; Tapper et al., 2004). In this assay, menthol was added to nicotine at a dose perceived to be smoking-relevant and produced a significant increase in nicotine reward-related behavior (Henderson et al., 2017).

Rat self-administration assays were also used to examine menthol’s effect on nicotine reward and reinforcement. Like conditioned place preference, these experiments are used to examine drug reward, but unlike conditioned place preference, drug delivery is contingent to rodents triggering a mechanism. In rat nicotine

12.5 MENTHOL ENHANCES NICOTINE REWARD BY ALTERING MIDBRAIN DOPAMINE NEURONS

intravenous self-administration assays, menthol was also reported to enhance nicotine intake (Biswas et al., 2016; Wang, Wang, & Chen, 2014). It is assumed that menthol itself is not rewarding as these studies also revealed that menthol at many doses failed to produce reward in mice using the conditioned place preference assay (Henderson et al., 2017). Together, these experiments clearly document that menthol enhances nicotine reward and reinforcement. More importantly, this suggests that the lowered quit rate observed with smokers of menthol cigarettes could be due to the fact that menthol makes nicotine more rewarding (more addictive). Of course, there are other features that likely underlie the reduced cessation rate as it was recently revealed that menthol also enhances nicotine withdrawal (Alsharari et al., 2015). Although reports of menthol enhancing nicotineinduced upregulation of β2* nAChRs were made for several brain regions (Alsharari et al., 2015) (Henderson et al., 2017), these studies were not done with concentrations of menthol or nicotine that produce changes in reward-related behavior. When upregulation assays were repeated using doses of nicotine and menthol sufficient to alter reward-related behavior (Henderson et al., 2017), there was only an enhancement in the nicotineinduced upregulation of α4α6β2* nAChRs in the VTA (see Fig. 12.3). Given that α4α6β2* nAChRs are the most sensitive to nicotine and selectively expressed on dopamine neurons in the midbrain, this suggests that the selective upregulation of these nAChRs would dramatically alter dopamine neuron excitability and

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signaling (discussed further below). This marks α4α6β2* nAChRs as a primary contributor to menthol’s ability to enhance nicotine reward and reinforcement.

12.5.2 Menthol Enhances Nicotine-Induced Changes in Dopamine Neuron Excitability Midbrain dopamine neurons exhibit spontaneous activity of two distinct types: one characterized by slow, regular spikes (tonic) and one characterized by bursts of activity (phasic). Nicotine, through its ability to upregulate nAChRs in a cell-specific manner (Nashmi et al., 2007), alters both the tonic and phasic activity of dopamine neurons (see Faure et al. (2014) for a full review). GABA neurons are critical for this observed nicotine-induced change in dopamine neuron firing. GABA neurons from the VTA and SNr provide inhibitory input to the VTA dopamine neurons that project to anterior brain regions. Following chronic exposure to nicotine, α4* nAChRs are upregulated on VTA and SNr GABAergic neurons (Nashmi et al., 2007). The upregulated α4* nAChRs on GABA neurons provide enhanced inhibitory signaling and decrease tonic dopamine neuron firing. However, acute nicotine exposure enhances GABAergic activity transiently, followed by a prolonged depression in activity due to the upregulated α4* nAChRs’ desensitization. This triggers increased dopamine neuron burst firing (Mansvelder, Keath, & McGehee, 2002) (summarized in Fig. 12.4). This likely originates from α4α6β2* nAChRs, as the nicotine-induced

FIG. 12.4 Nicotine is well characterized to enhance both dopamine and GABA neuron firing transiently (denoted by transparent gray shading). It is also known that following chronic exposure to nicotine (>7 days), GABA neurons rapidly desensitize and exhibit reduced firing frequencies (for up to 1 h). This results in a disinhibition of target dopamine neurons. This contributes to an increase in dopamine neuron excitability. Chronic treatment with menthol and nicotine prevents GABA neurons from desensitization yet dopamine neurons exhibit an enhanced increase in excitability. See Henderson et al. (2017).

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enhancement in dopamine neuron firing is dependent upon nAChRs that contain both α4 and α6 subunits (Engle et al., 2013; Liu et al., 2012). Menthol, combined with nicotine, was also found to alter dopamine neuron firing (Fig. 12.4). Menthol plus nicotine produces increases in nicotine-stimulated dopamine neuron firing frequency that is significantly greater than nicotine alone. Once again, this is likely due to the enhanced upregulation of α4α6β2* nAChRs (see Fig. 12.3A). Despite menthol enhancement of nicotine’s actions on dopamine neurons, menthol was found to reverse the actions of nicotine on GABA neuron firing. As previously mentioned, long-term exposure to nicotine causes GABA neurons to exhibit a sustained depression in activity when they are acutely exposed to nicotine (Fig. 12.4). In the case of menthol plus nicotine, GABA neuron firing frequency increases transiently but slowly returns to a state resembling baseline firing frequency. Thus, no signs of sustained depression in activity were observed. It has been shown that menthol (alone) reduces α4β2* nAChR desensitization (Henderson et al., 2016). This may explain how GABA neurons treated with exposed to long-term menthol and nicotine may fail to desensitize during nicotine applications since they have only α4 (non-α6)β2* nAChRs. To summarize, when we consider the characterized effects of nicotine on dopamine neuron firing, menthol enhances these changes in dopamine neuron firing that ultimately leads to enhanced excitability.

12.6 MENTHOL BY ITSELF ALTERS nAChRs ON MIDBRAIN DOPAMINE NEURONS Much of our knowledge of menthol’s actions comes from studies that examined menthol combined with nicotine. From these studies, it is not clear if menthol produces an effect on its own and is additive to that of nicotine or if menthol by itself causes distinct brain changes. To that end, several investigations have been completed to address this question.

12.6.1 Menthol Alone Upregulates nAChRs To gain a better understanding of how menthol produces enhanced nAChR upregulation in smokers, menthol was examined for its effect on upregulation in the absence of nicotine (Henderson et al., 2016). Here, it was found that menthol upregulated α4β2 and α6β2β3 nAChRs that were transiently transfected into neuro-2a cells. More importantly, this was achieved using a concentration of menthol (0.5 μM) that was much lower than the concentrations necessary for allosteric antagonism or

enhancement of Cys-loop receptors (see Table 12.1). In mice, upregulation of nAChRs was observed using menthol concentrations <2.5 μM (Henderson et al., 2016). Menthol also exhibited cell-specific upregulation of midbrain α4* nAChRs that differs from nicotine-induced upregulation. Nicotine robustly upregulates α4* nAChRs on SNr and VTA GABA neurons (Nashmi et al., 2007), but menthol upregulates α4* nAChRs on SNc and VTA DA neurons and does not upregulate SNr α4* nAChRs (Henderson et al., 2016). There also was a difference in the upregulated nAChRs when you compare menthol plus nicotine to menthol alone: nicotine plus menthol upregulates high-sensitivity nAChRs, while the menthol alone upregulates only lowsensitivity nAChRs (Fig. 12.5). This was observed only with in vitro preparations as the studies using mice could not provide information in regard to nAChR stoichiometry.

12.6.2 Menthol-Alone Abolishes Nicotine Reward-Related Behavior Interestingly, menthol exposure prior to nicotine prevents nicotine reward-related behavior in a conditioned place preference assay using mice (Henderson et al., 2016). In this assay, mice were treated with menthol alone for 10 days prior to the training for conditioned place preference. Therefore, the prevention of nicotine reward-related behavior likely resulted from menthol’s ability to upregulate low-affinity nAChRs. The result is that nicotine failed to activate midbrain dopamine and GABA neurons robustly due to the presence of lowaffinity and not high-affinity nAChRs being available.

12.7 SUMMARY Menthol, nicotine, and their combination cause several changes in the brain. Although menthol has been shown to act on several Cys-loop receptors, the longterm effects have only been characterized on nAChRs. Alone or together, nicotine and menthol upregulate brain nAChRs. The upregulation of highly nicotine sensitive nAChRs cause significant changes in dopamine reward signaling (summarized in Table 12.2). Most notably, these neurons exhibit increased excitability in response to nicotine. This likely is the reason why menthol, combined with nicotine, causes greater positive reward when compared to nicotine alone. Together, these changes highlight the fact that the reduced cessation rates for smokers of menthol cigarettes can be attributed, at least in part, to menthol-induced enhancement in nicotine reward.

MINI-DICTIONARY OF TERMS

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FIG. 12.5 Menthol, nicotine, and menthol plus nicotine alter (A) α6* and (B) α4* nAChR stoichiometry. This diagram represents findings from cultured neuro-2a cells transiently transfected with α6, β2, and β3 nAChR subunits or α4 and β2 nAChR subunits (Henderson et al., 2017, 2016). Menthol alone stabilizes low-sensitivity nAChRs (α6β2(non-β3) and α4(3)β2(2)). Nicotine and nicotine plus menthol, stabilize high-sensitivity nAChRs (α6β2β3 and α4(2)β2(3)). Blue dots denote the agonist binding site for nicotine and acetylcholine. The yellow dots denote the noncanonical binding site that has been recently characterized for ACh. TABLE 12.2 Comparison of Brain Changes Caused by Nicotine, Nicotine Plus Menthol, and Menthol Nicotine

Nicotine + menthol

α4* nAChR upregulation

VTAa, SNca, SNra,d, and striatumb

VTAa, SNca, SNra, striatumb, and prefrontal cortexb

VTAc, SNcc, striatumc, and prefrontal cortexc

α6* nAChR upregulation

VTAa and SNca

VTAa

VTAc and SNcc

α4α6* nAChR upregulation

VTAa and SNca

VTAa and SNca

Not yet examined

Menthol

Upregulation

Neuron baseline firing Dopamine neurons

Decreased firinga,d

Decreased firinga

Decreased firingc

GABA neurons

Increased firinga

Increased firinga

Not yet examined

Neuron excitability Dopamine neurons

Increased excitabilitya,d

Increased excitabilitya

Decreased excitabilityc

GABA neurons

Decreased excitabilitya

Increased excitabilitya

Not yet examined

a b c d

Henderson et al. (2017). Alsharari et al. (2015). Henderson et al. (2016). Nashmi et al. (2007).

MINI-DICTIONARY OF TERMS Conditioned place preference An assay of drug reward-related behavior that is noncontingent to an instrumental/animal reaction. Drug reward and reinforcement The study of addiction with animal models is complex. For this reason, assays are optimized to measure individual components of addiction: reward, reinforcement, reinstatement, withdrawal, etc. Electrophysiology The study of electric properties of cells and neurons. Nicotinic acetylcholine receptors (nAChRs) Cholinergic receptors that are sensitive to nicotine. These receptors are present in the peripheral and central nervous system. Acetylcholine (ACh) is the endogenous neurotransmitter of both nAChRs and muscarinic ACh receptors. Nucleus accumbens A region of the ventral striatum where the terminals from mesolimbic dopamine neurons terminate onto GABAergic medium spiny neurons. Prefrontal cortex The region of the cerebral cortex that covers anterior brain regions and is implicated in many aspects of cognitive behavior. Self-administration An assay of drug reinforcement-related behavior that is contingent to an action (choice) of the animal. Substantia nigra pars reticulata Midbrain region mostly consisted of inhibitory GABAergic neurons. Upregulation Upregulation is a complex process, but this term is most widely used to designate an increase in the number of a particular protein. This can occur with many proteins, but this is a hallmark feature of nicotine: nicotine increases the number (upregulates) of nAChRs. Ventral tegmental area Brain region close to the midline of the ventral midbrain and is the origin of dopamine projections of the mesolimbic and nigrostriatal pathways. This region is critical for reward circuitry.

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Key Facts of Menthol • Menthol is the only legal flavorant allowed in nonelectronic cigarettes in America. • Menthol is present even in 98% of the nonmenthol cigarettes sold. • Menthol is present at 3 mg/cigarette in menthollabeled cigarettes. • In nonmenthol cigarettes, menthol is present in trace amounts (<0.1 mg). • In addition to its use as a tobacco flavorant, menthol is used as a local anesthetic, decongestant, antipruritic, and flavorant for mint and peppermint flavored products (gum, candy, etc.). Summary Points • Both nicotine and menthol upregulate nAChRs on midbrain dopamine and GABA neurons. • When nicotine is combined with menthol, the upregulation that occurs is significantly greater than either alone. • Individually, nicotine increases phasic dopamine neuron firing, while menthol decreases this pattern of firing. • Combined menthol and nicotine enhance phasic dopamine neuron firing to a level that is greater than nicotine alone. • Ultimately, these changes increase dopamine neuron excitability and enhance nicotine reward.

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