Human Tobacco Smokers in Early Abstinence Have Higher Levels of β2* Nicotinic Acetylcholine Receptors than Nonsmokers

“The severity of nicotine dependence was assessed using the Fagerström Test for Nicotine Dependence (FTND) (Heatherton et al., 1991), nicotine withdrawal symptoms were assessed using the Minnesota Withdrawal Questionnaire (MWQ) (Hatsukami et al., 1984), and the urge to smoke (or craving) was assessed using the Urge to…”

“Because the 2*-nAChR is the initial site of action of nicotine, adaptive changes during acute abstinence may be associated with nicotine withdrawal symptoms that emerge within hours after the last cigarette and persist for 3– 4 weeks (Hughes and Hatsukami, 1986).”

“Because the reinforcing properties of nicotine have been linked specifically to nAChRs that contain the 2 subunit ( 2*-nAChRs) (Picciotto et al., 1998), an understanding of the adaptive changes in 2*-nAChR number during acute abstinence and their relationship to behavioral correlates of tobacco…”

“Despite the well known health risks of tobacco smoking, 24.9% of the United States population continues to smoke (Grant et al., 2004).”

“Although most withdrawal symptoms resolve after a few days of abstinence, craving (i.e., the urge to smoke) is a prominent feature of nicotine dependence (Tiffany and Drobes, 1991) and is a primary factor associated with relapse (Killen et al., 1997).”

“Postmortem studies of human brain have suggested that numbers of nAChRs are higher in smokers than in nonsmokers or former smokers (Benwell et al., 1988; Breese et al., 1997; Perry et al., 1999), and animals exposed to nicotine chronically exhibit increased levels of nAChRs in the brain (Marks et…”

“Greater 2*nAChR availability is not associated with changes in 2*-nAChR mRNA (Marks et al., 1992; Peng et al., 1994; Zhang et al., 1994) protein synthesis (Buisson and Bertrand, 2001), rate of receptor internalization, postendocytic trafficking, or lysosomal degradation (Darsow et al.”

“…that numbers of nAChRs are higher in smokers than in nonsmokers or former smokers (Benwell et al., 1988; Breese et al., 1997; Perry et al., 1999), and animals exposed to nicotine chronically exhibit increased levels of nAChRs in the brain (Marks et al., 1983, 1985, 1992; Schwartz and Kellar, 1983).”

“…that numbers of nAChRs are higher in smokers than in nonsmokers or former smokers (Benwell et al., 1988; Breese et al., 1997; Perry et al., 1999), and animals exposed to nicotine chronically exhibit increased levels of nAChRs in the brain (Marks et al., 1983, 1985, 1992; Schwartz and Kellar, 1983).”

“The interclass coefficient to assess inter-rater reliability (Rousson et al., 2002) ranged between 0.”

“Greater 2*nAChR availability is not associated with changes in 2*-nAChR mRNA (Marks et al., 1992; Peng et al., 1994; Zhang et al., 1994) protein synthesis (Buisson and Bertrand, 2001), rate of receptor internalization, postendocytic trafficking, or lysosomal degradation (Darsow et al.”

“…brain have suggested that numbers of nAChRs are higher in smokers than in nonsmokers or former smokers (Benwell et al., 1988; Breese et al., 1997; Perry et al., 1999), and animals exposed to nicotine chronically exhibit increased levels of nAChRs in the brain (Marks et al., 1983, 1985, 1992;…”

“…nAChR availability is not associated with changes in 2*-nAChR mRNA (Marks et al., 1992; Peng et al., 1994; Zhang et al., 1994) protein synthesis (Buisson and Bertrand, 2001), rate of receptor internalization, postendocytic trafficking, or lysosomal degradation (Darsow et al., 2005), but instead…”

“Functionally, greater 2*-nAChR availability in tobacco smokers likely represents greater numbers of desensitized and inactivated nAChRs (Wonnacott, 1990; Dani and Heinemann, 1996).”

“Although a species difference cannot be ruled out, this divergence may suggest that the ability of nicotine to regulate 2*-nAChR availability is age or sex dependent because the nonhuman primates were male adolescents and the smokers were male and female adults (Slotkin, 2002; Nguyen et al., 2003).”

“…that numbers of nAChRs are higher in smokers than in nonsmokers or former smokers (Benwell et al., 1988; Breese et al., 1997; Perry et al., 1999), and animals exposed to nicotine chronically exhibit increased levels of nAChRs in the brain (Marks et al., 1983, 1985, 1992; Schwartz and Kellar, 1983).”

“, 2003), enhanced maturation and transport through the secretory pathway to the cell membrane (Sallette et al., 2005), increased trans-”

“…it has also been suggested that the upregulation occurs as a consequence of increased assembly of 4 and 2 subunits in the endoplasmic reticulum (Nashmi et al., 2003), enhanced maturation and transport through the secretory path- way to the cell membrane (Sallette et al., 2005), increased…”

“…endoplasmic reticulum (Nashmi et al., 2003), enhanced maturation and transport through the secretory path- way to the cell membrane (Sallette et al., 2005), increased transport to the membrane (Harkness and Millar, 2002), and/or decreased receptor turnover (Peng et al., 1994; Wang et al., 1998).”

“Smokers (12 of 16) were assisted in quitting smoking using brief behavioral counseling based on Clinical Practice Guidelines and contingency management (Stitzer and Bigelow, 1985; Stitzer et al., 1986; Roll et al., 1996).”

“Because nicotine competes with [ 123I]5-IA binding for binding to nAChRs (Mukhin et al., 2000), we first studied nonhuman primates chronically treated with nicotine to estimate the time interval necessary for smokers to abstain from smoking (before [ 123I]5-IA SPECT imaging) to ensure that residual…”

“Although a species difference cannot be ruled out, this divergence may suggest that the ability of nicotine to regulate 2*-nAChR availability is age or sex dependent because the nonhuman primates were male adolescents and the smokers were male and female adults (Slotkin, 2002; Nguyen et al., 2003).”

“Although most withdrawal symptoms resolve after a few days of abstinence, craving (i.e., the urge to smoke) is a prominent feature of nicotine dependence (Tiffany and Drobes, 1991) and is a primary factor associated with relapse (Killen et al., 1997).”

“, 2003) and that radiolabeled nicotine rapidly clears from brain (Crooks and Dwoskin, 1997; Crooks et al., 1997).”

“…and taste reception, these findings may suggest that occupancy of 2-nAChR by nicotine in the sensorimotor cortex may contribute to some of the sensory cues or “tasting of cigarettes” that has been shown to play an important role in the relief of craving and the urge to smoke (Rose and Behm, 1994).”

“*p values 0.00625 are significant after Bonferroni’s correction. unlike a previous study that demonstrated a positive correlation between [ 3H]nicotine binding to nAChRs on polymorphonuclear lymphocytes and the number of cigarettes smoked per day (Benhammou et al., 2000).”

“Smokers (12 of 16) were assisted in quitting smoking using brief behavioral counseling based on Clinical Practice Guidelines and contingency management (Stitzer and Bigelow, 1985; Stitzer et al., 1986; Roll et al., 1996).”

“Although 2 subunits exist in several different combinations with 2, 3, 4, and 6 subunits, the 4 2 combination is most prevalent and also the most sensitive to upregulation by nicotine (Nguyen et al., 2003; Sallette et al., 2004).”

“…et al., 1994) protein synthesis (Buisson and Bertrand, 2001), rate of receptor internalization, postendocytic trafficking, or lysosomal degradation (Darsow et al., 2005), but instead appears to be attributable to occupancy of the nicotine binding site that bridges the / subunit interface of the…”

“…endoplasmic reticulum (Nashmi et al., 2003), enhanced maturation and transport through the secretory path- way to the cell membrane (Sallette et al., 2005), increased transport to the membrane (Harkness and Millar, 2002), and/or decreased receptor turnover (Peng et al., 1994; Wang et al., 1998).”

“Nicotine was administered orally using the dose escalation paradigm described previously (Pietila et al., 1998).”

“With the recent development of [ 123I]5-IA-85380 ([ 123I]5-IA), a radioligand that has specificity for the agonist binding site on 2*- nAChRs (Musachio et al., 1998, 1999; Horti et al., 1999), it is now possible to measure this site in living smokers during acute abstinence from smoking and to…”

“The apparent long residence time of nicotine on 2*-nAChR in brain is surprising given that the terminal half-life of nicotine is 1.6 h in macaques (Schoedel et al., 2003) and that radiolabeled nicotine rapidly clears from brain (Crooks and Dwoskin, 1997; Crooks et al., 1997).”

“With the recent development of [ 123I]5-IA-85380 ([ 123I]5-IA), a radioligand that has specificity for the agonist binding site on 2*- nAChRs (Musachio et al., 1998, 1999; Horti et al., 1999), it is now possible to measure this site in living smokers during acute abstinence from smoking and to…”

“The rapid pharmacokinetics were observed, however, after a single administration of a trace dose of intravenous nicotine, and the accumulation of nicotine and/or metabolites (Vainio and Tuominen, 2001) in brain may differ with protracted and repeated nicotine administration.”

“Smokers (12 of 16) were assisted in quitting smoking using brief behavioral counseling based on Clinical Practice Guidelines and contingency management (Stitzer and Bigelow, 1985; Stitzer et al., 1986; Roll et al., 1996).”

“…of [ 123I]5-IA-85380 ([ 123I]5-IA), a radioligand that has specificity for the agonist binding site on 2*- nAChRs (Musachio et al., 1998, 1999; Horti et al., 1999), it is now possible to measure this site in living smokers during acute abstinence from smoking and to determine the relationship…”

“Nicotine is highly lipophilic and, when radiolabeled, demonstrates very high levels of nonspecific brain uptake (Broussolle et al., 1989; Muzic et al., 1998).”

“The relationship between greater 2*-nAChR availability and nAChR function may vary in a region-dependent manner attributable to different subunit combinations that demonstrate increased agonist binding but differ in the effects on function (Rush et al., 2002).”

“Greater 2*- nAChR availability is not associated with changes in 2*-nAChR mRNA (Marks et al., 1992; Peng et al., 1994; Zhang et al., 1994) protein synthesis (Buisson and Bertrand, 2001), rate of receptor internalization, postendocytic trafficking, or lysosomal degradation (Darsow et al., 2005), but…”

“Three SPECT scans (30 min each) were obtained between 6 and 8 h of infusion, and plasma samples were collected immediately before and after the scan to quantify total parent and the free fraction of parent tracer in plasma ( f1, free fraction) (Zoghbi et al., 2001).”

“Nonhuman primates were imaged using methods described previously (Staley et al., 2000).”

“have high β2* receptor levels for at least seven days after stopping smoking [123].”

“Indeed, this hypothesis suggests that the increased depressive symptoms observed in some patients following acute cessation from smoking might be explained by the fact that the clearance of nicotine following smoking cessation coupled with persistent nAChR upregulation (Staley et al., 2006) results in increased abilit…”

“…in humans have also confirmed both the upregulation of a4b2 receptors after chronic NIC administration (smoking) and the termination of upregulation after approximately 21 days of smoking cessation (Mamede et al., 2007; Staley et al., 2006; Wullner et al., 2008; Kassiou et al., 2001).”

“Long-term nicotine administration, delivered in multiple forms (injection, minipump, drinking water, or self-administration), increases the numbers of 4 2* binding sites throughout the brain in experimental animal models and humans (Marks et al., 1983; Schwartz and Kellar, 1983; Benwell et al., 1988; Perry et al., 199…”

“Overnight abstinence was confirmed with breath CO levels 10 ppm. Plasma nicotine and cotinine levels were measured as previously described (Staley et al., 2006).”

“Using a radioligand with specificity for β2containing nAChRs, which enabled the use of single-photon emission computed tomography (SPECT) (Staley et al. 2006), it was found that chronic smokers have more cortical, striatal, and cerebellar β2* nAChRs than non-smokers (Staley et al.”

“Imaging studies of nicotine abuse report upregulation of the nicotinic receptor (Cosgrove et al., 2009; Mukhin et al., 2008; Staley et al., 2006; Wullner et al., 2008) and an imaging study of opiate abusers showed upregulation of the mu receptor (Zubieta et al., 2000).”

“Further, following cessation of smoking, unbound nAChRs are present at a higher level for as long as one week, and nAChR binding availability has been associated with self-reported withdrawal and craving (Cosgrove et al., 2009; Staley et al., 2006).”

“…2-FA and 5-IA, have demonstrated up-regulation of available a4b2* nAChRs in smokers compared to non-smoking participants in brain regions other than the thalamus (Brody et al., 2012; Cosgrove et al., 2009, 2012; Mamede et al., 2007; Mukhin et al., 2008; Staley et al., 2006; Wullner et al., 2008).”

“Upregulation is still observed in abstinent smokers at least 14 days after smoking cessation [63], and tolerance can also be observed without changes in nicotine binding [64].”

“As mentioned, the observed changes in VT/fP were similar to those observed with SPECT and 5IA as the radioligand (28).”

“the effects of tobacco use on nicotinic receptor saturation and availability [86–91].”

“Nicotine-induced upregulation of nAChR expression has been detected in the brains of rodents and human smokers (Marks et al., 1983; Breese et al., 1997; Staley et al., 2006; Nashmi et al., 2007).”

“nAChRs and commonly used in smoking studies [81,82].”

“However, chronic smoking also leads to long-term upregulation of high affinity nAChRs containing the b2 subunit (Staley et al., 2006), potentially increasing nAChR activity once nicotine-induced desensitization is relieved following abstinence.”

“SPECT imaging protocol was conducted as described previously (34).”

“Widespread upregulation of nAChRs, likely related to receptor desensitization from nicotine exposure, has been observed among smokers [70, 71] and, conversely, smoking cessation associated with decreases in regional nAChR densities [72].”

“This issue of smokers using nicotine/tobacco during the abstinence period of a brain imaging study has also been reported in prior studies (Esterlis et al. 2010a ; Staley et al. 2006), presumably related to difficulty in having tobaccodependent smokers remain abstinent for a prolonged period.”

“Upregulation of AChRs in humans has been detected by comparing [3H]nicotine binding to postmortem brains of smokers and non-smokers and in vivo by fMRI or PET imaging (Benwell et al., 1988; Breese et al., 1997; Brody et al., 2013, 2008, 2011, 2006; Cosgrove et al., 2009; Jasinska et al., 2014; Mamede et al., 2007; Per…”

“Recently, increased nAChRs were imaged in living human subjects who smoked (Staley et al., 2006).”

“While the terminal half-life of nicotine is ~ 2 hours [14], a recent imaging study [15] suggests that nicotine may reside on the nicotine receptor for a longer duration.”

“In a recent study, Staley et al. (2006) hypothesized that an abnormally high number of β2nAChRs in early abstinence may be responsible for continued tobacco usage.”

“The authors noted that after 6.8 6 1.9 d of tobacco abstinence, the uptake of 123I-5IA increased significantly throughout the cerebral cortex (26%;36%) in smokers ( 13 ).”

“…using 2-[ 18F]fluoro-A-85380 (binds 2* nAChRs) indicate that 2* nAChR upregulation in humans is saturated with only a few cigarettes smoked daily (Staley et al., 2006), probably because 2* nAChRs are saturated even at nicotine concentrations found in low-nicotine cigarettes (Brody et al., 2009).”

“One consequence of chronic nicotine exposure through tobacco smoke is an upregulation of high-affinity β2-containing nAChRs throughout the brain [13].”

“…would occur globally throughout the brain, as prior research by our group (Brody et al, 2006a, 2009a, 2011, 2013) and others (Cosgrove et al, 2009; Staley et al, 2006) demonstrates widespread effects of smoking when studying systems (eg, the nicotinic cholinergic system) that are widely…”

“In humans, smoking leads to an upregulation of α4β2* nAChRs in the brain (Benwell et al., 1988; Staley et al., 2006).”

“Previous studies revealed that 1 week of smoking cessation is required for all nicotine to leave the brain to accurately quantify β2*nAChR availability (25).”

“Plasma nicotine concentrations were assayed using reversed-phase highperformance liquid chromatography (HPLC) based on a modification of a previously described method (115, 116).”

“Due to the chronic nAchRs upregulation, abstinence from nicotine will dramatically increase the availability of unbound α4β2 nAchRs, which in turn leads to smoking craving (Staley et al., 2006) and the subsequent smoking relapse.”

“, 2006), nicotine dependence (Mukhin et al., 2008; Staley et al., 2006), major depressive disease (Saricicek et al.”

“The same study demonstrated that abstinence-induced increases in the numbers of unbound receptors correlated with smoking urges (26).”

“As ACh and DA Frontiers in Neural Circuits | www.frontiersin.org 4 February 2018 | Volume 12 | Article 3 are believed to serve antagonistic roles, increasing cholinergic tone through acetylcholinesterase inhibitors can suppress addictive symptoms (Staley et al., 2006).”

“Abstinent smokers compared to nonsmokers had higher levels of b2* nAChRs, which returned to baseline between 3e12 weeks, and elevated nAChR levels correlated with urge to smoke (Cosgrove et al., 2009; Mamede et al., 2007; Staley et al., 2006).”

“Though there are some differences, such as a subunit distribution in the thalamus, reviewed previously (Spurden et al. 1997), they are in general similarly distributed in rats and humans (Spurden et al. 1997; Turner and Kellar 2005; Staley et al. 2006).”

“Imaging studies showed that receptors remain upregulated even in the abstinence of smoking for more than seven days [139].”

“…nicotinic receptor saturation and availability in volunteers (Brody et al., 2006; Gallezot et al., 2005; Kimes et al., 2003; Shumway et al., 2007; Staley et al., 2005, 2006), only a few studies were performed in patients which show alterations in nAChRs densities in frontal lobe epilepsy (Picard…”

“In abstinent smokers, the level of free a4b2 nAChRs is associated with the desire to smoke [29].”

“Specifically, there is a higher availability of b2*-nAChRs in tobacco smokers during acute abstinence compared to never smokers in the cerebellum, striatum, and throughout the cortex (Mukhin et al. 2008; Staley et al. 2006).”

“This study was done in recently abstinent (~1 week) tobacco smokers, since the nicotine from tobacco competes with the radiotracer, and previous studies have shown that there is still upregulation of nAChRs at this time point in smokers without psychiatric illness (Staley et al. 2006).”

“Moreover, molecular imaging studies have shown that occupancy of the nicotinic acetylcholine receptor is associated with the subjective hedonic response of smoking (Brody et al. 2009a, 2011, 2006a, 2014, 2013; Cosgrove et al. 2009a; Cosgrove et al. 2012; Dubroff et al. 2015; Lotfipour et al. 2012; Staley et al. 2006).”

“Tobacco use in humans and chronic nicotine or tobacco smoke exposure in rodents leads to an upregulation of nAChRs in the brain (Benwell et al. 1988; Marks et al. 1983; Small et al. 2010; Staley et al. 2006).”

“Upregulation of a4b2* nAChRs by long-term exposure to nicotine has been suggested to play a role in the development/ maintenance of nicotine dependence and has been observed repeatedly both in vitro and in vivo in rodents and human smokers (Staley et al., 2006; Lester et al., 2009).”

“441 1992; Mao et al., 2008; Moretti et al., 2010; Marks et al., 2011), and based on the limited spectrum of the radioligand used to image the receptors in vivo that is also probably the case in primate brains (Kassiou et al., 2001; Staley et al., 2006; Wüllner et al., 2008).”

“…(Brody et al, 2013) and other studies linking nicotine (Marks et al, 2011; Yates et al, 1995; Zhang et al, 2002) and cigarette smoke (Mamede et al, 2007; Mukhin et al, 2008; Staley et al, 2006; Wullner et al, 2008) exposure with upregulation of a4b2* nAChRs in brain regions other than the thalamus.”

“Preclinical studies assessing nicotine administration in animals and postmortem human studies of smokers demonstrated β2-nAChR upregulation throughout the striatum, frontal cortex, anterior cingulate cortex, temporal cortex, occipital cortex, and cerebellum [73], suggesting greater levels of β2-nAChR desensitization a…”

“High test–retest reliability and validity were recently reported for 123I-5-IA SPECT (22), and 123I-5-IA SPECT has demonstrated significantly higher b2-nAChR availability in recently abstinent tobacco smokers than in age- and sex-matched nonsmokers (23).”

“SPECT molecular imaging studies of the nAChR a4b2* subtype have also shown that nAChR availability in smokers peaks after 1 wk of abstinence (22,24), suggesting the persistent occupation of nAChRs by nicotine.”

“Long-term exposure to nicotine leads to an activity increase, amount upregulation, or both of nicotine binding sites in the brain of smokers [153, 154] and rodents subjected to repeated nicotine administration [155, 156].”

“…Marks et al., 1992; Buisson and Bertrand, 2002) and has been confirmed in several systems including cultured cells and rodent and human tissues (Marks et al., 1983; Schwartz and Kellar, 1985; Benwell et al., 1988; Peng et al., 1994; Breese et al., 1997; Perry et al., 1999; Staley et al., 2006).”

“At one week of abstinence, β2*-nAChR availability in the sensorimotor cortex was negatively correlated with the urge to smoke to relieve withdrawal symptoms (Staley et al. 2006).”

“A common feature in both conditions is that of higher ACh concentrations compared to those in the normal population [20,30,31].”