These in vivo studies were an extension of much prior research, including human postmortem brain tissue studies, demonstrating that chronic smokers have increased nAChR density compared to non-smokers and former smokers. Additionally, many studies of laboratory animals have demonstrated upregulation of markers of nAChR density in response to chronic nicotine administration . In a previous study by our group comparing nAChR availability between smokers and nonsmokers , we explored the effect of many variables, including caffeine and marijuana use. Both heavy caffeine and marijuana use were exclusionary, such that participants drank an average of 1.3 coffee cup equivalents per day and only 12 % of the study sample reported occasional marijuana use. PET results indicated that caffeine and marijuana use had significant relationships with α4β2* nAChR availability in this group with low levels of usage. Based on these preliminary findings, we undertook a study of the effect of heavy caffeine or marijuana usage on α4β2* nAChR density in cigarette smokers.One hundred and one otherwise healthy male adults completed the study and had usable data. Participants were recruited and screened using the same methodology as in our prior reports , with the exception that this study only included Veterans. For smokers, the central inclusion criteria were current nicotine dependence and smoking 10 to 40 cigarettes per day, while for non-smokers, the central inclusion criterion was no cigarette usage within the past year. Heavy caffeine use was defined as the equivalent of ≥3 cups of coffee per day, and heavy marijuana use was defined as ≥4 uses of at least 1 marijuana cigarette per week. Exclusion criteria for all participants were as follows: use of a medication or history of a medical condition that might affect the central nervous system at the time of scanning, any history of mental illness, or any substance abuse/dependence diagnosis within the past year other than caffeine or marijuana diagnoses.There was no overlap between this study and prior research by our group. During an initial visit,cannabis drying racks screening data were obtained to verify participant reports and characterize smoking history. Rating scales obtained were as follows: the Smoker’s Profile Form , Fagerström Test for Nicotine Dependence , Beck Depression Inventory , Hamilton Depression Rating Scale , and Hamilton Anxiety Rating Scale .

An exhaled carbon monoxide level was determined using a MicroSmokerlyzer to verify smoking status. A breathalyzer test and urine toxicology screen were obtained at the screening visit to support the participant’s report of no current alcohol abuse or other drug dependencies. This study was approved by the local institutional review board , and participants provided written informed consent.Roughly 1 week after the initial screening session, participants underwent PET scanning following the same general procedure as in our prior reports . Participants from the smoker groups began smoking/ nicotine abstinence two nights prior to each PET session and were monitored as described previously , so that nicotine from smoking would not compete with the radiotracer for receptor binding during PET scanning. Caffeine/marijuana abstinence was initiated 12 h prior to PET scanning, so that acute ingestion/ intoxication would not affect study results. At 11 AM on the scanning day, participants arrived at the VA Greater Los Angeles Healthcare System PET Center, and smoking abstinence was verified by participant report and having an exhaled CO ≤ 4 ppm. Each participant had an intravenous line placed at 11:45 AM in a room adjacent to the PETs canner. At 12 PM, bolus-plus-continuous-infusion of 2-[ 1 8F]fluoro-3-azetidinylmethoxy pyridine was initiated, with 2-FA administered as an intravenous bolus in 5-ml saline over 10 s . Roughly, the same amount of 2-FA was also diluted in 60-ml saline, and 51.1 ml was infused over the next 420 min by a computer-controlled pump . 2- FA-specific activities were similar for the study groups . Groups did not significantly differ for injected or infused doses of 2-FA, or for specific activity . Thus, the amount of 2-FA administered as a bolus was equal to the amount that would be infused over 500 min . This Kbolus was effective for reaching an approximate steady state in recent studies by our group and collaborators . After initiation of the bolus-plus-continuous-infusion, participants remained seated in the room adjacent to the PET scanner for the next 4 h to allow the radiotracer to reach a relatively steady state in the brain. At 4 PM, PET scanning commenced and continued for 3 h, with a 10-min break after 90 min of scanning. Scans were acquired as series of 10-min frames. PET scans were obtained using the Philips Gemini TruFlight , a fully three-dimensional PET-CT scanner, which was operated in non-TOF mode. Reconstruction was done using Fourier rebinning and filtered back projection, and scatter and random corrections were applied. The mean spatial resolution for brain scanning is 5.0 mm by 4.8 mm . 2-FAwas prepared using a published method ; this radiotracer was developed as a ligand specific for β2*-containing nAChRs . A magnetic resonance imaging scan of the brain was obtained for each participant within a week of PET scanning on a 1.5-T Magnetom Symphony System scanner , in order to aid in localization of regions on the PET scans.

The MRI had the following specifications: three-dimensional Fourier-transform spoiled-gradient-recalled acquisition with TR = 30 ms, TE = 7 ms, 30 degree angle, 2 acquisitions, 256 × 192 view matrix. The MRI scanning procedure typically lasted ∼30 min. The acquired volume was reconstructed as roughly 90 contiguous 1.5-mm-thick transaxial slices. Blood samples were drawn during PET scanning for determinations of free, unmetabolized 2-FA and nicotine levels in plasma. For 2-FA levels, four samples were drawn as standards prior to 2-FA administration, and nine samples were drawn at predetermined intervals during PET scanning. 2-FA levels were determined using previously published methods . For plasma nicotine levels, blood samples were drawn prior to and following PET scanning. These samples were centrifuged, and venous plasma nicotine concentrations were determined in Dr. Peyton Jacob’s laboratory at UCSF, using a modified version of a published GC-MS method . The lower limit of quantification for this method was 0.2 ng/ ml. In addition to the participants described in this paper, 11 smokers completed study procedures but were excluded from the data analysis because their plasma nicotine levels were unacceptably high . This issue of smokers using nicotine/tobacco during the abstinence period of a brain-imaging study has been reported in prior studies by our group and others , presumably related to difficulty in having tobacco-dependent smokers remain abstinent for a prolonged period.To determine if the four study groups differed on demographic, rating scale, or substance-use variables, analyses of variance were performed with the variables as dependent measures and group as a between-subject factor. ANOVAs were also performed for the three groups of smokers for smoking-related variables . These analyses were performed to verify that groups differed on caffeine and marijuana use and to determine if groups had potentially confounding variables that would need to be considered when evaluating the PET data. For evaluating group differences in α4β2* nAChR availability, overall analyses of covariance were performed using Vt/fP values for each of the three ROIs as dependent measures, group as a between-subject factor, and education level as a covariate based on results of the above analysis demonstrating group differences for this variable. To clarify results of these overall tests, post hoc Student s t tests were performed to determine which between-group differences accounted for significant findings. Bonferroni corrections for multiple comparisons were applied to all statistical tests, with the ANCOVA results being corrected for the three regions tested and post hoc Student s t tests being corrected for the six group comparisons performed for each region. Results were considered significant if corrected results passed a threshold of P < 0.05. To maximize power, the means of left and right Vt/fP values for prefrontal cortex and thalamus were used in statistical analyses, along with values for the whole brainstem. For descriptive purposes,hydroponic cannabis system percent group differences in Vt/fP values were determined between the smoker groups and the group of non-smokers, and between smokers with and without heavy caffeine or marijuana use. Statistical tests were performed using SPSS Statistics version 23 .

The central study finding was that smokers with concomitant heavy caffeine or marijuana use have higher Vt/fp values in the brainstem and prefrontal cortex than smokers without such use. The study also replicated earlier work demonstrating higher Vt/fp values in the prefrontal cortex and brainstem of smokers than nonsmokers. Taken together, these findings indicate that smokers with concomitant heavy caffeine or marijuana use have greater nAChR up regulation than smokers without concomitant heavy use. The most straightforward and likely explanation for the central study finding is that smokers who use caffeine or marijuana heavily have more nicotine exposure than smokers without such use. This explanation is supported by study data demonstrating that smokers with concomitant heavy caffeine or marijuana use had higher exhaled CO and greater depth of inhalation levels at baseline than smokers without such use . Other data supporting this theory include research demonstrating that caffeine increase nicotine intake in laboratory animals and a study of smokers with heavy marijuana use who had altered lung permeability , which resulted in greater cigarette smoke exposure. Thus, smokers with concomitant heavy caffeine or marijuana use may have increased brain nicotine exposure due to altered smoking topography, effects of caffeine or marijuana on other aspects of nicotine absorption/intake, or both. While the smoker groups with heavy caffeine or marijuana use did have higher exhaled CO levels and greater depth of smoking inhalation than the smoker group without concomitant use, the absence of group differences in cigarettes per day and FTND scores indicate that explanations for increased nAChR availability other than greater nicotine exposure are possible. We are not aware of studies that would fully explain direct effects of caffeine or marijuana on nAChR availability; however, recent research has begun to elucidate interactions between nicotine and caffeine or marijuana on a cellular level, and future research could determine a mechanism by which caffeine or marijuana exposure directly affects nAChR availability. The specific regional findings here may have functional significance, given the roles of the brainstem and PFC in the mediation of addiction. For the brainstem, many studies demonstrate that addictive drugs acutely stimulate neurons originating in the brainstem leading to the ventral striatum to produce reward and for reviews. For the PFC, this region is known to mediate executive functions, such as attention, working memory, and decision-making , which are associated with drug use. Extensive prior research has examined associations between smoking-related symptoms and nAChR availability in the regions studied here without finding strong evidence for associations between these variables. Future research could utilize specific testing for functions of the brainstem or PFC to further evaluate the functional significance of increased nAChR availability in these regions in smokers. Study results also have clinical implications regarding the co-use of cigarettes and other drugs. Prior research examining smokers trying to quit has demonstrated that concomitant use of caffeine or marijuana predicts less likelihood of smoking cessation. Recent research by our group showed that greater nAChR availability was associated with less likelihood of smoking cessation during a quit attempt with nicotine or placebo patch administration. Taken together, our findings imply that smokers with heavy caffeine or marijuana use have greater exposure to nicotine, more upregulation of nAChRs, and more trouble quitting in smoking cessation programs than smokers without concomitant heavy drug use. Future brain imaging research in smokers with concomitant heavy drug use who undergo smoking cessation treatment could confirm this implication of the current study. This study had several limitations. First, we did not examine non-smokers with heavy caffeine or marijuana use to determine if study findings were independent of cigarette smoking. Future research with such non-smokers could determine if caffeine and marijuana use affect nAChR density directly or if the effect on nAChR density is mediated through greater nicotine exposure in smokers with heavy caffeine or marijuana usage. Second, while we did determine exhaled CO levels, depth of inhalation, reported cigarettes per day, FTND scores, and plasma nicotine levels at the time of scanning, we did not collect blood for plasma nicotine levels at baseline during normal cigarette smoking.