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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Am J Psychiatry. Author manuscript; available in PMC 2014 January 6.
Published in final edited form as:
PMCID: PMC3881431
NIHMSID: NIHMS521771

Reduced β2*-Nicotinic Acetylcholine Receptor Availability in Smokers with Schizophrenia

Deepak Cyril D’Souza, M.D.,1,2,3 Irina Esterlis, Ph.D.,1,3 Michelle Carbuto, B.A.,1,2,3 Maegan Krasenics, B.A.,1,2,3 John Seibyl, M.D.,1,4 Frederic Bois, Ph.D.,1,3 Brian Pittman, M.S.,2,3 Mohini Ranganathan, M.D.,1,2,3 Kelly Cosgrove, Ph.D.,1,3 and Julie Staley, Ph.D.1,2,3

Introduction

Individuals with schizophrenia have the highest rates of comorbid cigarette smoking (58–88%) compared with the general population (~ 23%)(1-4). The association between schizophrenia and tobacco smoking is stronger even after controlling for confounds such as institutionalization, socioeconomic status and medications (4-6). Smokers with schizophrenia smoke more cigarettes and favor stronger cigarettes suggesting that they are more dependent on nicotine than other smokers reviewed in (4). They also appear to extract more nicotine from cigarettes than smokers without mental illness as suggested by some (7, 8) but not all studies (9). The odds that smokers with schizophrenia will become heavy smokers are significantly greater than those of smokers from the general population reviewed in(4). Furthermore, a number of studies suggest that the risk for developing to schizophrenia is associated with higher rates of smoking (4, 5, 10). Finally, smoking cessation rates for schizophrenia patients are significantly lower than the general population and, to a lesser extent, in smokers with other mental illnesses reviewed in(4).

Nicotine, the primary addictive chemical in tobacco smoke, initiates its effects in the brain through nicotinic acetylcholine receptors. Nicotinic acetylcholine receptors containing β2 subunits are some of the most abundant in the brain, have the highest affinity for nicotine, and are the most common subtype in the striatal reward pathways. The β2*-nicotinic acetylcholine receptors are a critical neural substrate mediating the primary reinforcing effects of nicotine in the brain (11). Repeated exposure to nicotine or smoking has been consistently shown, in preclinical, post-mortem human and human imaging studies, to result in the upregulation of nicotinic acetylcholine receptors, i.e., increased availability, throughout the brain (12-19). The increases in β2*-nicotinic acetylcholine receptor binding sites following chronic nicotine treatment has been shown to result from an increase in the number of nicotinic acetylcholine receptors containing β2 subunit proteins (20).

Post mortem evidence suggests alterations in β2*-nicotinic acetylcholine receptors in schizophrenia (21, 22). Smokers with schizophrenia showed lower numbers of β2*-nicotinic acetylcholine receptors compared to comparison smokers in several brain regions including the hippocampus, cortex, and striatum (21). Furthermore, there was no difference in numbers of β2*-nicotinic acetylcholine receptors in these same regions between smokers and nonsmokers with schizophrenia (21), suggesting a failure to upregulate in smokers with schizophrenia. Finally, there is a link between the genes that encode the α4 and β2 subunits of the nicotinic acetylcholine receptor and heavy smoking in individuals with schizophrenia (23).

The availability of β2*-nicotinic acetylcholine receptors in vivo can be measured with [123I]-5-IA-5380 ([123I]5-IA) and single photon emission computed tomography (SPECT). ([123I]5-IA binds to β2-containing nicotinic acetylcholine receptors at the interface between the α4 and β2 subunits. While it has been traditionally thought that nicotinic acetylcholine receptors containing two α4 and three β2 subunits are the primary receptor subunit composition that upregulates in response to nicotine, there is increasing evidence to suggest that other subunits (α5, α6, β3) may combine with the α4 and β2 subunits and they may also play a role in upregulation of the receptor (24). Therefore, to allow for the possibility of other β2 containing subunit combinations, e.g., α4β2α5, we denote this as β2*- nicotinic acetylcholine receptor to identify these as β2-containing nicotinic acetylcholine receptors. [123I]5-IA has low nonspecific binding (25), acceptable dosimetry in human subjects with high brain uptake (26, 27), and high test-retest reproducibility (28). Imaging with [123I]5-IA SPECT in nonhuman primates and human subjects results in a binding pattern that is consistent with the established regional distribution of the β2*-nicotinic acetylcholine receptors and is highest in the thalamus and intermediate throughout the cortex and cerebellum (29). Using [123I]5-IA SPECT we have previously shown significantly higher β2*-nicotinic acetylcholine receptor availability in the striatum, cerebellum and throughout the cortex in recently abstinent smokers compared to nonsmokers (30) and that it takes 6-12 weeks for β2*-nicotinic acetylcholine receptor availability to normalize to nonsmoker comparison levels (18).

The primary goal of this study was to compare β2*-nicotinic acetylcholine receptor availability in smokers with schizophrenia relative to comparison smokers. Based on post-mortem findings (21, 22), smokers with schizophrenia were hypothesized to exhibit lower β2*-nicotinic acetylcholine receptor availability relative to age and sex-matched healthy comparison smokers. A secondary goal of the study was to examine the relationship between β2*-nicotinic acetylcholine receptor availability, the symptoms of schizophrenia and tobacco smoking craving and withdrawal.

Methods

Regulatory approvals

This study was conducted with the approval of the institutional review boards of the Yale University School of Medicine and the Veterans Affairs Connecticut Healthcare System. The use of the radioactive isotope was approved by the Yale Radiation Safety Committee. Magnetic Resonance Imaging (MRI) was performed with the approval of the Yale Magnetic Resonance Research Center. The radiotracer was administered under IND 61,156.

Subjects

Tobacco smokers with a diagnosis of DSM-IV schizophrenia (31) and nicotine dependence were compared to age and gender matched comparison smokers selected from a group of healthy tobacco smokers who were being studied concurrently. The comparison smokers were medically and psychiatrically healthy and did not have any lifetime diagnosis of substance abuse or dependence (excluding nicotine and caffeine).

Consent process in smokers with schizophrenia

Before signing the consent form, subjects had several meetings with study staff where detailed information was provided (see supplemental section) to ensure that they understood the study and were suitable candidates. Subjects were required to pass a questionnaire about the key risks of the study. Parents and family and non-research clinicians were involved in the process when available. The patient’s primary clinician (i.e. non-research clinician) was required to assent to patient’s participation.

Screening procedures in smokers with schizophrenia

Subjects underwent a medical examination by a research physician to exclude any major, unstable medical issues or neurological disorders as described in the supplemental section.

Inclusion/Exclusion Criteria for Smokers with Schizophrenia

Only subjects with a primary diagnosis of schizophrenia were included. Diagnosis was confirmed by Structured Clinical Interview for DSM-IV conducted by a research assistant and evaluation by research psychiatrist. Subjects with a diagnosis of substance abuse within the past month or substance dependence (with the exception of nicotine or caffeine) within the past 6 months prior to screening evaluation were excluded. Smoking status was confirmed by plasma cotinine levels > 150 ng/mL, urine cotinine levels > 100 ng/mL, and carbon monoxide levels > 11 parts per million at baseline. Nicotine dependence was evaluated using the Fagerström Test for Nicotine Dependence (32). Subjects were also screened to ensure clinical stability, which included a chart review and review of medications and evaluation by a research psychiatrist. Subjects who were deemed clinically unstable as evidenced by recent psychiatric hospitalization or emergency room visits, increase in clinic visits due to psychiatric symptoms, homicidality, suicidality, and/or grave disability were excluded. Subjects needed to be taking stable doses of antipsychotic medications for at least 12 weeks. Benzodiazepines were permitted on a PRN basis. Subjects taking tricyclic antidepressants, anticholinergics or selective serotonin reuptake inhibitors were excluded due to some evidence that these drugs may interfere with [123I]5-IA binding.

Subjects were screened for any MRI exclusions, such as ferrous metal in the body, cardiac pacemaker or severe claustrophobia. If a potential subject had any experience working with metal or had any eye injury involving metal, orbital x-rays were performed.

Smoking Cessation

Subjects were helped to abstain from smoking for at least 5 days prior to the SPECT scan day using brief behavioral counseling based on Clinical Practice Guidelines and contingency management. Approximately 1 week of tobacco smoking abstinence is required for nicotine to clear from the brain in order to not interfere with [123I]5-IA binding (19, 30). Eligible smokers with schizophrenia were hospitalized on the Clinical Neuroscience Research Unit (CNRU) of the Connecticut Mental Health Center a smoke-free unit, for the purposes of initiating, maintaining and confirming nicotine abstinence. Subjects were not allowed to use any drugs that could facilitate smoking abstinence including nicotine replacement products (NRTs), bupropion or varenicline. Prior to abstaining from nicotine, subjects were advised about the risks and benefits of quitting smoking and told that they would be monitored daily to ensure abstinence. A trained member of the research team met with subjects daily to counsel on strategies to cope with withdrawal symptoms and approaches to quit long-term. Counseling was paired with contingency management: subjects were paid $25 for the first day of inpatient abstinence, and payments were escalated by $25 every inpatient day as described in greater detail in the supplemental section. If subjects were able to complete the inpatient abstinence period prior to the scan, they were eligible to receive $800. Abstinence from smoking or any other nicotine products was confirmed by daily monitoring of breath carbon monoxide measurements and dipstick-urinary cotinine levels [NicAlert, (Nymox)]. At the end of the study subjects were provided with referrals to obtain smoking cessation treatment if they expressed a desire to quit smoking long-term.

Nicotine craving and withdrawal were evaluated using the Tiffany Urge to Smoke Questionnaire (33) and Minnesota Nicotine Withdrawal Questionnaire (34), respectively, at intake, during smoking cessation, and on SPECT scan day. Positive, negative, and general symptoms of psychosis were measured by the Positive and Negative Syndrome Scale (PANSS) (35) and the Scale for the Assessment of Negative Symptoms (SANS) (36). Depressive symptoms were assessed by the Montgomery-Asberg Depression Scale (MADRS) (37) and involuntary movements by the Abnormal Involuntary Movements Scale (AIMS)(38).

SPECT Scanning

After achieving abstinence for at least 5 days, subjects participated in one [123I]5-IA SPECT scan as described previously (39). [123I]5-IA was prepared by iododestannylation to give a product with radiochemical purity of >90%. Blood samples were collected and analyzed as described previously (40). In the hour preceding the radiotracer administration, all subjects received a 0.6-g saturated solution of potassium iodide or potassium perchloride tablet (for subjects allergic to shell fish) to protect their thyroid. [123I]5-IA was administered (~10mCi, IV) using the bolus plus constant infusion paradigm with a B/I ratio of 7.0 h. SPECT images were acquired using the three-headed PRISM 3000XP (Picker, Cleveland, Ohio) equipped with low energy ultra high-resolution (LEUHR) fan beam collimators. In order to perform non-uniform attenuation correction, a simultaneous emission transmission protocol (STEP) was acquired with a 20mCi [Co-57] line source at 5.5 h. Three emission scans (30 min each) were obtained between 6-8 h of infusion (time necessary for radiotracer to achieve equilibrium in the brain). Plasma samples were collected at the midpoint of second scan to quantify total free parent in plasma (40) so that inter-individual differences in metabolism and protein binding of [123I]5-IA can be corrected (41).

MRI Scan

Each subject participated in one MRI scan prior to SPECT scanning. MRI was performed on a Signa 1.5T system (General Electric Co, Milwaukee, Wisconsin). Axial images were acquired parallel to the anteroposterior commissural line with an echo time of 5 milliseconds, repetition time of 24 milliseconds, matrix 256 × 192, number of excitations of 1, field of view of 24 cm, and 128 contiguous slices with a thickness of 1.3 mm.

SPECT Image Processing

SPECT emission images were analyzed as described previously (39). SPECT emission images were filtered using a 3-dimensional (3D) Butterworth filter (order 10, cutoff frequency 0.24 cycle/pixel) and reconstructed using a filtered back projection algorithm with a ramp filter on a 128 × 128 matrix to obtain 50 slices with a pixel size of 2.06 × 2.06 × 3.56 mm in the x-, y-, and z-axes. MRIs were coregistered to the SPECT images to provide an anatomical guide for placement of the regions of interest using Medx (version 3.4) software (Medical Numerics, Inc., MD). A standard 3D volume of interest (VOI) was generated for each region and transferred to the coregistered SPECT image to determine regional radioactive densities. Regions of interest (ROIs) chosen were those known to contain β2*-nicotinic acetylcholine receptors and included frontal, parietal, anterior cingulate, temporal and occipital cortices, thalamus, striatum (an average of caudate and putamen), and cerebellum. The average of two raters is used for all analyses with interrater variability being <10% between raters across regions.

Regional β2*-nicotinic acetylcholine receptor availability was determined by VT/fp ((regional activity (kilobecquerel[kBq]/cc)/plasma free parent (kBq/mL)) (42), a highly reproducible outcome measure (39). VT/fp corrects for possible differences in radiotracer metabolism and plasma protein binding between groups and subjects, and is referred to as β2*-nicotinic acetylcholine receptor availability because only receptors that are “available” to be bound by the radiotracer are measured. Receptors that are already occupied (i.e., by residual nicotine, by a pharmacologically active metabolite [cotinine or nornicotine], or by endogenous neurotransmitter [acetylcholine]) are not available. Therefore, receptor availability is not a measure of all receptors. The outcome measure VT/fp is proportional to the binding potential (Bmax/Kd, in milliliters per gram), which is proportional to the receptor number (Bmax) at equilibrium, given the assumptions that there is no change in affinity (Kd) and that nondisplaceable (nonspecific and free) uptake does not differ between subjects or comparison groups.

Statistical Analyses

Statistical analyses were conducted using SAS version 9.1 (SAS Institute Inc., Cary, NC) and SPSS version 16.0 (SPSS Inc. Headquarters, Chicago, IL). Independent t-tests were used to examine demographic variables (e.g., smoking characteristics) between diagnostic groups. Multivariate analysis of variance (MANOVA) models examined differences in receptor availability (VT/fp) between patients and comparison smokers in brain regions (frontal and parietal cortices, striatum, hippocampus and thalamus) chosen a priori based on the post-mortem literature.

PANSS, MADRS, SANS and AIMS data in smokers with schizophrenia were examined descriptively using means, standard deviations and graphs. Each outcome was tested for normality using Kolmogorov-Smirnov test statistics and normal probability plots. The outcomes were approximately normal and assessed using linear mixed models with time (baseline day and scan day) included as a within-subjects explanatory factor. Regional β2*-nicotinic acetylcholine receptor availability was correlated with smoking characteristics (packs per day, Fagerström score, years smoked, craving Tiffany [desire/relief], withdrawal), in both groups and measures of schizophrenia (PANSS, SANS) and depression (MADRS) in smokers with schizophrenia with Spearman’s rho correlation coefficients. All results were considered statistically significant at P<0.05.

RESULTS

Fourteen smokers with schizophrenia were enrolled but two were not scanned; one failed to achieve smoking abstinence and one could not tolerate inpatient hospitalization. Demographics of the remaining 12 are presented in table 1. Though both male and female smokers were recruited, the only female subject who enrolled was unable to remain abstinent for the scan. The period of smoking abstinence varied from 5-7 days (5.57 ± 0.65) due to scan and inpatient scheduling constraints. An additional subject was not included in the analysis because his β2*-nicotinic acetylcholine receptor availability data was >3 SD above the mean. Data from the remaining 11 subjects were compared to age and gender-matched comparison smokers who were selected from a sample of subjects who were being concurrently scanned with [123I]5-IA SPECT (Table 2). At intake (table 2), smokers with schizophrenia reported greater nicotine dependence (Fagerström score; 6.3 ± 2.1 vs. 4.2 ± 3.0) and smoked a greater number of cigarettes per day than comparison smokers (21.9 ± 8.4 vs.16.9 ± 4.9), although these did not reach statistical significance (Fagerström score: t(20)=1.96, p=0.06, cigarettes per day: t(20)=1.71, p=0.10).

Table 1
Demographics of Tobacco Smoking Schizophrenia Subjects
Table 2
Smoking Characteristics of Smokers with Schizophrenia and Comparison Smokers

Confirmation of Smoking Abstinence

Breath carbon monoxide (F(1, 10)= 26.35, p = 0.0004) and urine cotinine levels (F(1, 10)= 87.91, p < 0.0001) declined over time, confirming that all smokers with schizophrenia were abstinent from tobacco smoking for at least 5 days prior to scanning (supplemental Fig. 1). Furthermore, both plasma and urine cotinine levels declined over time and there were no differences between smokers with schizophrenia and comparison smokers in either urine [(F(1, 20)= 0.22, p = 0.65), time (F(1, 12)= 252.3, p < 0.0001), group × time (F(1, 20)= 0.08, p = 0.78)] or plasma cotinine [group (F(1, 17)= 0.71, p = 0.41), time (F(1, 17)= 36.5, p < 0.0001), group × time (F(1, 17)= 1.25, p = 0.28)] levels (supplemental figure 2).

β2*-nicotinic acetylcholine receptor availability

There was a significant omnibus MANOVA effect of group (Hotelling’s Trace = 1.09, p=0.025). Smokers with schizophrenia had significantly lower β2*-nicotinic acetylcholine receptor availability in the parietal (22%, F(1, 20)=4.7; p=.04) and frontal (26%, F(1, 20)=9.4, p=.006) cortices and thalamus (21%, F(1, 20)=5.5, p=.03). However, there were no significant differences in striatum (17%; F(1, 20)=2.9, p=0.10) and hippocampus (13%; F(1, 20)=1.6, p=0.22) (Figure 1; table 3).

Figure 1
Group Differences in β2*-Nicotine Acetylcholine Receptor Availability in Smokers With Schizophrenia and Comparison Smokersa
Table 3
Regional β2*-nAChR Availability in Schizophrenia Smokers and Comparison Smokers

Smoking Behaviors

In smokers with schizophrenia, there were no statistically significant changes in scores on the Tiffany desire for smoking subscale (F(6, 54)= 0.72, p =0.63), the Tiffany relief from smoking subscale (F(6, 54)= 1.23, p =0.3) or the Minnesota Questionnaire for smoking withdrawal (F(6, 54)= 0.2, p =0.97) from the baseline state (smoking as usual) to the day of the SPECT scan (supplemental figure 3). On the day that subjects were scanned, smokers with schizophrenia reported greater desire to smoke cigarettes (t = −2.97, p = 0.01) and greater expected relief from smoking (t = −2.82, p = 0.01) (supplemental figure 4). There were no significant differences (F=.85, p=.37) in withdrawal symptoms in smokers with schizophrenia (4.6±4.9) and comparison smokers (2.7±4.8). All but one smoker with schizophrenia resumed smoking within minutes of completing all the study procedures.

Depression, schizophrenia symptoms, negative symptoms and dyskinesia

There were no statistically significant changes in total PANSS scores (F(1,10)= 2.14, p =0.17), MADRS scores (F(1,10)= 0.58, p =0.46) or SANS scores (F(1,10)= 0.14, p =0.72) (supplemental figure 5, supplemental table 1) between baseline (smoking as usual state) and the day of the SPECT sczan (5 or more days of smoking abstinence) among smokers with schizophrenia. Furthermore, while there were no changes in PANSS positive symptoms (F(1,10)= 2.91, p =0.12), there was a reduction in PANSS negative symptoms (F(1,10)= 5.71, p =0.038) and an increase in PANSS general symptoms (F(1,10)= 6.1, p =0.033) with smoking abstinence. Total AIMS scores increased significantly over time (F(1,10)= 5.71, p =0.038).

Relationship between β2*-nicotinic acetylcholine receptor availability and nicotine dependence

There were no significant correlations between the number of cigarettes smoked per day or nicotine dependence and β2*-nicotinic acetylcholine receptor availability in either smokers with schizophrenia or comparison smokers.

Relationship between β2*-nicotinic acetylcholine receptor availability and negative symptoms

There were significant negative correlations between β2*-nicotinic acetylcholine receptor availability in the parietal cortex, frontal cortex, thalamus, striatum and hippocampus, and negative symptoms of schizophrenia assessed after smoking abstinence (figure 2). This was evident with both the negative symptoms subscale of the PANSS and the SANS (table 4). Finally, negative symptoms assessed at baseline also negatively correlated with β2*-nicotinic acetylcholine receptor availability in the parietal cortex (Pearson’s r = −0.61, p = 0.046). There were negative correlations with the frontal cortex, temporal cortex, striatum and thalamus (Pearson’s r = −0.57 to −0.52) but these relationships did not reach statistical significance (p = 0.05 to 0.09).

Figure 2
Relationship Between Regional β2*-Nicotine Acetylcholine Receptor Availability & Negative Symptoms
Table 4
Relationship Between Regional β2*- Nicotine Acetylcholine Receptor Availability and Negative Symptoms

Discussion

This report provides the first in vivo evidence of lower β2*-nicotinic acetylcholine receptor availability in smokers with schizophrenia relative to comparison smokers during early abstinence. The magnitude of group differences range from 13-25% (effects size of 0.32-0.08) with the largest differences in descending order observed in the frontal cortex, parietal cortex, thalamus, striatum and hippocampus. These in vivo findings are consistent with the post-mortem findings of lower β2*-nicotinic acetylcholine receptors in smokers schizophrenia compared to comparison smokers (21, 22, 43), except for thalamus which failed to show significant group differences in post-mortem studies. Importantly, this study demonstrates an inverse correlation between negative symptoms and β2*-nicotinic acetylcholine receptor availability in smokers with schizophrenia, i.e., those individuals with lower β2*-nicotinic acetylcholine receptor availability had greater negative symptoms.

Lower β2*-nicotinic acetylcholine receptor availability in smokers with schizophrenia may reflect altered receptor functionality. Specifically, higher β2*-nicotinic acetylcholine receptor availability in tobacco smokers likely represents greater numbers of desensitized and inactivated nicotinic acetylcholine receptors (44, 45). The lower β2*- nicotinic acetylcholine receptor availability in smokers with schizophrenia may be related to abnormal desensitization or turnover of nicotinic acetylcholine receptors. It should be noted that nicotine-induced desensitization is one of the major mechanisms for nicotine addiction (46-48). Since maintained nicotinic acetylcholine receptor desensitization may be important for relieving nicotine withdrawal in human smokers (49), the reduced β2*-nicotinic acetylcholine receptors availability observed in smokers with schizophrenia might explain why they are more likely to be addicted to nicotine.

The reduced β2*-nicotinic acetylcholine receptor availability in smokers with schizophrenia may also reflect a deficit in nicotine-induced upregulation of high affinity nicotinic acetylcholine receptors as suggested by post-mortem studies. However, that conclusion cannot be drawn from the current study. Future in vivo studies comparing schizophrenic smokers to schizophrenic non-smokers and ex-smokers will need to be conducted.

Underlying differences in the β2 gene (CHRNB2) may also explain lower nicotinic receptor availability in smokers with schizophrenia. In a family-based association study of a Canadian sample, CHRNB2 combined with the α4 gene (CHRNA4) may be linked to schizophrenia (50). However, there was no significant association between CHRNA4 and CHRNB2, and schizophrenia in a Japanese population (51). CHRNA4 and CHRNB2 have also associated been with smoking among schizophrenics (23, 52).

Relationship between Negative Symptoms and β2*-nicotinic acetylcholine receptor availability

The relationship between β2*-nicotinic acetylcholine receptor availability and negative symptoms is specific and robust. Those individuals with lower β2*-nicotinic acetylcholine receptor availability had greater negative symptoms. This relationship was consistent across two separate measures of negative symptoms: the SANS and PANSS. There was no such relationship with depression or positive symptoms. The current finding of a negative correlation between β2*-nicotinic acetylcholine receptor availability and negative symptoms is consistent with the observation in some studies that the heaviest smokers with schizophrenia had the lowest number of negative symptoms (53). The literature on the relationship between nicotine dependence and symptoms is mixed; one study reported a link to both negative and positive symptoms, four found associations with one type of symptoms but not the others (53-56), and another four did not find any significant relationship (57-60). Smoking high nicotine cigarettes has been shown to decrease negative symptoms more than denicotinized cigarettes and these effects are specific, since no effects were observed on positive symptoms, depression or anxiety (61). Nicotine, which has been shown to increase burst firing of dopaminergic neurons and dopamine release (62-64), may reduce negative symptoms by correcting the cortical hypodopaminergia that possibly mediates negative symptoms (65). Thus, the present finding of a negative association between receptor availability and negative symptoms may contribute to understanding the high smoking rates in schizophrenia and the relationship between smoking and negative symptoms in this population. Finally, in the absence of any approved treatments for negative symptoms the results of this study should provide the impetus to test treatments based on a β2*-nicotinic acetylcholine receptor mechanism for negative symptoms.

Effect of Smoking Cessation on Symptoms

There was a small increase in dyskinesia which is not considered clinically significant and is well within the margin of test-retest variability of the Abnormal Involuntary Movements Scale (AIMS). Smoking cessation and short-term abstinence had no significant effects on positive symptoms, negative symptoms or depression ratings. The data from this small sample do not support the “self-medication” hypothesis according to which individuals with schizophrenia smoke to alleviate positive and negative symptoms and depression. Nevertheless, most smokers with schizophrenia resumed smoking immediately after completing the SPECT scan. Perhaps they relapsed to smoking to ‘self-medicate” feeling states or needs that are distinct from positive, negative symptoms or mood states, or that their drive to smoke is different from comparison smokers.

Relationship of β2*-nicotinic acetylcholine receptor availability to baseline smoking

Similar to observations in “healthy” smokers (30), there were no significant correlations between the number of cigarettes smoked per day or nicotine dependence and β2*-nicotinic acetylcholine receptor availability in several brain regions in smokers with schizophrenia.

Alternative explanations

There are several other explanations for the reduced β2*-nicotinic acetylcholine receptor availability in smokers with schizophrenia. It is possible that antipsychotic treatment, which the two groups were not matched for, may contribute to the group differences in β2*-nicotinic acetylcholine receptor availability. However, the region-specific reduction in β2*-nicotinic acetylcholine receptor availability observed in the current in vivo study argues against a generalized effect of antipsychotic exposure. Furthermore, preclinical studies suggest that chronic exposure to clinically relevant doses of several antipsychotics do not affect high-affinity nicotinic receptors in the brain (66). Similarly, human post-mortem studies suggest that the lower β2*-nicotinic acetylcholine receptor numbers observed in smokers with schizophrenia are most likely independent of antipsychotic exposure. Ongoing in vivo studies comparing medicated and unmedicated smokers with schizophrenia will conclusively address this issue.

Smokers with schizophrenia and comparison smokers may differ in metabolism of nicotine and its metabolite, cotinine. However, analysis of plasma and urine nicotine and cotinine levels on the SPECT scan day, which is an indication of nicotine clearing the brain (30), did not reveal significant differences in these levels between the two groups.

Endogenous acetylcholine may compete with the [123I]5-IA radioligand binding at the receptor site. In fact, increased endogenous acetylcholine after a high dose of the acetylcholinesterase inhibitor physostigmine to nonhuman primates resulted in a ~15% displacement of [123I]5-IA in the thalamus (67). However, the findings of lower receptor number in post-mortem study where acetylcholine is washed out, i.e., not a confound, argues against the explanation that endogenous acetylcholine may be lowering receptor availability in this study (21).

It is unlikely that the reduced β2*-nicotinic acetylcholine receptors availability in smokers with schizophrenia is due to a change in receptor affinity. Breese et al. conducted Scatchard analyses in their post mortem and suggested that the changes in nicotinic acetylcholine receptor levels were due to changes in total receptor numbers (Bmax) and not to a change in receptor affinity (Kd)(21).

Limitations

Whether these findings are a result of schizophrenia, smoking or some interaction between the two cannot be confirmed unless nonsmoking schizophrenia patients are studied. In post mortem studies (21), β2*-nicotinic acetylcholine receptor availability in smokers with schizophrenia and never smokers with schizophrenia differed only in the cortex. Furthermore, while nicotine dose (cigarettes smoked per day) was not correlated with availability of β2*-nicotinic acetylcholine receptors, the limited range of nicotine exposure (heavy smoking) in the current sample was not ideal to establish the relationship between dose and receptor availability. Future studies that include subjects with a wider range of nicotine exposure might be useful in determining whether there is any relationship between nicotine dose and receptor availability. Other study limitations include the lack of a psychiatric comparison group and the medication status of smokers with schizophrenia. Future work includes imaging an unmedicated cohort of subjects. Lastly, whether these changes are present at the onset of the illness or develops with time is not known. Subjects early in the course of their illness will need to be studied.

Conclusions

In conclusion, this study provides the first in vivo evidence to our knowledge of reduced β2*-nicotinic acetylcholine receptor availability and an inverse correlation between β2*-nicotinic acetylcholine receptor availability and negative symptoms in smokers with schizophrenia. Given the absence of any effective treatments for negative symptoms, the findings of this study suggest that medications targeting the β2*-nicotinic acetylcholine receptor system may be effective. Future studies will need to be conducted in unmedicated schizophrenia patients, nonsmoking schizophrenia patients, first episode psychosis and prodromal patients to clarify the effects of antipsychotics and stage of illness on nicotinic acetylcholine receptor availability and to determine whether there are reductions in nicotine-induced upregulation of nicotinic acetylcholine receptors in schizophrenia as suggested by post-mortem data.

Supplementary Material

01

Supplemental Figure 1: Measures of Smoking Abstinence in Smokers with Schizophrenia

Left panel shows breath carbon monoxide levels (in parts per million) at baseline (smoking as usual) and on the day of the scan (~7 days abstinence) in smokers with schizophrenia. There was a significant reduction in breath carbon monoxide levels with abstinence (F(1, 10)= 26.35, p=0.0004).

Right panel shows plasma cotinine levels (ng/ml) at baseline (smoking as usual) and on the day of the scan (~7 days abstinence) in smokers with schizophrenia. There was a significant reduction in plasma cotinine levels with abstinence (F(1, 10)= 87.91, p<0.0001).

Supplemental Figure 2: Plasma & Urine Cotinine in Schizophrenia and Comparison Smokers

Left panel shows urine cotinine levels (ng/ml) at baseline (smoking as usual) and on the day of the scan (~7 days abstinence) in smokers with schizophrenia and comparison smokers. There was no significant group effect (F=0.22, df=1, 20, p=0.65) or group-by-time interaction (F=0.08, df=1, 20, p=0.78).

Right panel shows plasma cotinine levels (ng/ml) at baseline (smoking as usual) and on the day of the scan (~7 days abstinence) in smokers with schizophrenia and comparison smokers.

There was no significant group effect (F=0.71, df=1, 17, p=0.41) or group-by-time interaction (F=1.25, df=1, 17, p=0.28).

Supplemental Figure 3: Smoking Behaviors in Smokers with Schizophrenia

Left panel shows change in the desire to smoke over time measured by the Tiffany Urge to Smoke Questionnaire desire for smoking subscale. There was no significant change over time. Middle panel shows the change in the expected relief from smoking a cigarette over time measured by the Tiffany relief from smoking subscale. There was no significant change over time.

Right panel shows change in smoking withdrawal over time measured by the Minnesota Nicotine Withdrawal Scale. There was no significant change over time.

Supplemental Figure 4: Group Differences in Smoking Behaviors

Left panel shows significant differences (t = −3.56, p=0.002) in desire for a cigarette measured by the Tiffany Urge to Smoke Questionnaire between smokers with schizophrenia (red filled diamonds) and comparison controls (blue filled circles).

Right panel shows significant differences (t = −5.90, p=0.000) in expected relief from smoking a cigarette measured by the Tiffany Urge to Smoke Questionnaire between smokers with schizophrenia (red filled diamonds) and comparison controls (blue filled circles).

Supplemental Figure 5: Symptoms In Smokers with Schizophrenia

A: Symptoms of schizophrenia measured by the Positive and Negative Syndrome Scale (PANSS) at screening (smoking as usual) and on the day of the scan (~7 day abstinence); no statistically significant differences.

B: Negative symptoms of schizophrenia measured by the Scale for the Assessment of Negative Symptoms (SANS) at screening (smoking as usual) and on the day of the scan (~7 day abstinence); no statistically significant differences.

C: Depression symptoms measured by the Montgomery-Åsberg Depression Scale (MADRS) at screening (smoking as usual) and on the day of the scan (~7 day abstinence); no statistically significant differences.

D: Significant increase (F(1, 10)= 5.71, p=0.038) in dyskinesia measured by the Abnormal Involuntary Movements Scale (AIMS) at screening (smoking as usual) and on the day of the scan (~7 day abstinence).

Supplemental Table 1: Symptoms in Smokers with Schizophrenia

Acknowledgements

The authors also wish to thank 1) Marina Picciotto, Ph.D., for her advice in preparing this manuscript and 2) Louis Amici, M.S., for his help with radiochemistry.

Grant support: (1) National Institute of Drug Abuse (R01 DA 022495 to DCD and JS),

(2) Department of Veterans Affairs Schizophrenia Research Center (DCD),

(3) National Alliance for Research in Schizophrenia and Depression (Young Investigator Award to IE),

(4) The Yale Center for Clinical Investigation (YCCI),

(5) Veteran Affairs Career Development Award (IE),

6) K01DA020651 (Cosgrove) and

7) K02DA021863 (Staley).

Footnotes

The results of this study were presented at the Annual meeting of American College of Neuropsychopharmacology meeting (December, 2010) and the International Congress on Schizophrenia Research (April, 2011).

Disclosure of Financial Relationships: Frederic Bois, Michelle Carbuto, Irina Esterlis, Maegan Krasenics and Brian Pittman report no financial relationships with commercial interests. John P. Seibyl consults to Bayer Healthcare, GE Healthcare and has equity in Molecular Neuroimaging, LLC which provides imaging services in preclinical and neurodegenerative trials for Pfizer, Lilly, Janssen, Genentech, Roche, Sepracor, Amgen, Sanofi, and Abbott. Mohini Ranganathan receives research grant support from Lilly. Dr. Cosgrove has a services contract with the Institute of Neurodegenerative Disorders and Molecular Neuroimaging group. Deepak Cyril D’Souza currently receives research grant support administered through Yale University School of Medicine from Astra Zeneca, Abbott Laboratories, Eli Lilly Inc., Organon, Pfizer Inc., Sanofi, and has equity in Pfizer.

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