Acetylcholine, Tobacco Smoking, Genes and Nicotinic Receptors
Phase: NA
Conditions: Smoking, Schizophrenia
Sponsor: Yale University

APPROVED BY THE YALE UNIVERSITY IRB 3/10/2021
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YALE UNIVERSITY 
HUMAN INVESTIGATION COMMITTEE
e-Application to Involve Human Subjects in Biomedical Research
100 FR 1e (2012-1)
For use with Electronic Protocol Submissions Only
HIC Protocol Number: 1210010989
Title of Research Project: 
Acetylcholine, Tobacco smoking, Genes and Nicotinic Receptors
Principal Investigator:                                                        Yale Academic Appointment:
Kelly Cosgrove, Ph.D.                                                           Associate Professor
E-mail: Kelly.cosgrove@yale.edu
Campus Phone: 203-737-6969
Campus address: 2 Church St South, Suite 511 New Haven, CT 06519
Protocol Correspondent (If different from PI): Jon Mikael Anderson
E-mail: jonmikael.anderson@yale.edu
Campus phone: 203-737-7074
Yale Cancer Center CTO Protocol Correspondent (If applicable):
E-mail:
Campus phone:
Faculty Advisor: (required if PI is a student, resident, fellow or other trainee)
Yale Academic Appointment:
Email:
Campus Phone:
   
Investigator Interests:
Does the principal investigator, or do any research personnel who are responsible for the design, 
conduct or reporting of this project or any of their family members (spouse or dependent child) 
have an incentive or interest, financial or otherwise, that may affect the protection of the human 
subjects involved in this project, the scientific objectivity of the research or its integrity? Note: 
The Principal Investigator (Project Director), upon consideration of the individual’s role and 
degree of independence in carrying out the work, will determine who is responsible for the 
design, conduct, or reporting of the research. 
See Disclosures and Management of Personal Interests in Human Research 
http://www.yale.edu/hrpp/policies/index.html#COI 
  Yes
x  No
Do you or does anyone on the research team who is determined by you to be responsible for the 
design, conduct or reporting of this research have any patent (sole right to make, use or sell an 
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invention) or copyright (exclusive rights to an original work) interests related to this research 
protocol?
  Yes
x  No
If yes to either question above, list names of the investigator or responsible person:
The Yale University Principal Investigator, all Yale University co-investigators, and all Yale 
University individuals who are responsible for the design, conduct or reporting of research must 
have a current financial disclosure form on file with the University’s Conflict of Interest Office. 
Yale New Haven Hospital personnel who are listed as con-investigators on a protocol with a 
Yale University Principal Investigator must also have a current financial disclosure form on file 
with the University’s Conflict of Interest Office. If this has not been done, the individual(s) 
should follow this link to the COI Office Website to complete the form:  
http://www.yale.edu/coi/  
NOTE: The requirement for maintaining a current disclosure form on file with the University’s 
Conflict of Interest Office extends primarily to Yale University and Yale-New Haven Hospital 
personnel.  Whether or not they are required to maintain a disclosure form with the 
University’s Conflict of Interest Office, all investigators and individuals deemed otherwise 
responsible by the PI who are listed on the protocol are required to disclose to the PI any 
interests that are specific to this protocol.
Billing Information: IRB Review fees are charged for projects funded by Industry or Other For-Profit 
Sponsors.  If this study is funded by Industry or Other For-Profit Sponsor, provide the Name and 
Address of the Sponsor Representative to whom the invoice should be sent.  Note: the PI’s home 
department will be billed if this information is not provided.
Send IRB Review Fee Invoice To:
Name:
Company:
Address:
SECTION I: GENERAL INFORMATION
1. Performing Organizations:  Identify the hospital, in-patient or outpatient facility, school or 
other agency that will serve as the location of the research.  Choose all that apply:
 
a. Internal Location[s] of the Study:
 Magnetic Resonance Research Center 
 Yale University PET Center
     (MR-TAC)   
 YCCI/Church Street Research Unit (CSRU)
 Yale Cancer Center/Clinical Trials Office (CTO)    
 YCCI/Hospital Research Unit (HRU)
 Yale Cancer Center/Smilow
 YCCI/Keck Laboratories
 Yale-New Haven Hospital
 Cancer Data Repository/Tumor Registry
 Specify Other Yale Location:
b. External Location[s]:
 APT Foundation, Inc.
 Haskins Laboratories
 Connecticut Mental Health Center
 John B. Pierce Laboratory, Inc.
 Clinical Neuroscience Research Unit (CNRU)  
Veterans Affairs Hospital, West Haven
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 Other Locations, Specify: 
 International Research Site 
(Specify location(s)):
2. Probable Duration of Project: State the expected duration of the project, including all 
follow-up and data analysis activities. 
9 years
      
3. Research Type/Phase: (Check all that apply)
a. Study Type
    
 Single Center Study
    
 Multi-Center Study
Does the Yale PI serve as the PI of the multi-site study? Yes 
No 
  
 
 Coordinating Center/Data Management
   
 
 Other: 
b. Study Phase
 N/A
    
 Pilot
 Phase I
 Phase II
 Phase III
 Phase IV
4.    Is this study a clinical trial? Yes 
No 
NOTE the current ICMJE (International Committee of Medical Journal Editors) definition of a 
clinical trial: “any research study that prospectively assigns human participants or groups of 
humans to one or more health-related interventions to evaluate the effects on health outcomes.” 
Health-related interventions include any intervention used to modify a biomedical or health-
related outcome (for example, drugs, surgical procedures, devices, behavioral treatments, 
dietary interventions, and process-of-care changes). Health outcomes include any biomedical or 
health-related measures obtained in patients or participants, including pharmacokinetic 
measures and adverse events”
If yes, where is it registered?
Clinical Trials.gov registry 
Other (Specify) 
Registration of clinical trials at their initiation is required by the FDA, NIH and by the ICMJE.
If this study is registered on clinicaltrials.gov, there is new language in the consent form and 
compound authorization that should be used.
For more information on registering clinical trials, including whether your trial must be 
registered, see the YCCI webpage, http://ycci.yale.edu/researchers/ors/registerstudy.aspx  or 
 contact YCCI at 203.785.3482)
5. Will this study have a billable service as defined by the Billable Service Definition?
Yes 
 No
If you answered "yes", this study will need to be set up in Patient Protocol Manager (PPM)
http://medicine.yale.edu/ymg/systems/ppm/index.aspx 
6. Are there any procedures involved in this protocol that will be performed at YNHH or one of 
its affiliated entities?  Yes _ __ No X  If Yes, please answer questions a through c and note 
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instructions below.  If No, proceed to Section II.
a. Does your YNHH privilege delineation currently include the specific procedure that 
you will perform?
 
b. Will you be using any new equipment or equipment that you have not used in the past 
for this procedure?
 
c. Will a novel approach using existing equipment be applied?
 
If you answered “no” to question 6a, or "yes" to question 6b or c, please contact the YNHH 
Department of Physician Services (688-2615) for prior approval before commencing with your 
research protocol.
SECTION II: RESEARCH PLAN
1. Statement of Purpose: State the scientific aim(s) of the study, or the hypotheses to be tested.
The acetylcholinergic (ACh) system is critically involved in learning, memory, arousal and 
attention, functions that are substantially impaired in many neuropsychiatric conditions.  For 
example, Alzheimer’s disease (AD) is associated with decreased cholinergic function, whereas 
recent evidence suggests increased and decreased cholinergic function in major depression and 
schizophrenia, respectively.  Cholinergic function is also fundamentally impaired in nicotine 
addiction. In vivo molecular imaging studies of muscarinic and nicotinic receptors have provided 
substantial contributions to our understanding of these disorders, but these contributions were 
limited by the lack of a method suitable to measure fluctuations in synaptic ACh level.
We recently developed and tested an innovative molecular imaging method in humans to detect 
changes in brain synaptic ACh levels, using single photon emission computed tomography 
(SPECT), the beta2-nicotinic acetylcholine receptor (β2-nAChR) radioligand [123I]5-IA-85380 
(5-IA) and the acetylcholinesterase (AChE) inhibitor, physostigmine, challenge. Results suggest 
elevated ACh levels induced by physostigmine lead to a reduction in the availability of nicotinic 
receptors for the binding of the radioligand. If validated, this paradigm might provide a very 
useful method to interrogate presynaptic cholinergic function in health and disease. To date, 
receptor imaging of the ACh system at Yale has been limited to 5-IA and SPECT. SPECT is 
known to have poorer resolution and less reliable quantification compared to positron emission 
tomography (PET). Furthermore, the slow kinetic of this radiotracer requires lengthy imaging 
sessions, which is not well tolerated in impaired populations.  [18F]NCFHEB (NCFHEB) has 
been recently introduced as a superior β2-nicotinic PET imaging agent (1, 2). This proposal 
outlines a series of studies aiming at testing the suitability of PET and NCFHEB to examine 
genetic polymorphisms that modulate nAChR availability and to detect changes in ACh synaptic 
levels, as well as to determine the effect of nicotine on nAChR availability.
This innovative paradigm will lay the groundwork for future studies that can directly interrogate 
ACh function in the living human brain. This is critical to the advancement of our understanding 
of ACh involvement in psychiatric and medical disorders.  
Additionally, a substantial literature body demonstrates that nAChRs and the cholinergic system 
dynamically control the mesolimbic DA system by enhancing, inhibiting, and filtering striatal 
DA release.  We have preliminary data suggesting tobacco smokers at 2 weeks of abstinence 
have blunted DA release compared to nonsmokers. We propose in Aim 7 to determine 1) 
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whether there is reduced DA release in smokers at 2 weeks of withdrawal vs. nonsmokers, 2) if 
the magnitude of DA release is correlated with alterations in ACh levels measured in Aim 4 and 
3) if alterations in DA and ACh predict relapse vulnerability.
Aim 1:  To determine the test-retest reproducibility of NCFHEB binding parameters in 10 
nonsmoking subjects. β2-nAChRs will be quantified twice in nonsmoking subjects. Based on 
preclinical evidence, we hypothesize within 20% difference of binding parameters between test 
and retest scans. 
CLOSED Aim 2: To determine if 2-nAChR availability is genetically predisposed in never 
smokers and smokers We propose to determine if the ANKK1 genotype 
(rs4938015/hCV38879) or CHRNA4 genotype determines 2-nAChR availability in adult never 
smokers. Specifically, 10 never smokers and 10 smokers at one week of abstinence will be 
imaged using NCFHEB PET and blood samples for DNA extraction will be collected from all 
subjects.
Hypothesis: Homozygotes (TT) will have higher 2-nAChR availability compared to age-, and 
sex matched heterozygotes that carry the C allele in the ANKK1 gene (rs4938015/ hCV38879). 
CLOSED Aim 3: To determine if the adaptive increase in 2-nAChR availability and 
change in 2-nAChR availability over the first month of abstinence in smokers is 
genetically predisposed. We will determine if ANKK1 or CHRNA4 genotypes are associated 
with changes in 2-nAChR availability in smokers over time recruited in aim 2. The same 
smokers from Aim 2 will be supported to maintain abstinence for 4-8 weeks. Smokers that 
successfully abstain for 4-8 weeks (~ 50%; e.g. 5 smokers) will be imaged a second time using 
NCFHEB and PET. The % change in 2-nAChR availability will be determined as the difference 
in NCFHEB binding [(VT/fp @7days of abstinence –VT/fp 4 wks abstinence)/VT/fp @7 days 
abstinence]. Blood samples for DNA extraction will be collected from all subjects. 
Hypothesis: Based on our preliminary data we hypothesize that smokers that carry the A allele 
for hCV16178933/rs2273504 or the G allele for hCV15953820/rs2236196 genotype will have 
higher 2-nAChR availability at 7-9 days of abstinence compared to never smokers matched for 
age and sex and genotype. Whereas smoker homozygotes (GG and AA respectively for 
hCV16178933/rs2273504 or for hCV15953820/rs2236196) will demonstrate similar 2-nAChR 
availability compared to never smokers.  We also hypothesize that CC homozygotes for the 
CHRNA4 rs2273502 will show a pronounced decrease in 2-nAChR availability while carriers 
of the T allele show no change in 2-nAChR availability will demonstrate the greatest change or 
normalization over 6 weeks of abstinence.
Aim 4: To measure the sensitivity of NCFHEB binding to changes in endogenous ACh 
levels in smoking and nonsmoking subjects. After baseline quantification of 2-nAChR 
availability, physostigmine will be administered as previously described (HIC#09100005837) 
and subjects will be scanned again with NCFHEB. We hypothesize there will be greater increase 
in ACh level (or greater reduction in radio tracer binding) in smoking as compared to 
nonsmoking subjects.  These results will be correlated with changes in mood and cognition 
associated with physostigmine administration. Up to 50 smoking and 50 nonsmoking subjects 
will participate. Smokers that are able to remain abstinent will have a second set of baseline and 
post-physostigmine scans with NCFHEB 4-8 weeks later.  This will allow us to determine 
whether ACh function changes over the course of abstinence.     
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CLOSED Aim 5: To measure the sensitivity of NCFHEB binding to changes in endogenous 
ACh levels in smoking and nonsmoking subjects with schizophrenia. After baseline 
quantification of 2-nAChR availability, at 1 week of smoking abstinence, physostigmine will be 
administered as previously described (HIC#09100005837) and subjects will be scanned again 
with NCFHEB. We hypothesize there will be greater increase in ACh level (or greater reduction 
in radio tracer binding) in smoking as compared to nonsmoking subjects with schizophrenia, but 
the extent of this change will be different than in controls.  These results will be correlated with 
changes in mood, negative and positive symptoms, and cognition associated with physostigmine 
administration. Up to 10 smoking and 10 nonsmoking subjects with schizophrenia will 
participate.   
Aim 6: To determine the efficacy of a bolus to infusion scan versus a bolus scan of 
NCFHEB with physostigmine challenge.  We will invite back never smokers and smokers who 
completed bolus scans in Aim 4 to complete one bolus to infusion scan with NCFHEB with 
physostigmine administration.  Smokers will complete the scan at approximately one week of 
abstinence from smoking. We will compare the results from this scan to their Aim 4 bolus scans 
to determine which administration better suits a physostigmine challenge.  Smokers who 
completed all 4 scans in Aim 4 will not be asked to return within the same year.
CLOSED Aim 7.  To determine amphetamine-induced DA release in tobacco smokers 
during acute withdrawal and in nonsmokers. Smoker and nonsmoker subjects from Aim 4 
will participate. Each subject will participate in up to 2 [11C]PHNO PET scans (ideally, the two 
PET scans will be carried out in the same day). Up to three hours before the second PET scan, 
amphetamine (0.5 mg/kg, PO) will be administered. In smokers, the scan will occur at 10-30 
days of smoking abstinence. We hypothesize that at 10-30 days of withdrawal amphetamine-
induced DA release will be blunted compared to healthy nonsmokers. 
CLOSED Aim 8. To determine the degree of occupancy of the β2*-nAChRs by nicotine 
after use of an e-cig compared to a tobacco cigarette using [18F]NCFHEB PET 
neuroimaging. Given recent evidence that experienced e-cig users can achieve similar 
plasma nicotine levels compared to tobacco smoking 6, 7 , we hypothesize there will not be a 
significant difference in β2*-nAChR occupancy by nicotine after use of an e-cig as compared 
to a regular cigarette.  We will also examine arterial plasma nicotine levels and compare to 
nicotine occupancy of the nAChRs between groups. 
                                                           
2.   Background: Describe the background information that led to the plan for this project.   
      Provide references to support the expectation of obtaining useful scientific data. 
The cholinergic system, and the beta2-nicotinic acetylcholine receptors (β2-nAChRs) 
specifically, have been implicated in many neuropsychiatric conditions, such as addiction, 
depression, schizophrenia and AD.(3-6) This involvement has been supported by molecular 
imaging studies of the β2-nAChR, including studies performed by our group at Yale.(7-13) The 
β2-nAChRs are the most abundant nAChRs in the brain.  Until very recently, β2-nAChR SPECT 
and PET radiotracers available for human use displayed slow kinetics, requiring prolonged 
scanning sessions(14, 15), and practically eliminated our ability to study severely impaired 
patients.(16, 17)  The recently introduced radiolabeled antagonist (-)-2-(6-(18)F-fluoro-2,3'-
bipyridin-5'-yl)-7-methyl-7-azabicyclo[2.2.1]heptane ([18F]NCFHEB or NCFHEB) has been 
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shown to have high binding potential at the β2-nAChRs and rapid kinetics (1) and is thus a very 
promising new radioligand.  The reproducibility of NCFHEB VT measurement in humans and the 
sensitivity of NCFHEB VT to changes in extracellular Ach concentrations have not yet been 
reported.  Here, we propose to test the reproducibility of NCFHEB in vivo binding in humans 
and to examine its vulnerability to changes in synaptic ACh. These critical studies will answer a 
fundamental question regarding use of NCFHEB and PET in the study of cholinergic systems 
and will have broad implications to the future studies of severe neuropsychiatric conditions 
(nicotine addiction, mood disorder, schizophrenia, AD), with high relevance to individual and 
public health.  If validated, such an imaging tool would have enormous potential to facilitate the 
development of innovative medicines aimed at modulating the cholinergic system.
Furthermore, since nicotine binds with high affinity to the 2-nAChRs and there is a high 
prevalence of smoking in the US, we will examine this system and its response to increases in 
endogenous ACh in healthy smokers and the associated cognitive and genetic factors. 
Evidence for Genetic Inheritance of Tobacco Smoking  A family history of tobacco smoking 
is a powerful predictor of smoking risk. Children of ever- or current- smokers are more likely to 
smoke, compared to children of never smokers (18-22). Smokers with a family smoking history 
are more likely to be persistent smokers than those with no history (23-26). Regular stable 
smoking in adolescence is related to having a current parent smoker relative to a former or never 
smoker parent (27). Failure to quit smoking is linked to having parents that smoked daily (19, 28, 
29). Parental smoking is related to a greater likelihood of developing nicotine dependence, 
higher numbers of cigarettes smoked per day, higher anxiety levels and a trend towards 
depression (24, 30). While all smokers experience anxiety in response to stressor, only family 
history positive smokers experience heightened craving for cigarettes (31). These data suggest 
that there is a biological difference between family history positive smokers and family history 
negative smokers that is likely transmitted between generations through shared genetics.
There is strong evidence to support genetic transmission of smoking between family members. 
Ever-smoking segregates in families following expectations consistent with a dominant 
Mendelian genetic factor with a frequency of 0.02. Studies of monozygotic and dizygotic twins 
reared together and apart suggest that there is 50-72% inheritance rate (26, 32-44). The 
concordance rate amongst monozygotic twins is higher than dizygotic twins, regardless of 
whether they were raised together or separately [reviewed by (45)]. Genetics contributes to the 
initiation of smoking behavior (25, 26, 34, 43) with heritability estimates of 0.11 and 0.78 
whereas shared and unique environmental effects range between 0.00 and 0.59 and 0.07 and 0.36 
respectively [reviewed by (46, 47)]. Persistence of smoking is also determined by genetics with 
heritability estimates between 0.04 and 0.86 [reviewed by (46, 47)].  An analysis of 17,500 
monozygotic and dizygotic twin pairs from 14 studies, estimated that genetics accounted for 
56%, whereas shared and unique environmental factors accounted for 24 and 20% of the liability 
for smoking initiation, and genetics accounted for 67%, whereas shared and unique 
environmental factors accounted for 2 and 31% of the liability for smoking persistence. 
Smoking behavior has been linked to chromosomes 1, 2, 4, 5, 6, 9, 10, 11, 14, 17, 18 and 21 (48-
51). A recent genome-wide association study of nicotine dependent individuals showed that 
single nucleotide polymorphisms (SNPs) nominate candidate genes coding for cell adhesion, 
enzymes, transcriptional regulators, neurotransmitters, and receptors and the regulation of DNA, 
RNA, and proteins are associated with successful abstinence from smoking (52). In marker based 
genetic studies, associations between smoking behavior and several candidate genes have been 
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A P P R O V E D B Y T H E Y A L E U NI V E R SI T Y I R B 3/ 1 0/ 2 0 2 1
Pa ge 8 of 6 8
e val uate d i ncl u di n g c yt oc hr o me p 4 5 0, d o pa mi ne rece pt or ( D 2) d o pa mi ne tra ns p orter, ser ot o ni n 
tra ns p orter a n d t he nic oti nic acet ylc h oli ne rece pt or [re vie we d b y ( 4 6)]. Varia nts i n ge nes occ ur 
d ue t o i nserti o ns, deleti o ns, varia ble n u m bers of re peats, a n d si n gle n ucle oti de p ol y m or p his ms 
( S N Ps) i n t he D N A se q ue nce.  S N Ps are t he m ost c o m m o n t y pe of p ol y m or p his m occ urri n g as 
fre q ue ntl y as o nce f or e ver y 2 0 0- 1 0 0 0 base pairs i n differe nt re gi o ns of t he ge n o me.  S N Ps ha ve 
bee n tar gete d beca use t he y ma y be use d eit her as s y n o n y m o us mar kers f or li n ka ge, a n d li n ka ge 
dise q uili bri u m ma p pi n g, or t he y ma y re prese nt f u ncti o nal m utati o ns t he msel ves. T his li mitati o n 
ma y be dealt wit h b y d oi n g a ha pl ot y pe a nal ysis of m ulti ple S N Ps. Ha pl ot y pes pr o vi de greater 
statistical p o wer c o m pare d t o S N Ps ( 5 3). 
Nic oti nic Acet ylc h oli ne Rece pt or ( n A C h R) Nic oti ne i nitiates its effects i n brai n t hr o u g h t he 
nic oti nic acet ylc h oli ne rece pt or ( n A C h R). Ne ur o nal n A C h R bel o n g t o a rece pt or fa mil y of 
li ga n d gate d i o n c ha n nel rece pt ors t hat i ncl u de t he G A B AA  a n d G A B AC , gl yci ne a n d 5- H T3 
rece pt ors. Ne ur o nal n A C h R are c o m prise d of 5 s u b u nits ( 5 4). Eac h s u b u nit s pa ns t he me m bra ne 
4 ti mes s uc h t hat t he sec o n d tra ns me m bra ne d o mai n f or ms t he wall of t he i o nic p ore t hat 
tra ns p orts Ca2 +. T o date, 1 2 ge nes c o di n g f or s u b u nits ass ociate d wit h t he ne ur o nal n A C h R ha ve 
bee n i de ntifie d fr o m t he ma m malia n ge n o me i ncl u di n g ,  [re vie we d b y ( 5 5)]. 
Ne ur o nal n A C h R c o m prise d of 7 a n d 9  are f u ncti o nal as m o n o meric rece pt ors w hic h are 
p har mac ol o gicall y c haracterize d b y l o w affi nit y f or nic oti nic a g o nists, a n d hi g h affi nit y f or -
b u n gar ot o xi n, w hile all ot her  s u b u nits (e. g. ) re q uire c o- 
e x pressi o n of  a n d  pairs a n d are disti n g uis he d b y t heir hi g h affi nit y f or nic oti nic a g o nists a n d 
l o w affi nit y f or - b u n gar ot o xi n ( 5 6, 5 7). Bi n di n g sites f or t he e n d o ge n o us ne ur otra ns mitter, 
acet ylc h oli ne, are hi g hl y c o nser ve d bet wee n s u b u nit t y pes w hereas nic oti ne has hi g her affi nit y 
f or t he 4 a n d 3  s u b u nits vers us t he 2, 3, 4, or 6 a n d 2 or 4 s u b u nits ( 5 8). Acet ylc h oli ne 
bi n ds t o heter o meric n A C h Rs at t he i nterface bet wee n 2, 3, 4, or 6 a n d 2 or 4 s u b u nit ( 5 9). 
T he fift h access or y s u b u nit, w hic h is n ot i n v ol ve d i n acet ylc h oli ne bi n di n g ma y be 2, or 4, or 
5 or 3 ( 5 9). T he 3 a n d 5 s u b u nits are li kel y part ners wit h 6 s u b u nits i n n A C h R f or mi n g a 
pe nta mer of 6435 t hat are l ocalize d t o d o pa mi ner gic cell b o dies ( 6 0). N A C h R 42 
rece pt ors de m o nstrate 4-f ol d l o wer a m plit u de w h ole cell c urre nts, a n d sl o wer ac ute 
dese nsitizati o n a n d f u ncti o nal r u n d o w n as c o m pare d t o 7-c o ntai ni n g n A C h R. Beta s u b u nits 
i nfl ue nce t he se nsiti vit y of 4 t o f u ncti o nal i nacti vati o n. T he sec o n d maj or i ntracell ular l o o p 
i nfl ue nces t he ac ute dese nsitizati o n of n A C h R ( 6 1).  Nic oti nic a g o nists ha ve hi g her f u ncti o nal 
p ote nc y at 4 c o ntai ni n g vers us 2 c o ntai ni n g b ut t he y ha ve hi g her bi n di n g affi nit y at 2 
c o ntai ni n g n A C h R( 6 2). Hi g h a n d l o w affi nit y 42n A C h R res ult fr o m t he asse m bl y of 4 a n d 2 
i nt o t w o disti nct st oic hi o metric arra n ge me nts (4)2 (2)3 hi g h affi nit y; a n d (4)3 (2)2, l o w 
affi nit y t hat differ si g nifica ntl y i n t heir f u ncti o nal a n d p har mac ol o gical pr o perties [re vie we d 
b y( 6 3)].  T he ( 4)2 (2)3 st oic hi o metr y is m ore se nsiti ve t o acti vati o n a n d u pre g ulati o n b y 
nic oti ne a n d dese nsitizes m ore sl o wl y. I m p orta ntl y, a g o nist bi n di n g t o n A C h R li kel y reflects 
m ulti ple states, i ncl u di n g t he resti n g a n d dese nsitize d states ( 6 2).
R ole of n A C h R S u b u nits i n Nic oti ne De pe n de nce 
T he rei nf orci n g pr o perties of nic oti ne are me diate d pri maril y t he heter o pe nta meric - n A C h R 
a n d t he h o m o meric 7- n A C h R. B ot h s u bt y pes ha ve bee n l ocalize d t o G A B A ne ur o ns i n t he 
ve ntral te g me ntal area, t he brai n re gi o n w here mes oli m bic d o pa mi ner gic ne ur o ns belie ve d t o be 
part of t he fi nal c o m m o n pat h wa y f or dr u g re war d ori gi nate. T hese G A B Aer gic ne ur o ns s y na pse 
o nt o t he d o pa mi ner gic ne ur o ns t hat als o ha ve 7- n A C h R a n d - n A C h R t hat are li kel y als o 
ass ociate d wit h 5 a n d/ or 6 s u b u nits ( 6 4). I m p orta ntl y, mice wit h o ut t he 2 
A P P R O V E D B Y T H E Y A L E U NI V E R SI T Y I R B 3/ 1 0/ 2 0 2 1
s u b u nit d o n ot self-

A P P R O V E D B Y T H E Y A L E U NI V E R SI T Y I R B 3/ 1 0/ 2 0 2 1
Pa ge 9 of 6 8
a d mi nister nic oti ne a n d als o ha ve n o hi g h affi nit y nic oti ne bi n di n g sites ( 6 5). I n kee pi n g, 
rest orati o n of 2 s u b u nits i n V T A ca uses rei nstate me nt of nic oti ne self-a d mi nistrati o n ( 6 6). 
I nteresti n gl y, t he 2 b ut n ot t he 7-s u b u nit is re q uire d f or nic oti ne-c o n diti o ne d place prefere nce 
( 6 7). Si nce 4 s u b u nits pre d o mi na ntl y c o-l ocalize  wit h t he 2 s u b u nit, t hese st u dies s u g gest t hat 
t he - n A C h R is a pri nci pal n A C h R res p o nsi ble f or t he rei nf orci n g pr o perties of nic oti ne. 
Alt h o u g h e vi de nce is acc u m ulati n g de m o nstrati n g t hat t he 5, 6 a n d 3 s u b u nits als o c o-l ocalize 
wit h 4 a n d 2 s u b u nits a n d f or m part of t he heter o pe nta mer. I n mice wit h 4 n A C h R s u b u nits 
c o ntai ni n g a si n gle p oi nt m utati o n i n t he p ore-f or mi n g M 2 d o mai n t hat re n ders 4* rece pt ors 
h y perse nsiti ve t o nic oti ne, t hat selecti ve acti vati o n of 4* wit h l o w nic oti ne d oses is s ufficie nt t o 
i n d uce rei nf orce me nt, t olera nce a n d se nsitizati o n t o nic oti ne, s u g gesti n g t hat t he  s u b u nit 
pla ys a critical r ole i n nic oti ne de pe n de nce.  
Ot her n A C h R s u b u nits are als o  i m plicate d i n nic oti ne de pe n de nce. 4 n A C h R ma y i nfl ue nce 
nic oti ne c o ns u m pti o n a n d rela pse d ue t o its relati o ns hi p t o t he e x pressi o n of a n xiet y relate d 
be ha vi ors ( 6 8). Nic oti ne wit h dra wal is preci pitate d b y dr u gs wit h prefere nces f or 
, 2, or 7 ( 6 9). 4 k n oc k o ut mice de m o nstrate d decrease d nic oti ne wit h dra wal 
s y m pt o ms w hereas 2 k n oc k o uts d o n ot ( 7 0).  T he 7 n A C h R i n t he V T A ha ve als o bee n 
li n ke d t o nic oti ne wit h dra wal s y m pt o ms ( 7 1, 7 2). Nic oti ne treate d 7 k n oc k o ut mice de m o nstrate 
n o nic oti ne wit h dra wal s y m pt o ms ( 7 3). F urt her e vi de nce s u p p orti n g a r ole f or - n A C h R i n 
nic oti ne de pe n de nce i ncl u des t he a bilit y of t he 7 rece pt or a nta g o nist met h ylcac o niti ne t o 
atte n uate t he a bilit y of nic oti ne t o re d uce I C S S (i ntracra nial self-sti m ulati o n) t hres h ol d ( 7 4).
Effects of C hr o nic Nic oti ne E x p os ure o n n A C h R It is well esta blis he d i n t he precli nical 
literat ure t hat c hr o nic nic oti ne e x p os ure ca uses a n a da pti ve i ncrease i n 2- n A C h R e x pressi o n 
( 7 5- 8 0).  I n p ost m orte m h u ma n brai n nic oti nic a g o nist bi n di n g is hi g her i n t he g yr us rect us 
( Br o d ma n area 1 1), hi p p oca m p us, t hala m us, mi d brai n ( 8 1, 8 2), striat u m, e nt or hi nal c orte x , 
cere bell u m ( 8 3) a n d prefr o ntal a n d te m p oral c orte x ( 8 4) of s m o kers vers us ne ver s m o kers. 
I ncrease d 2- n A C h R e x pressi o n after c hr o nic nic oti ne was rece ntl y de m o nstrate d i n n o n h u ma n 
pri mates usi n g S P E C T a n d [ 1 2 3I] 5-I A ( 8 5) [ Stale y et al., 2 0 0 6] a n d als o i n h u ma n t o bacc o 
s m o kers a bsti ne nt f or 4- 9 da ys [ Stale y et al., 2 0 0 6]. 
I n h u ma ns, nic oti ne bi n di n g i n e x-s m o kers ( > 2 m o nt hs) is si milar t o t hat of t he n o n-s m o kers 
s u g gesti n g t hat t he nic oti ne- me diate d u pre g ulati o n of a g o nist bi n di n g t o n A C h R is re versi ble 
( 8 1- 8 3). O ur o n g oi n g st u dies ev al u ati n g t he n or m aliz ati o n of a g o nist bi n di n g t o 2- n A C h R 
av ail a bility wit hi n-s u bject i n livi n g h u m a n s m o kers h ave reve ale d i nter-i n divi d u al differe nces 
i n t his n or m aliz ati o n wit h s o me s u bjects s h o wi n g decre ases of 1 4- 1 7 % over t he first m o nt h 
a n d ot hers s h o wi n g n o c h a n ge < 4 %. We h ave hy p ot hesize d t h at t hese differe nces i n rece pt or 
n or m aliz ati o n are ge netic ally deter mi ne d. We n o w h ave preli mi n ary d at a s u g gesti n g t h at t his 
differe nce i n n or m aliz ati o n is rel ate d t o t he C H R N A 4 rs 2 2 7 3 5 0 2 S N P. 
Ge nes Ass oci ate d wit h Nic oti ne De pe n de nce
C H R N A 4  T he  n A C h R s u b u nit is e nc o de d b y t he C H R N A 4 ge ne, t hat, has bee n ma p pe d t o 
c hr o m os o me 2 0 q 1 3. 2- 1 3. 3 ( 8 6). T he C H R N A 4 ge ne [ MI M 1 1 8 5 0 4] [ Nati o nal Ce nter f or 
Bi otec h n ol o g y I nf or mati o n, l oc us I D 1 1 3 7] has 6 e x o ns a n d is a p pr o xi matel y 1 7 k b i n size ( 8 7). 
T o date, 3 0 si n gle n ucle oti de p ol y m or p his ms ( S N Ps) ha ve bee n i de ntifie d o n t he C H R N A 4 ge ne, 
of w hic h, 1 4 were p ol y m or p hic ( 8 8). T he relati o ns hi p bet wee n 6 S N Ps [see Ta ble 2] a n d nic oti ne 
de pe n de nce defi ne d as t w o si bli n gs pl us at least o ne pare nt or a n ot her si bli n g wit h a n F T N D 
sc ore > 8 de m o nstrate d t hat rs 1 0 4 4 3 9 6 ( T allele) a n d rs 1 0 4 4 3 9 7 ( A allele), i n e x o n 5 of t he 
A P P R O V E D B Y T H E Y A L E U NI V E R SI T Y I R B 3/ 1 0/ 2 0 2 1
C H R N A 4 ge ne are pr otecti ve a gai nst nic oti ne de pe n de nce ( 8 8). M ore o ver, t he fa mil y base d 

APPROVED BY THE YALE UNIVERSITY IRB 3/10/2021
Page 10 of 68
Fig 1. A, Schematic representation of the six SNPs located on the CHRNA4 gene, showing their locations according to the 
information from the chromosome 20 genomic contig (GenBank accession number NT_011333). SNP1 and SNP2 in exon 5 are 
synonymous SNPs revealed by direct DNA sequencing. B, Schematic representation of the four SNPs located on the CHRNB2 
gene, showing their locations according to the information from the chromosome 1 genomic contig (GenBank accession 
number NT_004668): From Feng et al., Am J Hum Genet 75:112-121, 2004.
Table 2: Single Nucleotide Polymorphisms in Genes Encoding 42 nAChR
CHRNA4 SNPs
Gene Location
Base Change
Amino Acid Change
rs1044396
Exon 5
C -> T
Ser -> Ser
rs1044397
Exon 5
G -> A
Ala -> Ala
rs2273504
Intron
G -> A
rs2273502
Intron
C -> T
rs3827020
Intron
T -> C
rs2236196
3' UTR 
A -> G
CHRNB2 SNPs
rs2072658
5' UTR 
G -> A
A10160C
Intron 5
rs207660
3' UTR 
C -> T
rs2072661
3' UTR 
G -> A
Obtained from Feng et al., Am J Hum Genet 75:112-121, 2004
association test suggested that these 6 SNPs meet criteria for a haplotype block that is 
significantly associated with nicotine dependence and age-adjusted FTND (Fagerstrom Test for 
Nicotine Dependence Score). Thus, persons with the GCTATA haplotype of the CHRNA4 gene 
are protected from developing nicotine dependence.
An ethnic and gender-specific association of the CHRNA4 gene with nicotine dependence (89) 
such that in European Americans (EA) rs2273504 and rs 1044396 are significantly associated 
with smoking quantity and severity of nicotine dependence as determined by the Fagerstrom Test 
for Nicotine Dependence (FTND), whereas, SNPs rs3787137 and rs2236196 were associated 
with smoking quantity, FTND score and heaviness of smoking index (only rs2236196).  
Hutchison and colleagues have recently evaluated the CHRNA4 SNPs rs612249 and rs609387 
located in the 5’ promoter UTR region and also the 3’UTR SNP rs2236196 and the relationship 
to CHRNA4 mRNA, epibatidine binding in postmortem tissue (90), and nicotine sensitivity in 
living humans (91). In brief, these studies demonstrated that individuals with the AG (vs AA) 
genotype to RS2236196 demonstrated higher epibatidine binding [which agrees with our 
preliminary data [see section C.7] and greater sensitivity to the physiological and cognitive 
effects of smoking suggesting that CHRNA4 genotypes influence the etiology of nicotine 
dependence. And, individuals with GG RS612249 SNP reported greater subjective physiological 
effects after smoking three cigarettes and greater baseline CO. Recent studies have suggested an 
association between CHRNA4 rs 1044396 and nicotine-mediated attentional network function in 
the anterior cingulate cortex and parietal cortex (92) and CHRNA4 rs3746372  has been linked 
to the number of cigarettes smoked in schizophrenic smokers (93).
While there is some debate on how SNPs in the human genome translate to the rodent genome, it 
is interesting to note that a naturally occurring SNP (1587A to G) results in an alanine to 
threonine variation at the amino acid position 529 on the nascent 4 subunit in the last 1/3 of the 
cytoplasmic loop between the TM3 and TM4.  This SNP alters nAChR function including 
increased vulnerability to nicotine-induced seizures; ethanol withdrawal seizures; the effects of 
alcohol and nicotine on locomotor activity and is associated with lower nicotine consumption 
and preference (94). There were no differences in cytisine binding between variants, however 
cytisine is nonselective and labels multiple nAChRs, compared to 5-IA, which labels only 2- 
nAChRs. This variant also leads to a difference in the ratio of high and low affinity nAChR such 
that SS (AA or A carriers) mice have a greater fractions of desensitized 4- nAChR compared to 
the LS (GG) mice. Importantly, this variant is common in European Americans and uncommon 
in Asians and Africans (94-97).
 CHRNB2. The 2 subunit is encoded by the CHRNB2 [MIM 118507] [NCBI locus ID 1141] 
gene and has been mapped to chromosome 1q21.3 (98). The CHRNB2 gene has 6 exons with a 
G-C  rich promoter region that includes a presumptive neural-restrictive silencer element, an 
APPROVED BY THE YALE UNIVERSITY IRB 3/10/2021

A P P R O V E D B Y T H E Y A L E U NI V E R SI T Y I R B 3/ 1 0/ 2 0 2 1
Pa ge 1 1 of 6 8
u n us uall y l o n g 3’ u ntra nslate d re gi o n a n d is a p pr o xi matel y 1 2 k b ( 9 9). T o date 2 4 si n gle 
n ucle oti de p ol y m or p his ms ( S N Ps) ha ve bee n i de ntifie d f or t he C H R N B 2 ge ne alt h o u g h o nl y 4 
ha ve bee n f o u n d t o be p ol y m or p hic ( 8 8). T w o st u dies t hat ha ve assesse d t he relati o ns hi p 
bet wee n C H R N B 2 ge ne varia nts a n d nic oti ne de pe n de nce ha ve s u g geste d t hat t here is n o 
ass ociati o n ( 9 9, 1 0 0).  H o we ver, Fe n g a n d c ollea g ues rece ntl y de m o nstrate d t hat w hile 
i n di vi d ual S N Ps were n ot ass ociate d wit h nic oti ne de pe n de nce t he fa mil y base d ass ociati o n test 
s u g geste d t hat 4 S N Ps ( Ta ble 2) meet criteria f or a ha pl ot y pe bl oc k t hat de m o nstrates mar gi nall y 
si g nifica nt c orrelati o n wit h t he a ge –a dj uste d F T N D sc ore ( p = . 0. 0 6 5) ( 8 8). S pecificall y, pers o ns 
wit h t he G C C G ha pl ot y pe ma y be pr otecte d fr o m de vel o pi n g nic oti ne de pe n de nce.
C H R N A 5 T he C H R N A 5, C H R N A 3, a n d C H R N B 4 ge nes are cl ustere d o n c hr o m os o me 
1 5 q 2 4( 1 0 1). A rece nt st u d y has de m o nstrate d a str o n g ass ociati o n bet wee n a n o n-s y n o n y m o us 
S N P i n t he 5 - n A C h R s u b u nit ge ne (rs 1 6 9 6 9 9 6 8) a n d t he ris k f or nic oti ne de pe n de nce. T his 
varia nt res ults i n a c ha n ge i n a mi n o aci d 3 9 8 fr o m as para gi ne e nc o de d b y t he G allele, t o as partic 
aci d, e nc o de d b y t he A allele, w hic h alters t he c har ge of t he a mi n o aci d i n t he sec o n d 
i ntracell ular l o o p of t he 5 - n A C h R s u b u nit. I n di vi d uals h o m oz y g o us f or t he A allele 
de m o nstrate a 2-f ol d hi g her ris k of de vel o pi n g nic oti ne de pe n de nce o nce e x p ose d t o ci garette 
s m o ki n g ( 1 0 2). I m p orta ntl y, 5 - n A C h R s u b u nits c o m m o nl y fill t he fift h p ositi o n of t he 
heter o pe nta mer m ost c o m m o nl y c o m prise d of 4 a n d  2 rece pt ors ( 1 0 3)  
C H R N B 3 T he C H R N B 3 ge ne is l ocate d o n c hr o m os o me 8 p 1 1. 2. T o date 4 7 S N Ps ha ve bee n 
i de ntifie d. I n brai n 3 ca n ta ke t he fift h p ositi o n i n t he pe nta mer of 2- n A C h R, alt h o u g h it d oes 
n ot c o ntri b ute t o t he acet ylc h oli ne bi n di n g site it d oes alter c ha n nel f u ncti o n a n d i nfl ue nces 
a g o nist p ote nc y beca use t he y partici pate i n t he c o nf or mati o nal c ha n ges t hat g o al o n g wit h 
acti vati o n of t he rece pt or a n d dese nsitizati o n ( 1 0 4). T his ge ne has rece ntl y bee n ass ociate d wit h 
a hi g her ris k f or de vel o pi n g nic oti ne de pe n de nce ( 1 0 2). A n d C H R N B 3 (rs 4 9 5 0 a n d rs 1 3 8 0 6 0 4) 
has bee n li n ke d t o s u bjecti ve res p o nses t o t o bacc o s m o ki n g ( 1 0 5).
D R D 2 T a qI A  T he relati o ns hi p of t he D R D 2 Ta qI A p ol y m or p his m wit h t o bacc o s m o ki n g has 
bee n e xte nsi vel y st u die d. A meta-a nal ysis s u g gests t hat t here is a hi g her pre vale nce of t he D R D 2 
Ta q 1 A p ol y m or p his m, i n E ur o pea n A merica n s m o kers vers us ne ver s m o kers ( 1 0 6). M ore o ver 
t his relati o ns hi p see ms t o tra nsce n d acr oss m ulti ple et h nic gr o u ps wit h a n ass ociati o n of t he 
A 1/ A 1 ge n ot y pe wit h c urre nt s m o ki n g stat us i n Me xica n- A merica ns ( 1 0 7) vers us a hi g her 
ass ociati o n of t he A 2/ A 2 i n Ja pa nese e ver-s m o kers ( 1 0 8). C hi nese s m o kers wit h t he A 2/ A 2 
ge n ot y pe s m o ke d a greater n u m ber of ci garettes/ da y t ha n s m o kers wit h at least o ne A 1 allele 
( 1 0 9). D R D 2 Ta q 1 A s h o we d n o ass ociati o n wit h s m o ki n g stat us or q ua ntit y i n t he U K 
p o p ulati o n ( 1 1 0) or i n Africa n A merica ns ( 1 0 7).I n E ur o pea n A merica ns, t here a p pears t o be n o 
o verall ass ociati o n bet wee n t he D R D 2 Ta q 1 A p ol y m or p his m a n d s m o ki n g cessati o n ( 1 1 1). 
H o we ver, Ja pa nese w o me n s m o kers wit h t he A 1 allele are less li kel y t o q uit s m o ki n g t ha n t he 
A 2/ A 2 ge n ot y pe ( 1 1 2). Carriers of t he D R D 2- A 1 allele are m ore li kel y t o prese nt t o bacc o 
wit h dra wal s y n dr o me, s h orter late nc y peri o ds pri or t o rela pse a n d de m o nstrate greater res p o nse 
t o t he nic oti ne patc h ( 1 1 3- 1 1 5).  W hile t here a p pears t o be a n ass ociati o n bet wee n t he D R D2 
Ta q A 1 allele a n d t o bacc o s m o ki n g, t he f u ncti o nal rele va nce of t his ass ociati o n is still n ot 
u n derst o o d. T he Ta q 1 A p ol y m or p his m is l ocate d ~ 1 0 k b d o w nstrea m a n d u p t o 2 5 k b a wa y 
fr o m t he D R D 2 ge ne a n d is u nli kel y t o alter D R D 2 acti vit y directl y. H o we ver, t he D R D2 Ta q A 1 
allele has bee n ass ociate d wit h re d uce d D 2 rece pt or bi n di n g affi nit y a n d l o w striatal D2 rece pt or 
de nsities ( 1 1 3, 1 1 6, 1 1 7). F urt her m ore, a f u ncti o nal relati o ns hi p bet wee n D 2
A P P R O V E D B Y T H E Y A L E U NI V E R SI T Y I R B 3/ 1 0/ 2 0 2 1
 a n d n A C h R has 
rece ntl y bee n el uci date d. B ot h rece pt ors are k n o w n t o m o d ulate d o pa mi ne release i n t he striatal 
re war d pat h wa ys s uc h t hat acet ylc h oli ne bi n di n g t o n A C h R heter o-a ut orece pt ors e n ha nces D A 

A P P R O V E D B Y T H E Y A L E U NI V E R SI T Y I R B 3/ 1 0/ 2 0 2 1
Pa ge 1 2 of 6 8
release, w hile d o pa mi ne i nteracti o ns wit h pres y na ptic D2 a ut orece pt ors decrease D A release. A 
rece nt st u d y has de m o nstrate d t hat D2 a ut orece pt ors a n d n A C h R heter orece pt ors f or m 
heter o meric d o pa mi ne a ut orece pt or c o m ple xes t o m o d ulate t he efficac y of D A release ( 1 1 8). 
K n oc k o ut st u dies s u g gest t hat t here ma y be u p t o si x differe nt n A C h R s u b u nit c o m bi nati o ns t hat 
me diate D A release i ncl u di n g  62; 6 2(3); 42; 4562 462(3) a n d 45 2 ( 1 1 9)
A N K K 1 P oly m or p his m . Rece nt st u dies ha ve re veale d t hat t he D R D 2 Ta qI A ( Rs 1 8 0 0 4 9 7) site is 
l ocate d i n a ge ne a djace nt t o D R D 2 k n o w n as t he a n kr yi n re peat a n d ki nase d o mai n c o ntai ni n g 
A N K K 1 ( 1 2 0). T he a n k yri n re peat is o ne of t he m ost c o m m o n pr otei n- pr otei n i nteracti o n m otifs 
i n nat ure. Re peate d m o d ules of a b o ut 3 3 a mi n o aci ds occ ur i n a lar ge n u m ber of f u ncti o nall y 
di verse pr otei ns, i ncl u di n g tra nscri pti o nal i nitiat ors, cell c ycle re g ulat ors, c yt os keletal pr otei ns, 
i o n tra ns p orters, a n d si g nal tra ns d ucers. T he re peats t y picall y ha ve ne gati ve c har ge d s urface s o a 
p ol y m or p his m t hat c ha n ges fr o m ne gati ve t o ne utral or p ositi ve c ha n ge ma y c ha n ge t he 
f u ncti o nal r ole ( 1 2 1). T he Ta q 1 A p ol y m or p his m i n A N K K 1 ma y be i n li n ka ge dise q uili bri u m 
wit h u pstrea m p ol y m or p his m i n D R D 2 a n d lie wit hi n t he sa me ha pl ot y pe bl oc k t hat s pa ns t he 
o verla p bet wee n t he t w o ge nes ( 1 2 1). 
T he A N K K 1 ge ne c o des a pr otei n t hat has bee n li ke ne d t o t he t yr osi ne ki nase li ke ge nes ( T K L) 
t hat rese m ble t yr osi ne a n d seri ne ki nases a n d are si milar t o rece pt or i nteracti n g pr otei n ki nases 
( RI P K) a n d t he le uci ne ric h re peat ki nases.  P h os p h or ylati o n re prese nts t he maj or p ost-
tra nslati o nal m o dificati o n re g ulati n g n u mer o us li ga n d gate d i o n c ha n nels i ncl u di n g t he n A C h R 
( 1 2 2). S u b u nit p h os p h or ylati o n ca n alter t he affi nit y state of t he rece pt or f or a g o nist a n d t he 
f u ncti o nal c haracteristics of t he c ha n nel. T he D R D 2 Ta qI A s yste m ca uses a n a mi n o aci d 
s u bstit uti o n wit hi n t he 1 1 t h a n kri n re peat of t his ge ne ( Gl u 7 1 3 L ys), t hat ma y alter s u bstrate 
bi n di n g s pecificit y( 1 2 0). I n kee pi n g, Ta q 1 A ( A 1 carriers) are ass ociate d wit h decrease d D 2 
a ut orece pt or de nsit y, a n d c orres p o n di n g decrease i n a ut orece pt or f u ncti o n ( meas ure d b y 1 8F 
D O P A) ( 1 2 1).
 
O ur c ollea g ue, Dr J oel Geler nter has rece ntl y de m o nstrate d t hat A N K K 1 S N Ps are str o n gl y 
ass ociate d t o nic oti ne de pe n de nce i n E ur o pea n A merica ns a n d Africa n A merica ns. S pecificall y 
t he A N K K 1 is i n li n ka ge dise q uili bri u m wit h ot her varia nts t hat are ass ociate d wit h nic oti ne 
de pe n de nce as defi ne d b y D S M-I V criteria( 1 2 3). T his p he n ot y pe is partic ularl y br oa d a n d t h us 
ma y be relate d t o o ne or m ore of s m o ki n g be ha vi ors i ncl u di n g, t olera nce a n d wit h dra wal t o na me 
a fe w ( 1 2 3). T he relati o ns hi p bet wee n t he A N K K 1 ge ne a n d 2- n A C h R a vaila bilit y t hat we ha ve 
o bser ve d i n o ur pil ot a nal yses is n ot e ntirel y clear. H o we ver, it ma y be h y p ot hesize d t hat it ma y 
pla y a r ole i n deter mi ni n g t he p h os p h or ylati o n state of n A C h R s u b u nits, w hic h ma y m o d ulate 
dese nsitizati o n a n d rec o ver y of t he n A C h R rece pt or i n t he prese nce a n d a bse nce of nic oti ne 
( 1 2 4).  T yr osi ne ki nases re g ulate all n A C h R b ut t he f u ncti o nal c o nse q ue nces b y t he sa me ki nase 
fa mil y are s pecific f or eac h s u bt y pe a n d l ocati o n [re vie we d b y ( 1 2 5)]. T he 4 s u b u nit has bee n 
s h o w n t o be p h os p h orlate d i n sit u b y c A M P de pe n de nt pr otei n ki nase, ( P K A) a n d pr otei n ki nase 
C ( 1 2 2). P K A me diate d p h os p h or ylati o n of t he 4 s u b u nit e n ha nces its affi nit y f or t he 1 4- 3- 3 
c ha per o ne pr otei n res ulti n g i n i ncrease d e x pressi o n of t he 42 rece pt or( 1 2 6) ( 1 2 7). T he 
p h os p h or ylati o n state of ne ur o nal n A C h Rs a p pears t o re g ulate t heir rate of rec o ver y fr o m 
dese nsitizati o n a n d, rece pt ors rel y o n bala nce bet wee n p h os p h or ylati o n a n d de p h os p h or ylati o n 
state t o re gai n t heir a g o nist se nsiti vit y( 1 2 8). W hile it is k n o w n t hat t he 4 s u b u nit e xists i n 
p h os p h or ylate d a n d de p h os p h or ylate d states, t he relati o ns hi p bet wee n t hese states a n d rece pt or 
f u ncti o n are n ot yet k n o w n ( 1 2 2). A d diti o nal w or k re mai ns t o el uci date t he f u ncti o n of A N K K 1 
i ncl u di n g m ore e xte nsi ve e x pressi o n pr ofili n g a n d c haracterizati o n of t he f u ncti o nal si g nifica nce 
a n d l ocalizati o n at t he pr otei n le vels( 1 2 0) a n d its relati o ns hi p t o 
A P P R O V E D B Y T H E Y A L E U NI V E R SI T Y I R B 3/ 1 0/ 2 0 2 1
2- n A C h R a vaila bilit y.  

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In the present proposal we will explore the relationship of these and other SNPs (Aim 2 and 
3) that have been linked to nicotine dependence with 2-nAChR availability in never 
smokers and smokers to determine if these SNPs also convey genetic liability to 2-nAChR 
availability in relation to the regulatory effects of nicotine as well as relapse rates.
Nicotinic receptors and schizophrenia
Schizophrenia is associated with very high rates of tobacco addiction (80%) relative to 
the general population (20%) and other psychiatric disorders (129, 130). Compared to typical 
smokers, individuals with schizophrenia are reported to extract higher amounts of nicotine per 
cigarette and higher rates of smoking-related cardiovascular disease, pulmonary disease and 
associated mortality (131, 132). The high rate of tobacco smoking may reflect an attempt to 
“self-medicate” the negative symptoms, cognitive dysfunction, and antipsychotic-related side-
effects associated with schizophrenia (133-135).  Therefore, understanding the basis for high 
rates smoking in this population, a modifiable risk factor, might lead to increased life expectancy 
and quality of life, and may also provide the basis for developing drugs to target the symptoms of 
schizophrenia. 
Nicotine, the primary addictive and reinforcing constituent in cigarettes, has high affinity 
for the beta2-subunit containing nicotinic acetylcholine receptors (β2*-nAChRs). Evidence from 
postmortem (82), preclinical (10), and clinical (8, 9) studies demonstrates that chronic 
administration of nicotine and tobacco smoking increases the number of β2*-nAChRs in most 
brain regions (9, 136), which is commonly referred to as “upregulation”. However, post- mortem 
study suggests that smokers with schizophrenia fail to upregulate to the same extent as 
comparison smokers. A study that controlled for smoking status in postmortem samples of 
smokers and nonsmokers with and without schizophrenia shows that nonsmokers with and 
without schizophrenia have similar binding of [3H]-nicotine (symbolizing similar availability of 
β2*-nAChRs), but smokers with schizophrenia have lower [3H]-nicotine binding than smokers 
without schizophrenia. This suggests that smokers with schizophrenia do not upregulate nAChRs 
to the same extent as smokers without schizophrenia (137). We confirmed in vivo the 
postmortem findings of Breese et al. that smokers with schizophrenia had lower β2*-nAChR 
availability relative to smokers without schizophrenia (138).  Furthermore, we showed that β2*-
nAChR availability in individuals with schizophrenia correlated with negative symptoms (138).  
We followed up this evaluation with smokers and nonsmokers with schizophrenia and detected 
that although there is lower β2*-nAChR availability in schizophrenia, smokers with 
schizophrenia do upregulate as compared to nonsmokers. The study also showed that those 
smokers with schizophrenia who had lower β2*-nAChR availability, reported more negative 
symptoms and scored lower on tests of executive control. Given these recent developments, and 
the fact that the pharmaceutical companies are developing cholinergic medications for treatment 
of tobacco addiction, and possibly for treatment of cognitive deficits, it is first imperative to 
understand the cholinergic involvement in schizophrenia. Conducting an acetylcholine challenge 
via administration of an acetylcholinesterase inhibitor is an excellent way to examine whether 
there is cholinergic compromise in schizophrenia beyond lower receptor availability. We will 
correlate these results with measures of schizophrenia symptoms and cognitive performance in 
order to understand whether increasing endogenous acetylcholine may have effects on these 
variables.
Interaction of the Cholinergic and Dopaminergic Systems
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A substantial literature body demonstrates that nAChRs and the cholinergic system dynamically 
control the mesolimbic DA system by enhancing, inhibiting, and filtering striatal DA release. 
Nicotine releases DA by binding to β2*-nAChRs located on the mesolimbic DA neurons in the 
ventral tegmental area, resulting in neuronal firing and DA release in the nucleus accumbens and 
dorsal caudate. β2*-nAChRs are also critical for the reinforcing and motivational effects of 
nicotine, e.g., tying cues to drug consumption. Nicotine also “filters” DA release by modifying 
the sensitivity of DA synapses through its desensitization of the nAChRs. As reviewed in Exley 
and Cragg (2008), nicotine promotes DA release through both burst and tonic activity. Nicotine 
functions as an agonist and also as an antagonist via its desensitization/blocking actions and both 
may ultimately enhance DA neurotransmission. Thus, β2*-nAChRs are responsible for 
controlling the dynamic range of DA release. A recent preclinical study reported that chronic 
nicotine administration or deletion of the β2*-nAChRs (i.e., β2* knockout) reduced acute DA 
release. This suggests that chronic smokers may have a blunted stimulus-induced DA release 
compared to nonsmokers; however, this has not been examined. Several PET studies have 
demonstrated blunted amphetamine-induced DA release in the ventral striatum in both cocaine 
and alcohol dependent populations at approximately 2 weeks of withdrawal vs. controls. In the 
cocaine-dependent subjects, those with a more blunted drug induced DA release responded less 
well to a behavioral treatment that incorporated positive reinforcement. Their findings suggest 
that individuals with dysfunctional DA transmission are not able to “switch” well from drug-
reinforced behavior to more natural alternative rewards and this is supported by a preclinical 
study in which rats with a lesioned nucleus accumbens displayed an inability to choose greater 
magnitude delayed rewards vs. immediate rewards of lesser value. We have preliminary data 
suggesting tobacco smokers at 2 weeks of abstinence have blunted DA release compared to 
nonsmokers. We propose in Aim 7 to determine 1) whether there is reduced DA release in 
smokers at 2 weeks of withdrawal vs. nonsmokers, 2) if the magnitude of DA release is 
correlated with alterations in ACh levels measured in Aim 4 and 3) if alterations in DA and ACh 
predict relapse vulnerability. 
As mentioned, DA regulates the motivational properties of reinforcers including salience of 
stimuli and cue-reactivity. In landmark studies, Dr. Schultz illustrated that after learning the 
association between a cue and a reward, the DA neurons respond to the cue that occurs prior to 
the reward in anticipation of a future reward. This highlights the nature of cues because the cues 
themselves become reinforcing. In a smoker, the DA that is initially released as a result of 
smoking takes on the role of determining salience and connecting the smokers’ actions and 
rewarding feelings to environmental cues, e.g., the cigarette, lighter, ashtray, rest area where 
smoking occurs. A pack per day 20-year smoker has had 1,500,000 learning trials (puffs on a 
cigarette) that reinforce cues as stimuli for the physiological and rewarding effects of smoking. 
These associated cues elicit craving, and the magnitude of the reactivity to cues (craving, 
increase in heart rate, brain activation) may predict smoking cessation outcomes. Those who 
experience more craving in response to cues have a harder time quitting smoking. Functional 
magnetic resonance imaging (MRI) studies have found that during acute abstinence (24 h) there 
are increases in brain activations to smoking-related cues compared to when smoking as usual. 
PET studies have shown smoking-induced DA release that correlates with craving during acute 
withdrawal. While these studies confirm increased craving shortly after the last cigarette that is 
associated with DA release, few studies have systematically examined cue-induced reactivity 
over longer abstinence periods and none have examined the association between DA signaling 
and cue-induced reactivity and the relationship to relapse in human smokers. While many 
withdrawal related behaviors including significant mood impairment may improve over the first 
week of abstinence, cue-induced reactivity is long-lasting and may increase with abstinence. A 
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preclinical study demonstrated an incubation of nicotine-seeking behavior (i.e., cue-induced 
reinstatement) after extended nicotine self-administration. After self-administration, animals with 
a 7-day vs. 1-day enforced abstinence took longer to extinguish drug-seeking behavior and 
responded at significantly higher levels during the cue-induced reinstatement, and these 
behavioral changes were associated with dopaminergic molecular changes. Incubation of cue-
induced craving has also been shown in human tobacco smokers with different durations of 
abstinence (35 vs. 7 days). Critically, absolute craving levels in smokers do decrease over time, 
but cue-induced craving appears to increase. It is also known that DA D2 receptor drugs block 
cue-induced reinstatement in rodents and D3 receptor-specific drugs are being studied as 
treatments for smoking specifically for their potential to reduce relapse. We will determine 
relationships between drug-induced DA release and cue-reactivity during early phase abstinence 
and whether the magnitude of DA release predicts relapse vulnerability or change in cue 
reactivity. We predict that individuals who exhibit blunted drug-induced DA release will have a 
shorter latency to relapse and that their cue-reactivity will not be significantly reduced over time. 
While the focus in Aim 2 is on uncovering relationships between DA and cue-reactivity in 
smokers, DA also plays a powerful role in regulating mood and affect. Smoking is highly 
comorbid with Major Depressive Disorders, and many otherwise healthy smokers report 
significant changes in mood during acute withdrawal. Thus, in addition to cue-reactivity, we will 
examine relationships between DA transmission, mood and affect, and relapse vulnerability.
E-cigarettes
Tobacco smoking and secondhand smoke exposure has caused >20 million 
estimated premature deaths since 1965 and is the number one preventable cause of 
disease1. Although dependence rates have stabilized at ~18% for the general population, 
quit rates continue to be low2, 3. Nicotine is the primary addictive ingredient in tobacco 
smoke and binds with high affinity to the nicotinic acetylcholine receptors (β2*-nAChRs). 
Inhalation is the quickest way a drug can reach the brain and provide neurological effects; 
thus, when smoked, tobacco delivers nicotine to the pulmonary circulation and reaches the 
arterial circulation and brain within seconds after inhalation, leading to a rapid neurologic 
effect and a high level of addiction12, 13. Nicotine replacement products designed to help 
with smoking abstinence or to be used as replacement of smoked tobacco, have been 
developed in the form of patches, gum, lozenges, nasal spray, and inhaler. However, they 
deliver nicotine more slowly compared to smoked tobacco and may not be as rewarding to 
smokers11. Recently, electronic cigarettes (e-cigs) have gained popularity among seasoned 
tobacco smokers as well as tobacco-naïve users4. However, due to quick growth and 
popularity of these products and ever-changing design, it has proved difficult to apply 
regulatory process to e-cigs. The present study aims to add to the small pool of controlled 
studies examining biological and physiological effects of e-cigs.
 E-cigs are battery-operated devices that heat and aerosolize a liquid that typically 
contains nicotine. Although early studies of e-cigs suggested that nicotine delivery was 
ineffective14, 15, later studies found that nicotine can be delivered to the blood in 
significant amounts 6, 7, 16, 17. Further, advances in product technology have led to the 
development of more sophisticated e-cigs (i.e. personal vaporizers) with larger batteries 
and tank systems that are capable of producing larger amounts of aerosol and greater 
nicotine delivery as compared to earlier e-cig products 6, 7. Although there is little 
available data informing the addictive nature of e-cigs, the few available published studies 
suggest that e-cigs have lower addictive potential compared to smoked tobacco16, 18-20. 
Receptor imaging has been successfully utilized to examine mechanisms of tobacco 
addiction through evaluation of β2*-nAChR availability as a consequence of tobacco smoking21-
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23 as well as occupancy by nicotine in multiple previous studies8, 10, 24. Findings suggest that 
typical smokers maintain near complete receptor occupancy throughout the day, and that lower 
receptor occupancy levels lead to craving. As an example of our work, we compared β2*-nAChR 
occupancy between the Nicotrol nicotine inhaler and a regular tobacco cigarette using [123I]5-IA 
and SPECT imaging. We determined that using the inhaler leads to an average 55.9 ± 6.4% 
occupancy by nicotine of the β2*-nAChRs, which was significantly less compared to the 67.6 ± 
14.1% occupancy after use of a tobacco cigarette11. Further, we detected that arterial plasma 
nicotine concentration was significantly lower after use of the inhaler as compared to a regular 
cigarette. We observed a significant reduction in withdrawal but not craving symptoms 
associated with the use of the inhaler. These findings suggest that the nicotine occupancy after 
use of the inhaler may be at the degree of attenuating nicotine withdrawal; however, greater 
saturation of the receptors may be required to relieve nicotine craving symptoms. These data also 
suggest the nicotine inhaler has lower potential for abuse and addiction compared to a regular 
cigarette.  
In the present study, we will use [18F]NCFHEB, a radioligand that has high affinity for 
the β2*-nAChRs and has been shown to be sensitive to increases in nicotine25, and PET to 
conduct a critical study of nicotine’s occupancy of the β2*-nAChRs after use of an e-cig and 
relate to clinical data to better elucidate the molecular actions and addictive potential of e-cigs. 
Furthermore, we propose to conduct a novel examination of nicotine concentration in arterial 
plasma after use of an e-cig and a regular cigarette and relate these findings to the brain and 
clinical data. While the prior findings suggest differences in addictiveness between e-cigs and 
tobacco, the amount of nicotine delivered to the brain from an e-cig is not known and the 
relationship to tobacco smoking craving and withdrawal is not elucidated. Identifying e-cigs’ 
mechanisms of action in the brain, potential addictive nature, and the relationship between 
mechanism and behavior is thus the critical step in evaluating these products for potential 
regulation by the FDA.  
[18F]NCFHEB PET imaging. 
The most commonly used β2-nAChRs ligands to date, 5-IA for SPECT and 2-[18F]-F-A-85380 
for PET, have high affinity for this receptor but their utility for challenge studies in psychiatric 
populations is likely to be sub-optimal due to slow tracer kinetics. For example, 6 hrs are 
required for 5-IA to reach equilibrium in the brain, followed by 2 additional hours of scanning. 
For challenge studies, a 16+ hr scan day is required. Recent advancements have made possible 
the synthesis of NCFHEB for use in PET. NCFHEB activity uptake is similar to β2-nAChRs 
distribution in the brain, with higher uptake in the thalamus (Fig  3) and is superior to the 
previously discussed radioligands - it has similar affinity for the β2-nAChRs and it displays faster 
kinetics making it possible to conduct a single imaging study in 90mins (139).This radioligand is 
safe for use in humans in vivo (140), and the Yale PET Center has initiated its production. 
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 In the current proposal, we propose the first steps at assessing NCFHEB binding measure 
reproducibility in humans and sensitivity to drug-induced changes in endogenous ACh in order 
to establish the first paradigm to interrogate ACh function in vivo in humans. We predict that we 
can replicate and improve upon the findings obtained with 5-IA SPECT, which will enable future 
studies to include psychiatric populations through the use of a better tracer (NCFHEB) and a 
higher resolution method with superior quantification (PET). 
Preliminary data
β2-nAChR involvement in mood disorder. We conducted a preliminary evaluation of β2-
nAChR involvement in MDD in living human depressed and control subjects, as well as in 
postmortem tissue (141). Twenty–three nonsmokers with MDD (8 acutely depressed and 15 
remitted) and 23 age and sex-matched controls each participated in one 5-IA SPECT scan. 
Receptor availability was lowest in the acute MDD group (amygdala, p=0.02, hippocampus 
p=0.01, thalamus p=0.04 and mean cortex p=0.04). The remitted MDD group also showed lower 
receptor availability (p=0.01). However, in post-mortem data where endogenous ACh is not 
present, there appears to be no difference in receptor availability between depressed (n=15) and 
age- and sex-matched control (n=15) subjects. Therefore, it is hypothesized that high ACh levels 
associated with depressed mood competed with 5-IA binding at the β2-nAChRs, and we thus 
quantified lower β2-nAChR availability. Further experimentation is required to confirm these 
findings. 
We are presently conducting a similar evaluation of β2-nAChR involvement in patients with 
bipolar disorder (BD). Fifteen 15 depressed BD nonsmokers (5 unmedicated, 10 medicated (41.6 
± 13.1yrs)) and 15 controls (40.8 ± 13.1yrs) participated in one 5-IA SPECT. We found a 
significant effect of diagnosis (Hotelling's Trace F=4.7, p=0.002) with an average 29% lower β2-
nAChR availability in BD as compared to control subjects in frontal, parietal, anterior cingulate, 
temporal, and occipital cortices, thalamus, striatum, and cerebellum. There was a negative 
correlation between symptoms of depression and β2-nAChR (p=.04) only in unmedicated BD 
individuals. These baseline receptor availability studies provide important information on the 
neurochemistry of mood disorders, but limit our interpretation and future directions.  
Fig. 3. Kinetics in of [18F]NCFHEB, 
[18F]NCFHEB and 2-[18F]F-A85380 in left 
and right thalamus, caudate/putamen, and 
cerebellum of the porcine brain. Data 
represent standardized uptake values, SUV, 
(6SD). For the thalamus the time of peak 
uptake is given. * and denote significant
differences between the SUV for the 
particular time points in between. *P < 0.05 
vs. 2-[18F]F-A85380,P < 0.05 vs.
[18F]NCFHEB. 
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β2-nAChR involvement in schizophrenia. Smoking and nonsmoking subjects with 
schizophrenia (n=31) and age-, smoking- and sex-matched comparison subjects (n=31) 
participated in one [123I]5-IA-85380 single photon emission computed tomography (SPECT) 
scan to quantify β2*-nAChR availability. Psychiatric, cognitive, nicotine craving and mood 
assessments were obtained during active smoking as well as smoking abstinence. There were no 
differences in smoking characteristics between smokers with and without schizophrenia.  
Subjects with schizophrenia had lower β2*-nAChR availability relative to comparison group, and 
nonsmokers had lower β2*-nAChR availability relative to smokers. However, there was no 
smoking by diagnosis interaction. Relative to nonsmokers with schizophrenia, smokers with 
schizophrenia had higher β2*-nAChR availability in limited brain regions.  In smokers with 
schizophrenia, higher β2*-nAChR availability was associated with fewer negative symptoms of 
schizophrenia and better performance on tests of executive control.  Chronic exposure to 
antipsychotic drugs was not associated with changes in β2*-nAChR availability in schizophrenia. 
Conclusions: Although subjects with schizophrenia have lower β2*-nAChR availability as 
compared to comparison group, smokers with schizophrenia appear to upregulate in the cortical 
regions. Lower receptor availability in smokers with schizophrenia in the cortical regions is 
associated with a higher number of negative symptoms and worse performance on tests of 
executive function; suggesting smoking subjects with schizophrenia who upregulate to a lesser 
degree may be at risk for poorer outcomes.  
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5-IA SPECT imaging of β2-nAChR involvement in MCI and early AD. Our colleagues(11) 
conducted an evaluation of β2-nAChR involvement in early AD and mild cognitive impairment 
(MCI). They detected a global decrease in β2-nAChR as a function of group. No region-specific 
differences were detected. Due to poor 
resolution of SPECT, they were 
constrained to evaluation of larger brain 
regions and were not able to attain correct 
information in regions such as the 
hippocampus. The availability of 
NCFHEB would considerably increase our 
ability to measure regional change and to 
correlate with the clinical state.  
Furthermore, the availability of a method 
to quantify the acute response to AChE 
administration would provide a more 
functional evaluation of ACh function 
compared to the static measurement of 
baseline receptor availability.  Imaging 
ACh changes in the brain in humans. 
Competition between neurotransmitter and 
radioligands has provided a very useful 
method to assess synaptic changes in 
dopamine, but such an approach has not 
been applied to ACh. We examined 
whether 5-IA SPECT is sensitive to 
increases in extracellular levels of ACh in 
human subjects, when induced by the 
administration of physostigmine. Such 
sensitivity has been previously 
demonstrated in non-human primates by 
Fujita and colleagues, who detected a 
significant displacement of 5-IA in the 
thalamus after a physostigmine 
challenge(142). In our study, six healthy 
subjects participated in one 5-IA SPECT 
study.  We used a bolus plus constant 
infusion method to establish and maintain a 
state of binding equilibrium at the level of 
the receptors. After three 30-min baseline 
scans at 6h (early interval), physostigmine 
(1.5mg) was administered IV over 60 min, 
and nine additional 30-min scans were 
collected during the next 6h (late interval).  
We observed a significant reduction in BPF 
(=Bmax/KD; specific binding) after 
physostigmine (25 ± 15% reduction in 
cortical regions, 15 ± 11% thalamus, 16 ± 14% in striatum, and 35 ± 34% in cerebellum; p<.05; 
Figures 4 and 5). It has been previously established that, in the absence of challenge, no 
significant change in 5-IA BPF is observed during these time intervals (143).  These data 
Figure 5. Thalamic β2-nAChR availability (BPF, equal to Bmax/KD) before 
(shaded bars) and after (solid bars) physostigmine injection for each 
subject. Percent displacement of 5-IA for subjects 1-6 was 21%, 18%, 
9%, 11%, 41%, 2%, respectively
Figure 4.  Regional [123I]5-IA-85380 binding potential (BPF) in thalamus 
(circles), striatum (triangles) and prefrontal cortex (squares) measured 
during [123I]5-IA-85380 ([123I]5-IA) constant infusion in healthy 
volunteers. The first three scans were obtained starting at 6 hours post 
beginning of tracer infusion, when a state of equilibrium is achieved, and 
provided the baseline binding potential.  Following completion of the 
baseline scans, the AChE inhibitor physostigmine was administered i.v. 
(1.5 mg over 1 h, arrow). At the end of the physostigmine infusion, 
scanning was resumed up to 14 h.  BPF values measured after the 
physostigmine infusion were significantly reduced compared to the 
baseline values. As physostigmine has negligible affinity for β2-nAChRs, 
the decrease in β2-nAChR availability might be due to physostigmine 
induced elevation in ACh in the vicinity of β2-nAChR receptors and 
binding competition or allosteric modulation reducing the binding 
availability of the receptors for the radiotracer. 
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replicate in humans the previously published observation in baboon (142) and suggest that 
physostigmine-induced increases in ACh compete with 5-IA for binding to β2-nAChRs. 
Several limitations of these studies should be acknowledged. 1) While bolus plus constant 
infusion studies have demonstrated no systematic changes in VT between the early and late 
intervals in the absence of pharmacological challenges, suboptimal equilibrium conditions have 
been observed with this slow radiotracer during long scans, which is a source of noise in the 
evaluation of the physostigmine effect (143); 2) The limited resolution of SPECT and the low 
count rate toward the end of the infusion is a source of noise.  Both of these limitations will be 
addressed by moving to PET (better resolution, more accurate quantification), by using a fast 
radiotracer NCFHEB, and a single bolus injection; 3) The physostigmine effect on 5-IA VT 
might be due to factors other than increase in synaptic ACh.  Although physostigmine has no 
appreciable affinity for β2-nAChRs(144) (making a direct displacement by physostigmine 
unlikely), we cannot rule out that a brain-penetrant metabolite of physostigmine might directly 
interact with this receptor, or that some other nonspecific factors might be at play. Thus, it is 
imperative to verify that the effect can also be observed with an AChE inhibitor not chemically 
related to physostigmine.  
It should be clarified that other validations studies will be required before proposing that this 
paradigm provides a measure of changes in synaptic ACh. Studies in nonhuman primates will be 
needed to assess the correlation between changes in extracellular ACh measured with 
microdialysis and decrease in NCFHEB BP.  Our collaborators Drs. Laruelle(145) and 
Carson(146) have demonstrated that in nonhuman primates the magnitude of changes in 
extracellular DA measured with microdialysis following an amphetamine challenge are 
correlated with the magnitude of the decrease in striatal DA D2 receptors BP measured with PET 
or SPECT, respectively.  These experiments provided a critical validation of the molecular 
imaging measurement of DA tone (147).  In the current study, we propose to replicate and extend 
the human 5-IA SPECT physostigmine data with NCFHEB PET.  In future extensions of this 
work, we will propose combined PET/microdialysis studies in nonhuman primates. 
[11C]PHNO PET brain imaging.  [11C]PHNO has been developed as a dopamine D2/3 agonist 
ligand for PET brain imaging.  Its novelty lies in the measurement of the high affinity, 
functionally active D2 receptor, whereas most D2/3 ligands are antagonists and thus measure both 
the high and low affinity D2 receptors.  Dopamine is expected to bind preferentially to the high 
affinity state D2 receptors, which are thought to be the functionally important receptors.  
Typically, the most widely used ligand to measure endogenous dopamine release with PET has 
been [11C]raclopride, which is an antagonist, and thus measures both high and low affinity D2 
receptors. Differences in brain uptake between the two ligands have been examined with both 
ligands binding in areas with high D2/3 receptors (e.g., caudate and putamen).  In addition, 
[11C]PHNO showed uptake in the ventral striatum and globus pallidus, while [11C]raclopride had 
uptake in dorsal striatum.  Recently, a first report was published on the success of measuring d-
amphetamine-induced dopamine release using [11C]PHNO PET and the authors suggest this 
tracer may have some improvements in measuring endogenous dopamine release over the typical 
D2/3 ligands. They reported a 25% change in striatal BP after amphetamine administration. This 
is a significantly greater change than obtained with amphetamine-induced dopamine release 
measured with [11C]raclopride where average changes in striatal BP are between 10-15%. 
Amphetamine-induced DA release. Due to the difficulty of obtaining nicotine- or tobacco 
smoking-induced dopamine release, the majority of PET studies examining changes in synaptic 
DA levels have relied on amphetamine.  Amphetamine, as opposed to nicotine, acts directly at 
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the DA-ergic system and is a potent reuptake inhibitor of dopamine and a dopamine releaser.  
While nicotine results in 200% increases in dopamine release as measured with microdialysis, 
amphetamine results in up to 1000% increases in dopamine release.  Therefore, there is a much 
larger signal to detect, which has been reliably measured in many PET studies.  In the current 
study, we will use amphetamine-induced DA release to measure DA neurotransmission. 
3.   Research Plan: Summarize the study design and research procedures using non-technical 
language that can be readily understood by someone outside the discipline. Be sure to distinguish 
between standard of care vs. research procedures when applicable, and include any flowcharts of 
visits specifying their individual times and lengths. 
. 
3.1  Overall plan:
Aim 1:  To evaluate the test-retest reproducibility of NCFHEB binding paramenters in 10 
healthy subjects. 
CLOSED Aim 2: To determine if 2-nAChR availability is genetically predisposed in 10 
never smokers and 10 smokers at one week of abstinence with NCFHEB.
CLOSED Aim 3: To determine if the adaptive increase in 2-nAChR availability and 
change in 2-nAChR availability over the first month of abstinence in smokers is 
genetically predisposed (the same 10 smokers from Aim 2) with NCFHEB. 
Aim 4: To measure the sensitivity of NCFHEB binding to changes in endogenous ACh 
levels in up to 50 smoking and 50 nonsmoking subjects. 
CLOSED Aim 5: To measure the sensitivity of NCFHEB binding to changes in endogenous 
ACh levels in up to 10 smoking and 10 nonsmoking subjects with schizophrenia. 
Aim 6: To determine the efficacy of a bolus to infusion scan versus a bolus scan of 
NCFHEB with physostigmine challenge.
CLOSED Aim 7:  To determine amphetamine-induced DA release in tobacco smokers 
during acute withdrawal and in nonsmokers with [11C] PHNO.
CLOSED Aim 8. To determine the degree of occupancy of the β2*-nAChRs by nicotine 
after use of an e-cig compared to a tobacco cigarette using [18F]NCFHEB PET 
neuroimaging in 10 subjects.
3.2 Subject recruitment. 
We have an established program to recruit healthy controls. For the present study, subjects will 
be recruited through our program as well as flyers, public advertisement (newspaper, radio, 
internet postings), and word of mouth. Dr. Cyril D’Souza has an established program to recruit 
subjects with schizophrenia. He will recruit them through nearby treatment programs, CMHC, 
clinicians, word of mouth and advertisements.
3.3 Screening for eligibility. 
After completing the informed consent process, subjects will have a physical and neurological 
examination. The following lab tests will be performed at screening to exclude medical illnesses: 
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complete blood count (CBC) and differential, chemistries, kidney function tests (creatinine, 
BUN, urinalysis), liver function tests, and TSH.  A urine drug screen and a pregnancy test (for 
women) will be done at screening and before radiotracer administration. The psychiatric 
assessment will include a psychiatric history, a structured clinical interview (SCID), and 
assessment of subsyndromal depression with the Center for Epidemiological Studies Depression 
Scan – CES-D and subsyndromal anxiety with Speilberger State-Trait Inventory (STAI). 
Aim 1, Aim 2, Aim 4/5,  Aim 6, and Aim 7 (nonsmokers) 

smoked < 40 cigarettes in lifetime

urinary cotinine levels 0-30 ng/mL both at intake evaluation and on scan day
Aims 2, 3 Aim 4/5, Aim 6, Aim 7, and Aim 8  (smokers) 

have a Fagerstrom Test for Nicotine Dependence (FTND) rating of at least 3 

have been smoking cigarettes on a daily basis for at least 1 year 

carbon monoxide levels > 8 ppm during intake evaluation

plasma nicotine levels > 10 ng/mL  during intake evaluation

plasma cotinine levels of > 50  ng/mL during intake evaluation

wish or willing to quit smoking for up to 8 weeks (except for Aim 8 for which only 
nontreatment seeking smoking will be asked to participate who are willing to abstain 
from smoking for up to 1 week to do the study)
Aim 5 (subjects with schizophrenia)

have been on stable antipsychotic monotherapy treatment for at least 1 month

if smoker, willing to abstain from smoking for about 1 week

if on anxiolytics, willing to abstain on PET scan days
3.4 Assessments:
All participants will be screened initially using a telephone screen that will include questions to 
evaluate medical history, personal and familial psychiatric and smoking history.  
3.4a. General Intake Assessments 
1.
Demographic Questionnaire This questionnaire will obtain: (1) basic demographic 
information including age, gender, marital status, employment status, occupation, (2) 
alcohol/drug history, (3) family history of alcohol/drug use, depression, anxiety, and smoking 
history.
2.
Medical History This questionnaire will obtain a basic medical history (personal and 
family) including past or current conditions such as neurological, endocrine, cardiovascular, 
renal, liver, and thyroid pathology. Current body weight and current medications will also be 
assessed. 
3.
Medical Assessments will include a physical exam by a state licensed physician 
(overseen by Dr. David Matuskey), a complete blood count, blood urea nitrogen, creatinine, 
fasting blood sugar, electrolytes, liver function tests, thyroid function tests  (including T3, T4, 
T3RU, estimated free T4), thyroid stimulating hormone levels, urine toxicology, EKG, and 
urinalysis. Female subjects will have serum pregnancy tests. All EKGs are read by a state 
licensed cardiologist and all abnormal MRIs will be reviewed by a state licensed 
neuroradiologist.
4.
Illicit Drug/Pregnancy Screen A urine sample will be collected to determine current illicit 
drug use (for all potential subjects). In addition, urine samples will be collected for the intake 
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visit and on each MR and PET scan day to confirm that the subject is not pregnant.  Note: the 
urine pregnancy test will not be required prior to the MRI if the serum pregnancy test was done 
within 1 week prior to the MR imaging session.
5.
Structured Clinical Interview for DSM-IV Axis I Disorders The psychotic screening and 
depression sections of the Structured Clinical Interview for DSM-IV Axis I Disorders (SCID) 
will be used to determine whether subjects meet exclusion criteria for diagnosis of psychotic 
disorders and major depression (148).
3.4b.  Mood Measures 
We may of mood and anxiety at intake and also on the PET scan day including the following.
1. 
Center for Epidemiological Studies Depression Scale (CES-D) The CES-D (149) is a 20-
item self-report instrument, which has been extensively used in both clinical and nonclinical 
populations to measure the frequency and severity of depressive symptoms over the past week. 
The CES-D, which has been used to document the severity of depressive symptoms in adults 
(150) and has been shown to be a sensitive measure of negative affect in smokers (151), will be 
used in the proposed studies to exclude for presence of major depression, and to measure level of 
mild depressive symptoms commonly noted in tobacco smokers.
2. 
Anxiety: The State-Trait Anxiety Inventory (152) is a 40-item, self-report measure, 
comprised of two subscales. The State-Anxiety scale is 20 items and assesses transitory states 
characterized by feelings of tension, apprehension, and heightened autonomic reactivity. The 
Trait-Anxiety scale is 20 items and assesses stable individual differences in anxiety proneness.
3.
Impulsivity: Barratt Impulsiveness Scale (BIS; (153)) is a 30 item self-report instrument 
designed to assess the personality/behavioral construct of impulsiveness. 
 
3.4c. Smoking Measures
We may obtain smoking measures at intake and also on the PET scan day, which may include 
the following. 
1.Fagerstrom Test for Nicotine Dependence (FTND).(154) This will be used to measure the 
severity of nicotine dependence.  It is a 6-item scale with an internal consistency of .61 and its 
total score is closely related to biochemical measures of intensity of smoking.  A score of at least 
3 is necessary for inclusion in the study.
2. Smoking History. This questionnaire will assess basic smoking status and history such as 
number of years smoked, number and length of quit attempts, reasons for quitting, and second 
hand smoke exposure.  
3. Nicotine Withdrawal Checklist.(155) This measures the severity of eight withdrawal 
symptoms on 5-point Likert scales.  
4. Tiffany Questionnaire of Smoking Urges (QSU).(156) The QSU-brief is a 10-item 
questionnaire that evaluates the structure and function of smoking urges.  Subjects indicate on a 
likert-type scale how strongly they agree or disagree with each statement with a score of 1 
(strongly agree) to 7 (strongly disagree).  This characterizes ‘urges to smoke’ into a negative 
affect related to relief of withdrawal symptoms and positive affect related to expectancy of 
reinforcement.  
5.  GLMS – set of questionnaires in which subjects rate sensations related to smoking such as 
craving on a scale from “no sensation” to “strongest imaginable.”
6.  LHS – set of questionnaires in which subjects rate hedonic reaction to sensations from 
smoking on a scale from “most disliked sensation imaginable” to “most liked sensation 
imaginable.”  
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3.4d Cognitive Measures
We may obtain these measures at baseline and up to two times on the PET days.
1. Cogstate Battery (30 minutes) – This computerized test battery will assess memory and 
cognition.  The tasks may include:
a. International Shopping List Task – a computerized task to assess verbal learning and memory.
b.  Groton Maze Learning Task – a computerized task to assess executive function and spatial 
problem solving.
c.  Detection Task – a computerized task to assess psychomotor function and speed of 
processing.
d.  Identification Task – a computerized task to assess visual attention and vigilance.
e.  One Card Learning Task – a computerized task to assess visual learning and memory.
f.  One Back Task – a computerized task to assess attention and working memory
2. Probabilistic Reward Task: The PRT has been successfully used to assess reward 
responsiveness (166-168). In each trial, subjects choose which of two difficult-to-differentiate 
stimuli was presented. Stimuli consist of simple cartoon faces (diameter: 25 mm; eyes: 7 mm) 
presented in the center of the monitor. At the beginning of the trial, the face has no mouth. 
After a given delay, either a straight mouth of 11.5 mm (“short mouth”) or 13 mm (“long 
mouth”) is presented for 100 ms. Subjects are instructed to press an appropriate button to 
decide whether a long or small mouth had been presented.  Unbeknownst to subjects, correct 
identification of one stimulus (“rich stimulus”) is rewarded three times more frequently 
(“Correct! You won 20 cents”) than the other (“lean”) stimulus. In healthy controls, this 
reinforcement schedule leads to a response bias (i.e., a preference for the more frequently 
rewarded stimulus). The degree of response bias toward the more frequently reinforced 
alternative will be used for operationalizing sensitivity to reward.
3. Conditioned Hallucinations Task: participants’ thresholds for detection (75% likelihood of 
detection) of a tone embedded in white noise will be determined using the maximum-
likelihood-based QUEST62 method, with detection and non-detection signaled by button-
press.  Target auditory stimuli will be of the following easily-discernible63 frequencies: 500, 
1000, 1500, 2000, and 2500 Hz. All target auditory stimuli will be paired with a unique visual 
pattern (colors: white, blue, orange, red, green, matched for luminance and contrast) present 
for the duration of the auditory stimulus presentation.  All auditory and visual stimuli will be 
randomized across participants and across assessments.  Given participant responses, 
individualized psychometric functions will be estimated, corresponding to likelihood of 
detection as a function of target stimulus intensity, and from this, estimations of the intensities 
at which individuals will report detection of the target stimulus at rates of 50% and 25% will 
be determined. Over twelve blocks, participants will be presented first with stimuli played at 
their detection threshold, and then increasingly with sub-threshold and absent stimuli. This is 
meant to foster implicit learning and subsequent testing of the association between light and 
tone.  Participants will respond by button-press, and during the train/test period will hold down 
response buttons to indicate degree of confidence in their answer, guided by an on-screen 
visual-analog scale.  Conditioned Hallucination trials will be taken to be those during which 
participants report the presence of the target tone despite its absence.
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4. Cold Pressor Task
Subjects may be asked to participate in the Cold Pressor Task.  The cold pressor task (CPT) 
is a stress task used to measure pain sensitivity and pain tolerance.  This task will be used to 
determine alterations in pain thresholds as a result of nicotine use.  Participants will immerse 
their hand (up to the wrist) for up to 3 minutes in the experimental (ice-cold temperature 0-
4°C)) and control (room temperature (20°C)) conditions.  Physiological measure (heart rate, 
blood pressure and subject responses (stress, mood) will be collected 5 minutes before, 1 
minute into, and immediately after the CPT.
3.4.e Schizophrenia assessments
Scale for the Assessment of Negative Symptoms (SANS), the Positive and Negative Symptom 
Scale (PANSS), the Abnormal Involuntary Movements Scale (AIMS), the Montgomery-Asberg 
Depression Scale (MADRS) for depression to assess any mood or behavioral changes
3.5  Experimental Methods
MRI.
Within approximately 6 months of the PET study, anatomical MRIs will be acquired at the Yale 
University MRI Center. Subjects will be taken through a ferromagnetic metal detector before 
entering the scan room. The purpose of the MRI scan is to direct the region of interest placement 
on the lower resolution PET images.  The T1 weighted images will be acquired on a 3 Tesla 
Siemens Scanner.  There will also be an additional resting state scan with subjects in the scanner, 
eyes open, fixating on a cross.  Smokers who participate in Aims 4 and 7 may return for a second 
MRI with resting state at 4-8 weeks of smoking abstinence.
Physostigmine challenge (same paradigm as HIC# 09100005837) 
Physostigmine will be administered as follows.  Glycopyrrolate, a cholinergic antagonist that 
does not have central side-effects, will be administered prior to physostigmine challenge to block 
physostigmine peripheral side-effects (e.g. nausea). Subjects will receive 200 mcg /ml x 1 ml of 
glycopyrrolate through an IV.  Physostigmine administered i.v. has a short half life of 20 min 
with peak plasma levels 20-30 min post administration.  The same dose of physostigmine will be 
administered as in the preliminary data: 1.5 mg/hr for 1 hr. Vital signs, including systolic and 
diastolic blood pressure, heart rate and respiration rate, will be monitored before physostigmine 
and then at 10, 20, 30, 60 min after the beginning of the infusion and then hourly until the end of 
the study day. Subjects will be questioned before, during, and after physostigmine challenge 
about potential adverse reactions typical to this medication (nausea, upset stomach, etc., as in 
Risks section). [If there is a significant and persistent drop in subjects’ heart rate (>15% for at 
least 1 min), the IV physostigmine infusion will be stopped but the subject will continue to be 
monitored and will be discharged at the discretion of the study doctor. In emergency situation, 
PET center protocol will be followed accordingly (on file with HIC). 
Amphetamine challenge
Dextroamphetamine sulfate is the dextro isomer of the compound d,l-amphetamine sulfate, a
sympathomimetic amine of the amphetamine group. It is an FDA approved drug available for the
treatment of narcolepsy and attention deficit hyperactivity disorder (maximum approved total
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daily dose of 5-60 mg). After the first [11C]PHNO scan, dextro-amphetamine (0.5 mg/kg, PO) 
will be given in increments of 5 or 10 mg, as it is available in 5 mg and 10 mg tablets, and will 
be rounded down to approximate 0.5 mg/kg total dose without exceeding it. Total dextro-
amphetamine dose will not exceed 50 mg per scan. A second transmission and emission scan 
will be acquired approximately 2.5-3 hours post amphetamine identical to the methods outlined 
previously. The timing of the second [11C]PHNO administration and subsequent PET scanning 
(i.e., 2.5-3 hours) corresponds to the time of maximum plasma concentration of amphetamine as 
stated in the respective FDA-approved product labeling. EKG and frequent BP monitoring will 
occur throughout the study and until the vital signs are within normal limits. Supplemental 
oxygen will be provided via nasal cannula if necessary.  If the systolic BP reaches or exceeds 
200 mmHg for more than 5 minutes, the study doctor will take the appropriate clinical measures 
in order to lower the BP, which may include phentolamine administration (5 mg IV over 10 min) 
or other appropriate measures.
Following the post-amphetamine PET scan, subjects will be assessed (EKG and vital signs) by 
one of the research nurses.  Subjects will be discharged when vital signs are within normal limits 
and when behavioral changes (if any) are found to be not clinically significant by the MD 
attending to the subject at the PET Center. If subjects experience any adverse events, they will be 
treated until they become asymptomatic, prior to discharge.
Behavioral response to amphetamine will be measured by self-ratings with a simplified version 
of the Amphetamine Interview Rating Scale (van Kammen and Murphy, 1975).  Four items will 
be investigated: euphoria ("feel good"), alertness ("feel energetic"), restlessness ("feel like 
moving") and anxiety ("feel anxious").  Self ratings will be obtained by analog scales at the 
following times relative to the d-amphetamine administration: -5 minutes, 0, and hourly 
thereafter until end of the second scan. 
Cigarette challenges (same procedures as in protocol HIC 0804003655)
Subjects will participate in one or more PET scans during which they will participate in 4 
possible smoking challenges: Smoking an e-cig with no nicotine, an e-cig with a low dose of 
nicotine (6mg/mL-10mg/mL), an e-cig with a high dose of nicotine (24 mg/mL-36mg/mL), or 
smoking a regular cigarette. Subjects will participate in a different challenge for each of their 
PET scans.  For the e-cig challenges, subjects will not be aware of the amount of nicotine in the 
e-cig. Subjects will abstain from smoking for ~1 week prior to each challenge day. After each 
PET scan, subjects will return to smoking for at least 1 week. They will then need to abstain 
from smoking for ~1 week prior to participating in their next PET scan. Because of this off-and-
on smoking cessation, only non-treatment-seeking smokers will be asked to participate in this 
study. We have employed this procedure previously for protocol HIC 0804003655. 
Use of e-cig: Based on the findings of a recent study that showed use of e-cigs led to similar 
plasma nicotine levels as tobacco smoking within 5 minutes of use7, we plan to use an e-Go type 
e-cig battery (3.3 V, 1000 mAh) with 1.5 ohm dual-coil 510-style cartomizer and a 70/30 
propylene glycol vegetable glycerin e-liquid with nicotine concentrations of approximately 
0mg/mL, 6mg/mL-10mg/mL, and 24mg/mL-36mg/mL. Subjects will be instructed to puff on the 
e-cig once every 30 seconds in a manner as they normally would puff on a cigarette for 10 puffs 
total. A mouthpiece to measure puff topography may be used.
Use of regular cigarette: To standardize research methodology, Camels brand cigarettes will be 
used for all subjects. This brand is not prevalent with the smokers who participate in our studies 
and any subject who has used this brand prior will be excluded. Subjects will be instructed to 
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puff every 30 sec for a total of 10 puffs via the puff device (CreSS from Plowshare 
Technologies, USA), which provides measures of depth of puff inhalation and volume. 
Blood Samples: On PET scan days, blood samples will be collected prior to radiotracer 
administration and at multiple time points after e-cig and tobacco cigarette use to determine 
nicotine concentrations in the blood, which will be assayed using reversed-phase HPLC 49. 
PET
PET scans may be performed on the High Resolution Research Tomograph (HRRT, 2-3 mm 
resolution) or another similar camera.  Venous catheters will be used for i.v. administration of 
the radiotracers, venous blood sampling of AChE activity, for the administration of 
glycopyrrolate and physostigmine, and for venous blood sampling of physostigmine and 
amphetamine PKs.  A radial artery catheter will be inserted by an experienced physician before 
the NCFHEB PET scans to draw arterial blood samples for metabolite analysis and for 
determination of the fraction of plasma radioactivity unbound to protein. At the beginning of 
scan, the subject’s head will be immobilized and a transmission scan will be obtained for 
attenuation correction. PET scans will be acquired using bolus or bolus to infusion 
administration of up to 8 mCi of [18F]NCFHEB or up to 10 mCi of [11C]PHNO and subjects will 
be scanned for up to 4 hours. Dynamic images of radioactivity concentration are reconstructed 
with corrections for attenuation, normalization, random events, scatter, and deadtime. Subject 
motion is corrected automatically on an event-by-event basis with the Vicra motion tracking 
system.  Vital signs (blood pressure, pulse and respiration) are collected prior to and during each 
PET scan. Urine pregnancy test will be again administered on the PET scan day prior to the 
initiation of any imaging procedures. Smoking abstinence, when appropriate, will also be 
confirmed for smoking subjects prior to PET scanning. 
PET scanning will then proceed as following for each aim: 
Aim 1. Subjects will be asked to come to the PET center on two separate days to participate in 
one NCFHEB PET scan each time to assess test retest reproducibility of binding parameters 
measured with the radiotracer. 
CLOSED Aim 2.  Subjects will participate in one NCFHEB PET scan day.  
CLOSED Aim 3. Subjects from Aim 2 who are able to continue smoking abstinence will be 
asked to come back for another NCFHEB PET scan after about 4-8 weeks of smoking 
abstinence.
Aim 4.  Baseline NCFHEB PET imaging will be conducted followed by administration of 
physostigmine. Preferably, this will be done on the same day. However, at times there is not 
enough radiotracer or subject is not able to tolerate a longer scan day. Therefore, some subjects 
may complete the study over two separate days (preferably within 1 month apart based on the 
availability of PET scanning times and subject’s schedule). 
CLOSED Aim 5.  Baseline NCFHEB PET imaging will be conducted followed by 
administration of physostigmine. Preferably, this will be done on the same day. However, at 
times when there is not enough radiotracer or when a subject is not able to tolerate a longer scan 
day. Therefore, some subjects may complete the study over two separate days (preferably within 
1 month apart based on the availability of PET scanning times and subject’s schedule). 
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Aim 6.  Subjects from Aim 4 may be asked to come back for another PET scan with 
physostigmine administration.  Up to 8mCi of NCFHEB will be administered as a bolus plus 
constant infusion for the duration of the scan (up to 4 hours).  Once equilibrium is reached 
(approximately two hours into the scan) glycopyrrolate and physostigmine will be administered 
and subjects will be scanned for approximately two more hours. A transmission scan will be 
acquired.
CLOSED Aim 7. Subjects from Aim 4 will be asked to come back for two [11C]PHNO scans 
with amphetamine administration.  Up to 10 mCi of PHNO will be administered.  After the first 
scan, subjects will take amphetamine (0.5mg/kg) by mouth, and will be imaged again with 
PHNO.  If technical difficulties arise the second PET scan will be scheduled as soon as possible 
(within 5 weeks). In that case, subjects may be asked to re-take amphetamine on the day of the 
rescheduled PHNO scan, approximately 3 hrs prior to the scan.  Amphetamine dose will not 
exceed 50mg per scan.  Subjects will not be allowed to drive home.
CLOSED Aim 8. Subjects who previously participated in Aim 4 or 6, or new subjects may 
complete this portion of the protocol as long as their yearly radiation dose allows. For Aim 8, 
subjects will participate in one or more PET scans during which they will participate in 4 
possible smoking challenges: Smoking an e-cig with no nicotine, an e-cig with a low dose of 
nicotine (6mg/mL-10mg/mL), an e-cig with a high dose of nicotine (24 mg/mL-36mg/mL, or 
smoking a regular cigarette. Subjects will participate in a different challenge for each of their 
PET scans.. For each scan, up to 8mCi of NCFHEB will be administered as a bolus plus constant 
infusion for the duration of the scan (up to 4 hours).  Once equilibrium is reached (approximately 
two hours into the scan), subjects will use a cigarette or e-cigarette (order will be predetermined 
ahead of time and subjects will be told what they will be using for each scan.  However, if 
subjects are using the e-cig they will not be told which nicotine concentration they will receive). 
A transmission scan will be acquired.
Control subjects may participate in more than 1 aim. For example, nonsmoking subjects may 
complete Aim 1 and if they choose, participate in Aim 4. Thus, subjects may participate in up to 
4 NCFHEB PET scans and up to 2 PHNO PET scans for this protocol per year.  Subjects who 
complete 4 NCFHEB and 2 PHNO scans may return after 365 days to complete more scans 
under additional aims in this protocol.  
3.6 Image analysis.
NCFHEB time activity curves will be extracted for each subject in the following brain regions: 
amygdala, caudate, cerebellum gray matter, cerebellum white matter, corpus callosum, anterior 
cingulate, posterior cingulate, frontal cortex, hippocampus, occipital cortex, pallidum, parietal 
cortex, putamen, temporal cortex, and thalamus. Sabri and colleagues found that NCFHEB 
distribution volume  (VT) can be estimated in humans using the one-tissue compartmental 
model. (139) We will perform and compare several quantification methods (such as one and two 
tissue compartment model, Logan, and multilinear analysis) on the test/retest dataset to define 
the optimal method for VT derivation.  The selected method will be used for the specific aims 2 
studies.
β2-nAChRs are present in all brain regions, so that no practical region of reference can be used to 
define nonspecific binding. The definition of the nonspecific binding would require displacement 
studies, which are not proposed in this application.  In the thalamus, where the density of B2-
nAChR is the most abundant, the contribution of nonspecific binding to the total NCFHEB VT is 
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A P P R O V E D B Y T H E Y A L E U NI V E R SI T Y I R B 3/ 1 0/ 2 0 2 1
Pa ge 3 0 of 6 8
s mall i n r hes us m o n ke y ( 2 0 %) ( u n p u blis he d data), s o t hat si g nifica nt c ha n ges i n t hala mic 
N C F H E B V T ca n be reas o na bl y ascri be d t o c ha n ges i n s pecific bi n di n g.  I n ot her re gi o ns s uc h as 
c orte x, t he c o ntri b uti o n of t he n o ns pecific bi n di n g t o V T is lar ger (a b o ut 6 0 %, u n p u blis he d data), 
t h us gl o bal V T c ha n ges i n t hese re gi o ns s h o ul d be i nter prete d caref ull y.
F or P H N O sca ns, T he pri mar y o utc o me meas ure is t he bi n di n g p ote ntial ( B P N D ), w hic h i n t ur n is 
pr o p orti o nal t o t he a vaila ble rece pt or c o nce ntrati o n ( B a vail), gi ve n t hat t here is n o c ha n ge i n 
affi nit y ( K D ) a n d t hat n o n dis placea ble ( n o ns pecific a n d free) u pta ke d oes n ot differ bet wee n 
s u bjects a n d st u dies.  We will e xa mi ne t he re gi o ns- of-i nterest liste d bel o w wit h t he cere bell u m 
use d as a refere nce re gi o n beca use it is de v oi d of D 2/ 3 rece pt ors.
We o btai n a n M RI (as pre vi o usl y descri be d) t o use as a g ui de t o place o ur re gi o ns of i nterest.  
T his is beca use we ca n defi ne a nat o mical re gi o ns of i nterest o n t he M RI, w hic h pr o vi des 
str uct ural i nf or mati o n a n d t he n we ca n a p pl y t hese re gi o ns of i nterest t o t he P E T sca ns.  T he P E T 
sca ns al o ne are n ot s ufficie nt t o i de ntif y brai n re gi o ns. T he P E T i ma ge sets are ali g ne d a n d 
reslice d t o yiel d i ma ges i n t he sa me pla nes a n d s patial s yste m as t he M RI i ma ges usi n g A A L 
te m plate.  Pri mar y R OIs will be ca u date, p uta me n, gl o b us palli d us, s u bsta ntia ni gra, mi d brai n, 
t hala m us, hi p p oca m p us, ci n g ulate, fr o ntal a n d occi pital c ortices, as well as ra p he a n d a m y g dala.  
Ot her R OIs will be e xa mi ne d p ost h oc  t o assess ra di otracer bi n di n g.  Cere bell u m will be t he 
refere nce re gi o n. 
3. 7  St atistic al a n al ysis.  
3. 7 a P o wer A n alysis i n Never S m o kers:  Ass u mi n g Har d y- Wei n ber g e q uili bri u m, t he allele 
fre q ue nc y pre dicts t hat 3 0 will be h o m oz y g o us ( C C); 2 5 will be heter oz y g o us ( C T) a n d 5 will be 
h o m oz y g o us ( T T) f or t he A N K K 1 alleles, a n d 1 1 will be h o m oz y g o us ( A A) 2 9 will be 
heter oz y g o us ( A G) a n d 2 0 will be h o m oz y g o us ( G G) f or t he C H R N A 5 alleles. I n a o ne- wa y 
A N O V A a nal ysis, sa m ple sizes of 3 0, 2 5, a n d 5 are o btai ne d fr o m t he t hree ge n ot y pes of 
A N K K 1 w h ose mea ns are t o be c o m pare d. T he t otal sa m ple of 3 0 s u bjects ac hie ves 0. 9 6 p o wer 
t o detect differe nces a m o n g t he mea ns vers us t he alter nati ve of e q ual mea ns usi n g a n F test wit h 
a 0. 0 5 si g nifica nce le vel f or t hala m us. T he size of t he variati o n i n t he mea ns is re prese nte d b y 
t heir sta n dar d de viati o n w hic h is 1 7. 4 9. T he c o m m o n sta n dar d de viati o n wit hi n a gr o u p is 
ass u me d t o be 3 2. 3 0. T he ot her p o wers are 0. 9 5, 0. 8 7, a n d 0. 9 9 f or A N K K 1 f or t he brai n re gi o ns 
of striat u m, cere bell u m a n d c orte x, res pecti vel y. We ha ve e n o u g h p o wer t o detect t he effect wit h 
t he pr o p ose d sa m ple size. We ai m t o recr uit at least 1 0 s u bjects t hat are h o m oz y g o us f or T T; if 
we d o n ot ac hie ve o ur g oals f or t he rarer ge n ot y pe b y t he e n d of year 3; we will scree n s u bjects 
pri or t o i ma gi n g t he m i n year 4.
3. 7. b P o wer A n alysis i n S m o kers : T he gr o u p mea ns of 2
A P P R O V E D B Y T H E Y A L E U NI V E R SI T Y I R B 3/ 1 0/ 2 0 2 1
- n A C h R a vaila bilit y f or t he 
heter oz o g otes of h C V 1 6 1 7 8 9 3 3/rs 2 2 7 3 5 0 4 or h C V 1 5 9 5 3 8 2 0/rs 2 2 3 6 1 9 6 are hi g her i n s m o kers 
t ha n n o n-s m o kers i n t he brai n re gi o ns of striat u m, cere bell u m a n d mea n c orte x, b ut n ot t hala m us. 
We ca n ac hie ve 0. 8 1 p o wer t o detect a differe nce of 1 1. 7 i n striat u m bet wee n t he n ull h y p ot hesis 
t hat b ot h gr o u p mea ns are 9 7. 5 a n d t he alter nati ve h y p ot hesis t hat t he mea n of n o n-s m o ki n g 
gr o u p is 8 5. 8 wit h esti mate d gr o u p sta n dar d de viati o ns of 1 4. 9 a n d 1 0. 8 a n d wit h a si g nifica nce 
le vel (al p ha) of 0. 0 5 0 0 0 usi n g a o ne-si de d t w o-sa m ple t-test f or h C V 1 6 1 7 8 9 3 3/rs 2 2 7 3 5 0 4. T he 
ot her p o wers are 0. 9 7, 0. 9 5 f or h C V 1 6 1 7 8 9 3 3/rs 2 2 7 3 5 0 4 a n d f or cere bell u m a n d mea n c orte x, 
res pecti vel y. T he differe nces d o n ot prese nt i n t he h o m oz y g ote s u bjects. We ca n als o ac hie ve 
0. 9 8 t o 0. 9 9 f or h C V 1 5 9 5 3 8 2 0/rs 2 2 3 6 1 9 6 i n t he brai n re gi o ns of striat u m, cere bell u m a n d mea n 
c orte x.

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Genotype quality control – genotyping error and missing genotype We will check the genotypes 
for the SNPs via the method of departures from Hardy-Weinberg equilibrium (HWE) developed 
by (157), and excluded genotyping errors as a likely cause of the disequilibrium. Genotyping 
errors and missing genotype/allele calls will be carefully examined. Those with genotyping 
errors will be assigned to be missing.
3.8a Contingency Reinforcement for Smoking Cessation (healthy controls)
We plan to image smokers whom have been abstinent from smoking for approximately 1 week 
(with and without schizophrenia), 2 weeks (controls only) and 6 weeks (controls only). 
Contingency management techniques have been successfully used to reduce CO levels in non-
treatment seeking smokers by a number of investigators (158-160). 
Subjects will set a quit date and prior to the quit date they meet with the research staff who will 
provide them with brief advice on quitting smoking based on AHCPR guidelines (The Smoking 
Cessation and Clinical Practice Guideline Panel and Staff, 1996). They will be advised about the 
risks and benefits of quitting smoking and told that they will be monitored daily to ensure 
abstinence.  Subjects will be informed of payment schedules (see below) for CO levels indicating 
abstinence and also given information on how quit rates in the first week of smoking cessation 
predicted sustained abstinence.  We use CO levels < 11 ppm to define abstinence from cigarettes.  
During the first week of abstinence we will obtain CO levels from subjects up to twice daily. In 
addition, we will obtain urine samples once daily to measure cotinine levels (a nicotine metabolite). 
For each contingency management appointment, subjects will get $10 if their CO levels are less 
than 11ppm and urine cotinine levels are less than 100 ng/ml.  
3.8b Contingency Reinforcement for Smoking Cessation (subjects with schizophrenia)
Because it is more difficult for smokers with schizophrenia to quit smoking, they may choose to 
participate in this part of the study as an inpatient at CNRU. This was previously done in our 
HIC27532. Therefore, smokers with schizophrenia consenting to the study will be admitted to 
the CNRU, for approximately 1 week prior to PET scanning to ensure abstinence from 
smoking, this is Day 1 of the study. All antipsychotic medications will be continued. While we 
will not ask subjects to change antipsychotic medications, anticholinergics agents like cogentin, 
that interfere with radioligand binding will be suspended for a duration of the study with the 
approval of the subject’s non-research clinician.  Patients will not be responsible for paying the 
costs associated with this admission, whether the subject completes the study or discontinues 
early. Ratings of psychiatric symptoms and a Battery of Neuropsychological Assessments will 
be obtained by the research staff from screening until the SPECT scan. In addition, a research 
staff member will visit the subjects daily for behavioral counseling to assist with smoking 
cessation while inpatient. Expired air carbon monoxide (CO<10ppm) and daily dipstick-urinary 
cotinine levels will be monitored to confirm abstinence from smoking. However, in case CO is 
found >10ppm or positive urine cotinine is detected on the day of PET scan, which means that 
subject has smoked a cigarette, the scan may be cancelled and subject may be discharged from 
the study.
3.9 Genotyping
3.9.a Source of DNA DNA will be extracted from blood using standard “salting out” methods. 
Using these kits we expect to obtain from 10-30 µg of DNA from each sample, with a failure rate 
of about 3%. Each genotype will require between 1 and 15 ng of DNA, so this expected yield 
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will be sufficient for the genotyping described here. Using this method, we have obtained DNA 
of suitable quality for SNP, STR, and VNTR genotyping. DNA yields from blood are at least an 
order of magnitude higher than saliva.
3.9.b SNP Genotyping Methods The ABI TaqMan system (model 7900 detection device) will be 
used for SNP genotyping. This instrument uses probes with two dyes on opposite ends of a target 
sequence oligonucleotide to recognize SNP polymorphisms. One dye is a reporter dye, the other 
a quencher. When the probe is intact, the quencher suppresses fluorescence from the reporter; 
when the quencher and reporter are separated, the reporter emits a fluorescence signal. When the 
probe hybridizes exactly to its complement, the 5’ exonuclease activity of Taq polymerase 
cleaves the probe and allows the signal to be detected. The Taqman system uses two probes to 
detect a SNP, one complementary to each allele. An advantage of the Taqman system is that ABI 
offers detection reagents for many polymorphic systems pre-synthesized and tested, “on 
demand.” Detection reagents for other variants are ordered “on demand” through a user-friendly 
WWW interface.
3.9.c Quality Assurance for Genotyping In each 96-well DNA set, we include two blank lanes 
as contamination controls and a CEPH control of known genotype. In addition, at least 8% of 
genotypes will be repeated and if any discrepancies are noted, the entire cohort will be 
regenotyped.
3.10 Future directions
As discussed above, the successful demonstration of a significant reduction in NCFHEB VT by 
both AChE inhibitors in humans follows preclinical studies in nonhuman primates, aiming at 
further validation of this paradigm as a noninvasive and functional measure of pre-synaptic ACh 
function.  The availability of this paradigm at Yale and in other centers has the potential to 
greatly extend our understanding of ACh function in health and disease. 
         4.  Genetic Testing    N/A
A. Describe
i.
the types of future research to be conducted using the materials, specifying if 
immortalization of cell lines, whole exome or genome sequencing, genome-wide 
association studies, or animal studies are planned
- One 10 mL tube of blood will be collected for DNA for testing of  
polymorphisms in genes of interest to nicotine dependence including ANKK1 and 
CHRNA4.
ii. the plan for the collection of material or the conditions under which material will 
be received
-as above, a blood sample will be collected at intake to test polymorphisms of 
interest for nicotine dependence. 
iii. the types of information about the donor/individual contributors that will be 
entered into a database
- the genetic polymorphism results will be entered. The results of this testing will 
be confidential, will not be entered into the subject’s medical record, and will not 
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be made available to the subject.
iv. the methods to uphold confidentiality
- The results of genetic testing in locked file cabinets and by separating the personal 
identifying information of the subjects from the genetic information.
B. What are the conditions or procedures for sharing of materials and/or distributing for 
future research projects?
No sharing is planned.
C. Is widespread sharing of materials planned?
No.
D. When and under what conditions will materials be stripped of all identifiers?
It will not. 
E. Can donor-subjects withdraw their materials at any time, and/or withdraw the identifiers 
that connect them to their materials?  Yes.
i.
How will requests to withdraw materials be handled (e.g., material no longer 
identified: that is, anonymized) or material destroyed)?
Subjects will be informed that their material has been anonymized.
 
F. Describe the provisions for protection of participant privacy
 Risks associated with genetic testing will be minimized by keeping the results of genetic 
testing in locked file cabinets and by separating the personal identifying information of 
the subjects from the genetic information.
G. Describe the methods for the security of storage and sharing of materials 
The results of genetic testing in locked file cabinets and by separating the personal 
identifying information of the subjects from the genetic information.
5. Subject Population Provide a detailed description of the targeted population of human    
       subjects for this research project.
Healthy controls and healthy smokers will be recruited from the community through 
advertisements as approved by the Yale University Human Investigations Committee (HIC).  
Interested individuals contacting the clinic by phone in response to advertisements are told that 
the information they give over the phone is written down and discussed by the research team.  
They are advised that if they do not enroll in research with the clinic the information is 
destroyed, and that if they do, it becomes part of their research chart.  A phone screen is 
completed after they give verbal authorization.  If an individual appears to meet enrollment 
criteria and is interested in participating, a face-to-face interview is conducted.  A release of 
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information is obtained for review of any available historical and clinical data.  A written 
authorization form is also obtained from each subject, permitting the research team to use, create, 
or disclose the subject's PHI for research purposes.  The nature of the project, procedures, 
relative risks and benefits, and alternatives to participation in the project are discussed with the 
individual.  Following this discussion, the individual is given a copy of the consent form to 
review, and any questions are answered. We will seek written consent from all participants.
Subjects with Schizophrenia will be recruited who are clinically stable, and meet criteria for 
schizophrenia according to the DSM-IV, and who are between 18-60 years of age, on stable 
monotherapy antipsychotic treatment and who are able to give written informed consent will be 
included.
6. Subject Classifications: Will subjects who may require additional safeguards or other 
   considerations be enrolled in the study? If so, identify the population of subjects requiring 
   special safeguards and provide a justification for their involvement.
   a. Is this research proposal designed to enroll children who are wards of the state as potential 
   subjects? 
 Yes  
 No (If yes, see Instructions section VII #4 for further requirements) 
7. Inclusion/Exclusion Criteria: What are the criteria used to determine subject inclusion 
or    exclusion?
General inclusion criteria: 

men and women, aged 18-60 years

who are able to read and write 

who are able to give voluntary written informed consent

have no current uncontrolled  medical condition such as neurological, cardiovascular, 
endocrine, renal, liver, or thyroid pathology 

have no history of a neurological or psychiatric disorder (DSMIV Axis 1 and 2) other 
than schizophrenia in schizophrenia subgroup

have not regularly used any prescription, herbal or illegal psychotropic medications (e.g. 
antidepressants, antipsychotics, anxiolytics, ecstasy) in the past 6 months (controls) that 
in the PI’s determination puts the subject at increased risk or interferes with the study 
outcome.

Subjects with schizophrenia have not used any herbal or illegal substances in the past 6 
months (medication inclusion listed below in Aim 5)

drink less than <21 drinks/week for women and less than <35 drinks per week for men 

have not used marijuana in the past 30 days and have not met criteria for dependence  in 
the past 2 years

If female, not pregnant or breast feeding

If female of childbearing age, must use an acceptable method of birth control, as 
determined by the principal investigator

do not suffer from claustrophobia or any MR contradictions

willing to donate blood for genetic studies

willing to be followed up monthly after study participation via phone or email contact
General exclusion criteria:
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
Presence of acute or unstable medical or neurological illness. Subjects will be excluded 
from the study if they present with any history of serious medical or neurological illness 
or if they show signs of a major medical or neurological illness on examination or lab 
testing including history of seizures, head injury, brain tumor, heart, liver or kidney 
disease, eating disorder, diabetes.

Presence of an Axis I diagnosis other than nicotine dependence and schizophrenia (for 
schizophrenia subgroup) in the past 2 years

Regular use of any psychotropic drugs including anxiolytics and antidepressants and 
other over-the-counter medications and herbal products within the last year, and none 
within the last month for healthy controls per the PI’s discretion.  The PI will take a 
number of factors into consideration on a case-by-case basis including type of 
psychotropic drug used, frequency, and dose.

For subjects with Schizophrenia, use of SSRI’s (Paxil, Prozac Zoloft, Lexapro and 
Celexa) and use of tricyclic anti-depressants, except for a minimal dose used to treat 
anything other than depression, per the Investigator’s discretion.

Pregnancy/Breast feeding

Subjects with a pacemaker or other ferromagnetic material in body.

Subjects with a sitting pulse rate >100 bpm will be excluded

Subjects with hypertension defined as sitting systolic blood pressure of >160 mmHg 
and/or sitting diastolic blood pressure of >100 mmHg will be excluded.  Those 
individuals with hypertension that is well controlled by medication (e.g., within the above 
mentioned range) are not excluded

Specifically, we will exclude subjects who have any active clinically significant deviation 
from the normal range in their electrocardiogram (EKG).  However, subjects who have 
abnormalities in their EKG but the condition has been present for a while and the study 
cardiologist has evaluated and feels comfortable with the condition, would not be 
excluded on the basis of their cardiac condition.  Examples of conditions that may meet 
these criteria (e.g., condition has been present for a while) include but are not limited to 
T-wave abnormalities, atrial fibrillation, prolonged PR interval, and right bundle branch 
block. 

Subjects with an allergy to salicylates

Subjects with history of prior radiation exposure for research purposes within the past 
year such that participation in this study would place them over FDA limits for annual 
radiation exposure. This guideline is an effective dose of 5 rem received per year.

Subjects with current, past or anticipated exposure to radiation in the work place

Blood donation within eight weeks of the start of the study.

History of a bleeding disorder or are currently taking anticoagulants (such as Coumadin, 
Heparin, Pradaxa, Xarelto).
8. How will eligibility be determined, and by whom? 
Eligibility to participate will be determined by the PI of this study after completion of the 
medical and psychiatric evaluation of the potential participant.
9. Risks: Describe the reasonably foreseeable risks, including risks to subject privacy, 
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     discomforts, or inconveniences associated with subjects’ participation in the research. 
Risks from this study include 1) risks associated with radiation exposure, 2) risks associated with 
MRI, 3) intravenous lines and blood drawing, 4) arterial catheter, 5) nicotine withdrawal 
symptoms, 6) genetic testing, 7) IV physostigmine, 8) administration of physostigmine to 
subjects with schizophrenia 9) Risks from D-Amphetamine 10) Risk from E-Cigarettes 11) Risk 
from Cognitive Testing 
1. Risks Associated with Radiation 
The Yale University Radioactive Drug Research Committee (YURDRC)  will review the use of 
radiation in this research study, and no subjects will be enrolled until RDRC approval is 
obtained. This research study involves exposure to radiation from [18F]NCFHEB  and 
[11C]PHNO PET scanning. This radiation exposure is not necessary for medical care and is for 
research purposes only. 
For each individual [18F]NCFHEB PET scan, subjects will receive up to ≤ 8 mCi of 
[18F]NCFHEB, plus transmission scans. This is equal to an effective dose equivalent of 0.635 
rem per injection. For each individual [11C]PHNO PET scan, subjects will receive up to ≤ 10 
mCi of [11C]PHNO, plus transmission scans. This is equal to an effective dose equivalent of 0.27 
rem per injection.
The maximum amount of radiation per year an individual subject will receive in this study is 
from up to 4 injections of ≤ 8 mCi each of [18F]NCFHEB, up to 2 injections of ≤ 10mCi each of 
[11C]PHNO, plus transmission scans. This includes the fact that some subjects may have to come 
back on a separate day for baseline and physostigmine scans. Four [18F]NCFHEB injections and 
two [11C]PHNO injections is the maximum each subject may receive from participation in this 
study.
Although each organ will receive a different dose, the maximum amount of radiation exposure 
subjects will receive per year from this study is equal to an effective dose equivalent of 3.08 rem 
for a total of up to 32 mCi of [18F] NCFHEB in 4 injections of  ≤ 8mCi each and 20 mCi of [11C] 
PHNO in 2 injections of ≤ 10mCi each. This calculated value is used to relate the dose received 
by each organ to a single value.
The amount of radiation subjects will receive in this study is below the dose guidelines established 
by the FDA and monitored by the Yale University Radioactive Drug Research Committee for 
research subjects. This guideline sets an effective dose limit of 5 rem per year.
2.  MRI
MR carries a risk for subjects who have pacemakers, metal pieces, aneurysm clips, or other 
contraindications for MR.
Magnetic resonance (MR) is a technique that uses magnetism and radio waves, not x-rays, to 
take pictures and measure chemicals of various parts of the body.  The United States Food and 
Drug Administration (FDA) has set guidelines for magnet strength and exposure to radio waves, 
and we carefully observe those guidelines. 
 
Subjects will be watched closely throughout the MR study.  Some people may feel 
uncomfortable or anxious.  If this happens, the subject may ask to stop the study at any time and 
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we will take them out of the MR scanner.  On rare occasions, some people might feel dizzy, get 
an upset stomach, have a metallic taste or feel tingling sensations or muscle twitches.  These 
sensations usually go away quickly but we will ask subjects to tell the research staff if they have 
them.  
There are some risks with an MR study for certain people.  If subjects have a pacemaker or some 
metal objects inside their body, they may not be in this study because the strong magnets in the 
MR scanner might harm them.  Another risk is the possibility of metal objects being pulled into 
the magnet and hitting a subject.  To reduce this risk we require that all people involved with the 
study remove all metal from their clothing and all metal objects from their pockets.  We also ask 
all people involved with the study to walk through a detector designed to detect metal objects.  It 
is important to know that no metal can be brought into the magnet room at any time.  Also, once 
subjects are in the magnet, the door to the room will be closed so that no one from outside 
accidentally goes near the magnet.
We want subjects to read and answer very carefully the questions on the MR Safety 
Questionnaire related to their personal safety. We will be sure that subjects have read the MR 
Safety Questionnaire and tell us any information they think might be important.  
This MR study is for research purposes only and is not in any way a clinical examination.  The 
scans performed in this study are not designed to find abnormalities.  The primary investigator, 
the lab, the MR technologist, and the Magnetic Resonance Research Center are not qualified to 
interpret the MR scans and are not responsible for providing a diagnostic evaluation of the 
images.  If a worrisome finding is seen on a subject’s scan, a radiologist or another physician 
will be asked to review the relevant images.  Based on his or her recommendation (if any), the 
primary investigator or consulting physician will contact the subject, inform them of the finding, 
and recommend that they seek medical advice as a precautionary measure.  The decision for 
additional examination or treatment would lie solely with the subject and their physician.  The 
investigators, the consulting physician, the Magnetic Resonance Research Center, and Yale 
University are not responsible for any examination or treatment that a subject receives based on 
these findings.  The images collected in this study are not a clinical MR exam and for that 
reason, they will not be made available for diagnostic purposes.
3. Blood Drawing and IV line Insertion
Drawing blood and inserting an intravenous line (IV) into an arm vein are safe and standard 
medical procedures. Sometimes a bruise will occur at the puncture site and rarely a blood clot or 
infection will occur in the vein. Certain individuals may feel light-headed during venipuncture. 
The volume of blood collected during this study, may include screening laboratories, MRI and 
PET scans, will be approximately 32 tablespoons. This is not expected to have any serious 
negative effects on a study participant. 
4.  Arterial Catheter
On the NCFHEB PET scan days a radial arterial catheter will be inserted. Arterial sampling may 
be associated with mild-to-moderate pain, hematoma, inflammation, or bruising at the puncture 
site. If this occurs, these signs and symptoms will dissipate over time, usually 24 to 72 hours 
after the event. In rare instances blocking of the artery, poor healing, or infection at the catheter 
insertion site may occur. Certain individuals may feel light-headed during arterial catheter 
placement.
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5.  Nicotine Withdrawal
Smokers that quit smoking may experience symptoms of nicotine withdrawal such as craving 
cigarettes, mild anxiety, restlessness, irritability, difficulty concentrating, loss of energy, and 
excessive hunger. These are typical symptoms that people experience when they stop smoking 
and they can be uncomfortable but they are not life threatening. 
6.   Genetic Testing
Under some circumstances, it can be a risk for genetic information about the subject to be 
known. Variation in some genes is known to be directly related to risk for certain illnesses and 
drug dependence, or may in the future be shown to be related to illness. Since the results of these 
genetic tests may allow prediction of risk of illness in some cases, we will keep the results 
confidential (only scientists working on this research project will know the results). We will not 
make any of our laboratory results available to the subject, nor will we add them to their medical 
record. (If the participant wants to know their risk for genetic diseases, we will refer them to a 
genetic counselor.) The DNA samples will not have the subject’s name on them.
7.  IV Physostigmine
IV physostigmine is a cholinergic agent. The dose proposed in this protocol is less than that used 
for treatment. 
Side-effects from physostigmine include nausea, vomiting, diarrhea, anorexia, dizziness, 
headache, stomach pain, sweating and dyspepsia, pain, itching, burning, or swelling, or a lump 
under the skin where the shot is given. Rarely, more serious side-effects may include allergic 
reaction such as itching or hives, swelling in the face or hands, swelling or tingling in the mouth 
or throat, chest tightness, trouble breathing; increased watering in the mouth, severe nausea, or 
vomiting; increase in volume or frequency of urination, or severe diarrhea; seizures; slow 
heartbeat, dizziness, or fainting. In order to reduce these effects, we will administer 
glycopyrrolate, prior to physostigmine administration and control food intake. Furthermore, 
physostigmine will be administered in the presence of a doctor who will monitor side-effects, 
including reduced heart rate, and will be available throughout the study. In the case of serious 
side-effects, the study may be terminated and appropriate measures, which may include call to 
911, will be taken.
8. Administration of physostigmine and glycopyrrolate to subjects with schizophrenia
There are few studies with Physostigmine in schizophrenia patients (161, 162). It has been used 
primarily as a probe of cholinergic function to study schizophrenia, tardive dyskinesia and 
mania. Overall, it appears that physostigmine either improves or has no effects on symptoms in 
schizophrenia. In a more recent study with the related acetylcholine esterase inhibitor 
Rivastigmine, there appeared to be no benefit or worsening of either cognitive deficits or 
symptoms in schizophrenia patients (163, 164). In contrast to the studies with physostigmine and 
Rivastigmine, Rowntree et al. (165) observed that  Diisopropylfluorophosphonate, a centrally 
active cholinesterase inhibitor, produced worsened symptoms among a 30% of schizophrenia 
patients studies. The increase in symptoms in contrast to a lowering of symptoms has been 
interpreted as a rebound phenomenon.  In summary, the evidence available suggests that 
physostigmine will either have no effects or may improve symptoms in schizophrenia patients.  
If schizophrenia patients do experience any worsening of symptoms we will manage those 
symptoms as we usually do: with a range of supportive and pharmacological measures.
Since patients with schizophrenia may already be on an anticholinergics e.g., benztropine or 
triphenhexydl, for the treatment of antipsychotic-induced motor side effects making them more 
sensitive to the peripheral anticholinergic effects of glycopyrrolate, we will withhold standing 
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anticholinergics for 24 hrs prior to each physostigmine test day in those patients who are taking 
anticholinergics.  Withholding standing anticholinergics for 24 hours is likely to be well-
tolerated.
9.  Risks of oral d-amphetamine 
Risks of amphetamine administration include both medical and psychiatric risks.
The frequent somatic side effects of d-amphetamine administration are cardiovascular 
(hypertension, palpitations, tachycardia, bradycardia, orthostasis). General effects such as 
sweating, feeling warm or cold, nausea, diarrhea, muscle and abdominal cramping, have been 
reported frequently. Behavioral effects in this dose range are increased level of alertness, 
talkativeness, restlessness, agitation, mood changes (usually euphoria) and anxiety. In our 
experience, these effects are generally transient and well tolerated. This dose of amphetamine 
has not been reported to induce psychotic symptoms in non-schizophrenic subjects. Infrequently 
blurred vision, headaches and chest tightness, and changes in EKG have been reported. There is 
a rare risk of permanent neurological damage and death as a result of cardiac arrest or stroke.
Psychiatric or behavioral side effects:  General behavioral effects of amphetamine in this dose 
range are increased level of alertness, talkativeness, restlessness, agitation, mood changes 
(usually euphoria) and anxiety.  In our experience, these effects are generally transient and well 
tolerated.  This dose of amphetamine has not been reported to induce psychotic symptoms in non 
schizophrenic subjects and we confirm this observation. 
10.  Risks of E-Cigarette Use
The physiologic effects of e-cigarette use have been evaluated in human subjects in 9 studies.  
The following physiologic effects were associated with acute exposure to e-cigarette aerosols: 
-Mouth and throat irritation and dry cough at initial use (which decreased in severity with 
continued use), 
-No change in the following biomarkers: complete blood count (CBC), lung function, cardiac 
function, inflammatory markers, carbon monoxide (CO) level, plasma nicotine level, and heart 
rate
-Reduced fractional exhaled nitric oxide (FeNO) and increase in respiratory impedance and 
respiratory flow resistance 
Based on available data regarding the short term risks of using e-cigarettes, these products 
appear to be less harmful than tobacco cigarettes. They will be administered to tobacco smokers 
only, who are already dependent on tobacco cigarettes.  
10.
Risk from Cognitive Testing
The risk from Cognitive Testing is very minimal, there is a small risk for subjects who have a 
history of seizures due to flashing lights in the Conditioned Hallucination Task, however a 
history of seizures is an exclusion criteria. 
11. Minimizing Risks: Describe the manner in which the above-mentioned risks will be  
    minimized. 
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1.  The dose of radiation will be submitted for approval to the Yale University Radioactive 
Drug Research Committee (YURDRC).  All scans will be done in the presence of medical 
supervision and trained staff in an institution specifically designed to support imaging studies. In 
the event of serious medical complications, the PET scan facilities have immediate access to or 
consultation with specialized medical units at the Yale-New Haven Hospital. Preparation of 
radiopharmaceuticals and performance of PET scans will be by radiochemists, physicians, and 
technologists of the Department of Diagnostic Radiology, Yale University School of Medicine. 
These professionals are qualified by training and experience in the safe use and handling of 
radiopharmaceuticals. Subjects will be asked about their previous radiation exposure and those 
who have had research exposure within the past year will be excluded if their cumulative annual 
exposure (including the present study) exceeds FDA limits.   The information on the previous 
radiation exposure of study subjects will be notified to the study doctor. 
No PET studies will be performed on pregnant or potentially pregnant women, as confirmed by 
pregnancy testing during evaluation and on each scan day before initiation of any scan 
procedures. If subjects are breastfeeding they will not be able to participate in this research 
study. 
2.  The risks of bruising, clotting, and infection will be minimized by having venipuncture 
performed by trained and experienced personnel using aseptic technique. To avoid injury due to 
fainting, the venous catheters will be inserted when the subjects are recumbent. The blood draws 
during PET scanning sessions will be obtained from the already inserted catheter, to minimize 
discomfort.
3.  Risks of radial artery cannulation are minimized by having the procedure performed by an 
experienced physician. Pain is minimized by local anesthesia. Bleeding is prevented by local 
pressure applied for a minimum of 15 minutes after catheter removal. Subjects will have their 
hand and finger blood supply examined after arterial cannulation and again following catheter 
removal. Also, subjects will be asked to abstain from using aspirin or other NSAIDs for 7-10 
days prior to arterial line insertion and 7-10 days following arterial line removal. Subjects will be 
provided a 24 hour emergency physician telephone number to call if they encounter pain, 
discoloration, numbness, tingling, coolness, hematoma, inflammation, or any other unusual 
symptoms in the wrist or hand, or fever, chills or drainage from the vascular puncture sites, 
following the procedure. In addition, if an emergency arises at the time of cannulation or 
scanning, 911 will be called, and the subject will be sent to the Emergency Department for 
evaluation and treatment. Nurses will provide the subjects an instruction sheet documenting 
problems to watch for and procedures to follow should such problems occur. Infection is avoided 
by adequate cleansing of the skin prior to intravascular line insertion.
4.  All subjects will be screened for any metallic objects other MR contraindications that they 
may be holding or have implanted in their bodies using a questionnaire and all potential subjects 
with contraindications for MR will be excluded. This questionnaire will be repeated immediately 
before each measurement to insure that no metallic materials are brought into close proximity of 
the magnet, where they might be pulled toward the magnet.  For additional security, subjects will 
be taken through a ferromagnetic metal detector immediately before going to the scan room.
Effective screening to exclude subjects who would be placed at a greater risk. This includes 
medical history, physical examination, and screening studies (blood, urine and ECG) performed 
before starting studies.  A state-licensed physician or an Advanced Practice Registered Nurses 
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(APRN’s) will conduct all medical screenings.
In case of an adverse event, the PI, under the advice of the study collaborators Drs. Matuskey 
and Carson, will evaluate the adverse event and determine whether the adverse event affects the 
Risk/ Benefit ratio of the study and whether modifications to the protocol (at Risks to Subjects) 
or consent form (at Risks and Inconveniences) are required.
5.  For risks associated with oral d-amphetamine administration
Medical side effects: Subjects will be screened for absence of significant medical history and 
current medical conditions with a complete medical history, physical examination, routine blood 
tests, urine toxicology and EKG. Inclusion in the study will be limited to individuals who are 
between the ages of 18-55. Patients will be excluded if they have any h/o severe medical or 
neurological illness, any clinically significant brain abnormality, insulin dependent diabetes, a 
history of cardiovascular disease, or hypertension. Patients will be excluded if they have recently 
donated blood.  Administration of oral d-amphetamine will take place at the PET center by a 
research nurse, with a physician on site.  The research nurse will report vital signs to the physician 
prior to administering the amphetamine.
 If several of the subject’s blood pressure readings are recorded at >100 or <60 for diastolic BP 
or >160 or <90 for systolic BP while at rest, they will be evaluated by the MD.  The study may 
be cancelled at the discretion of the MD after evaluation.  Any automated blood pressure results 
that are abnormal will be repeated manually. The manual reading will be the official reading. 
Constant EKG and frequent BP monitoring will occur until the vital signs are within normal 
limits. If the systolic BP reaches or exceeds 200 mmHg for more than 5 minutes, an infusion of 
phentolamine (5 mg IV, over 10 min) or other appropriate measures may be initiated to control 
the blood pressure response.  The study physician will be notified if those parameters are reached 
and he/she will supervise the treatment.  
In case of chest pain, chest tightness or other symptoms suggestive of cardiac ischemia, the 
experiment may be cancelled and an EKG will be obtained to rule out angina (ST segment 
elevation or depression as compared to the baseline EKG). Appropriate treatment will be 
initiated.
Upon discharge, patients will be given the phone numbers of the study physicians and will not be 
allowed to drive home.  They will either arrange a ride or a taxi.
For Data and Safety Monitoring Plan templates, see 
http://www.yale.edu/hrpp/forms-templates/biomedical.html 
Data Safety Monitoring Plan:
1. Personnel responsible for the safety review and its frequency:
The principal investigator will be responsible for monitoring the data, assuring protocol 
compliance, and conducting the safety reviews at the specified frequency which must be 
conducted at a minimum of every 6 months (including when reapproval of the protocol is 
sought).  During the review process, the principal investigator (monitor) will evaluate whether 
the study should continue unchanged, require modification/amendment, or close to enrollment. 
Either the principal investigator, the IRB or Safety Monitoring Committee (DSMC) have the 
authority to stop or suspend the study or require modifications.
2. The risks associated with the current study are deemed moderate for the following 
reasons: 
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1. We do not view the risks associated with the __ radiotracers [18F]NCFHEB and 
[11C]PHNO_ as minimal.
2. We do not view the risks associated with the combined use of ________and_________ as 
minimal.
3. Given the now established safety and validity of the current _________ in our prior work, 
we do not view the proposed studies as high risk.
4. Given our experience with the combined co-administration_________, we do not view 
the proposed studies as high risk.
Although we have assessed the proposed study as one of moderate risk, the potential exists for 
anticipated and/or unanticipated adverse events, serious or otherwise, to occur since it is not 
possible to predict with certainty the absolute risk in any given individual or in advance of first-
hand experience with the proposed study methods. Therefore, we provide a plan for monitoring 
the data and safety of the proposed study as follows:
3. Attribution of Adverse Events:
Adverse events will be monitored for each subject participating in the study and attributed to the 
study procedures / design by the principal investigator Kelly Cosgrove, Ph.D. according to the 
following categories:
a.) Definite: Adverse event is clearly related to investigational procedures(s)/agent(s).
b.) Probable: Adverse event is likely related to investigational procedures(s)/agent(s).
c.) Possible: Adverse event may be related to investigational procedures(s)/agent(s).
d.) Unlikely: Adverse event is likely not to be related to the investigational 
procedures(s)/agent(s).
e.) Unrelated: Adverse event is clearly not related to investigational procedures(s)/agent(s).
4. Plan for Grading Adverse Events:
The following scale will be used in grading the severity of adverse events noted during the study:
1. Mild adverse event
2. Moderate adverse event
3. Severe 
5. Plan for Determining Seriousness of Adverse Events:
Serious Adverse Events:
In addition to grading the adverse event, the PI will determine whether the adverse event meets 
the criteria for a Serious Adverse Event (SAE).  An adverse event is considered serious if it:
1. is life-threatening OR
2. results in in-patient hospitalization or prolongation of existing hospitalization OR
3. results in persistent or significant disability or incapacity OR
4. results in a congenital anomaly or birth defect OR
5. results in death OR
6. based upon appropriate medical judgment, may jeopardize the subject’s health and may 
require medical or surgical intervention to prevent one of the other outcomes listed in 
this definition, OR
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7. adversely affects the risk/benefit ratio of the study
An adverse event may be graded as severe but still not meet the criteria for a Serious Adverse 
Event.  Similarly, an adverse event may be graded as moderate but still meet the criteria for an 
SAE.  It is important for the PI to consider the grade of the event as well as its “seriousness” 
when determining whether reporting to the HIIRB is necessary.
6. Plan for reporting serious AND unanticipated AND related adverse events, anticipated 
adverse events occurring at a greater frequency than expected, and other unanticipated 
problems involving risks to subjects or others to the IRB
The investigator will report the following types of adverse events to the IRB: a) serious AND 
unanticipated AND possibly, probably or definitely related events; b) anticipated adverse events 
occurring with a greater frequency than expected; and c) other unanticipated problems involving 
risks to subjects or others.
These adverse events or unanticipated problems involving risks to subjects or others will be 
reported to the IRB within 48 hours of it becoming known to the investigator, using the 
appropriate forms found on the website.
7. Plan for reporting adverse events to co-investigators on the study, as appropriate the 
protocol’s research monitor(s), e.g., industrial sponsor, Yale Cancer Center Data and 
Safety Monitoring Committee (DSMC), Protocol Review Committee (PRC), DSMBs, study 
sponsors, funding and regulatory agencies, and regulatory and decision-making bodies.
For the current study, the following individuals, funding, and/or regulatory
agencies will be notified:
□ All Co-Investigators listed on the protocol.
□ Safety Monitoring Committee (DSMC)
□ National Institutes of Health
The principal investigator Kelly Cosgrove, Ph.D. will conduct a review of all adverse events 
upon completion of every study subject. The principal investigator will evaluate the frequency 
and severity of the adverse events and determine if modifications to the protocol or consent form 
are required.
12.
Statistical Considerations: Describe the targeted number of subjects and the 
statistical 
     analyses that support the study design. 
Never Smokers: In a one-way ANOVA analysis, sample sizes of 30, 25, and 5 are obtained from 
the three genotypes of ANKK1 whose means are to be compared. The total sample of 30 subjects 
achieves 0.96 power to detect differences among the means versus the alternative of equal means 
using an F test with a 0.05 significance level for thalamus. The size of the variation in the means 
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is represented by their standard deviation which is 17.49. The common standard deviation within 
a group is assumed to be 32.30. The other powers are 0.95, 0.87, and 0.99 for ANKK1 for the 
brain regions of striatum, cerebellum and cortex, respectively. We have enough power to detect 
the effect with the proposed sample size. We aim to recruit at least 10 subjects that are 
homozygous for TT; if we do not achieve our goals for the rarer genotype by the end of year 3; 
we will screen subjects prior to imaging them in year 4.
Smokers: The group means of b2-nAChR availability for the heterozogotes of 
hCV16178933/rs2273504 or hCV15953820/rs2236196 are higher in smokers than non-smokers 
in the brain regions of striatum, cerebellum and mean cortex, but not thalamus. We can achieve 
0.81 power to detect a difference of 11.7 in striatum between the null hypothesis that both group 
means are 97.5 and the alternative hypothesis that the mean of non-smoking group is 85.8 with 
estimated group standard deviations of 14.9 and 10.8 and with a significance level (alpha) of 
0.05000 using a one-sided two-sample t-test for hCV16178933/rs2273504. The other powers are 
0.97, 0.95 for hCV16178933/rs2273504 and for cerebellum and mean cortex, respectively. The 
differences do not present in the homozygote subjects. We can also achieve 0.98 to 0.99 for 
hCV15953820/rs2236196 in the brain regions of striatum, cerebellum and mean cortex.
Genotype quality control – genotyping error and missing genotype We will check the genotypes 
for the SNPs via the method of departures from Hardy-Weinberg equilibrium (HWE) developed 
by (157), and excluded genotyping errors as a likely cause of the disequilibrium. Genotyping 
errors and missing genotype/allele calls will be carefully examined. Those with genotyping 
errors will be assigned to be missing.
SECTION III: RESEARCH INVOLVING DRUGS, BIOLOGICS, RADIOTRACERS, PLACEBOS AND 
DEVICES
           If this section (or one of its parts, A or B) is not applicable, state N/A and delete the rest of the 
section.
.  DRUGS, BIOLOGICS and RADIOTRACERS
13. Identification of Drug ,Biologic or Radiotracer: What is (are) the name(s) of the drug(s),
biologic(s) or radiotracer(s) being used? Identify whether FDA approval has been granted and for what 
indication(s). 
[18F]NCFHEB, IV, radioactivity dose of no more than 8 millicuries for one injection and PET.   
RDRC approval to use [18F]NCFHEB to image the nAChRs will be obtained from the Yale University 
RDRC for this protocol. 
[11C]PHNO, IV, radioactivity does of no more than 10 millicuries for one injection. [11C]PHNO has 
been used in humans and has been shown to be safe and well tolerated after its administration to healthy 
subjects or patients.  No serious adverse effects are expected from tracer doses, which is one thousand 
fold ≤ the pharmacological used during the therapeutic trials of PHNO.  The Yale RDRC has approved 
its use at the Yale University PET Center. 
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Physostigmine is a parasympathomimetic, specifically, a reversible cholinesterase inhibitor which 
effectively increases the concentration of acetylcholine at the sites of cholinergic transmission. Its 
mechanism is to prevent the hydrolysis of acetylcholine by acetylcholinesterase at the transmitted sites 
of acetylcholine. This inhibition enhances the effect of acetylcholine, making it useful for the 
treatment of cholinergic disorders and myasthenia gravis. More recently, physostigmine has been used 
to improve the memory of Alzheimer’s patients due to its potent anticholinesterase activity.
d-Amphetamine, dose 0.5 mg/kg, PO to healthy controls so no IND necessary per 21 CFR 312.2(b).
All protocols which utilize a drug, biologic or radiotracer not approved by, but regulated by, the FDA 
must provide the following information:  
   
a.   What is the Investigational New Drug (IND) number assigned by the FDA? 
b.  Who holds the IND? 
c. All protocols which utilize a radiotracer not approved by, but regulated by the FDA must 
provide the IND number: _______________
Alternatively, use of the investigational radiotracer may be under RDRC/RSC oversight: (check 
if appropriate)_____________
For all investigational radiotracers, attach a copy of the RDRC/RSC application ( for 
radioisotopes used in the PET Center, PET Center personnel may complete this step)
Go to http://rsc.med.yale.edu/login.asp?url=myApps.asp.  When you have logged in, complete 
the application and attach a copy to this submission.     
Alternatively, an exemption from IND filing requirements may be sought for a clinical
 investigation of a drug product that is lawfully marketed in the United States. If there is no IND and
 an exemption is being sought, review the following categories and complete the category that applies
 (and delete the inapplicable categories):
Exempt Category 1
The clinical investigation of a drug product that is lawfully marketed in the United States can be
exempt from IND regulations if all of the following are yes:
i.   The intention of the investigation is NOT to report to the FDA as a well-controlled study in support
     of a new indication for use or to be used to support any other significant change in the labeling for
     the drug.  
 Yes  
 No
ii. The drug that is undergoing investigation is lawfully marketed as a prescription drug product, and
      the intention of the investigation is NOT to support a significant change in the advertising for the
      product. 
 Yes  
 No
iii.  The investigation does NOT involve a route of administration or dosage level or use in populations
      or other factor that significantly increases the risks (or decreases the acceptability of the risks)   
      associated with the use of the drug product. 
 Yes  
 No
iv. The investigation will be conducted in compliance with the requirements for institutional (HIC)   
      review and with the requirements for informed consent of the FDA regulations (21 CFR Part 50  
and 21 CFR Part 56). 
 Yes  
 No
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v.   The investigation will be conducted in compliance with the requirements regarding promotion and 
charging for investigational drugs. 
 Yes  
 No
Exempt Category 2 (all items i, ii, and iii must be checked to grant a category 2 exemption)
 i. The clinical investigation is for an in vitro diagnostic biological product that involves one or
more of the following (check all that apply):
 Blood grouping serum
 Reagent red blood cells 
 Anti-human globulin
 ii. The diagnostic test is intended to be used in a diagnostic procedure that confirms the
diagnosis made by another, medically established, diagnostic product or procedure; and
 iii. The diagnostic test is shipped in compliance with 21 CFR §312.160.
Exempt Category 3
 The drug is intended solely for tests in vitro or in laboratory research animals if shipped in 
accordance with 21 CFR 312.60
Exempt Category 4
 A clinical investigation involving use of a placebo if the investigation does not otherwise 
require submission of an IND.
1. Background Information: Provide a description of previous human use, known risks, and 
data addressing dosage(s), interval(s), route(s) of administration, and any other factors that 
might influence risks. If this is the first time this drug is being administered to humans, 
include relevant data on animal models. 
[18F]NCFHEB has been administered I.V. to human subjects previously by collaborator Osama 
Sabri, Ph. D., at the Department of Radiopharmacy, Institute of Interdisciplinary Isotope 
Research, Leipzig, Germany. 
Whole body dosimetry of [18F]-(-)-NCFHEB was performed in volunteers.  The subjects were 
sequentially imaged up to 7h post i.v. injection of 353.7±10.2 MBq of[18F]- (-)-NCFHEB on a 
SIEMENS Biograph16 PET/CT-system with 9 bed positions (BP) per frame,1.5-6min/BP, CT-
attenuation correction and iterative reconstruction. All relevant organs were defined by volumes 
of interest. Exponential curves were fitted to the time-activity-data. The ODs were calculated 
using the adult male model with OLINDA. The ED was calculated using tissue weighing factors 
as published in the ICRP 103/2007. The highest OD was received by the urinary bladder 
(80.2±37.8), followed by liver (44.7±5.4) and kidneys (38.6±5.1). 
Preclinical Characterization of [11C]PHNO
The radiosynthesis of [11C]-(+)-PHNO ([11C]PHNO) has been described in detail. The specificity 
of [11C]PHNO binding was demonstrated in preclinical studies that included ex vivo and in vivo 
studies in rodents and cats. Ex vivo biodistribution studies in rat brain demonstrated that 
[11C]PHNO crossed the blood-brain barrier readily and had an appropriate regional brain 
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distribution for a radiotracer that maps dopamine D2 receptors.  Recent in vivo imaging studies 
showed that [11C]PHNO is a D2/D3 agonist, but D3 preferring, and it displayed unusually high 
binding in globus pallidus (GP), which is not observed with [11C]raclopride (a D2-like antagonist 
with the highest binding in dorsal striatum).  Most recently, studies showed that 95% of 
[11C]PHNO binding in substantia nigra (SN) is due to the binding to D3, not to D2. Furthermore, 
[11C]PHNO showed marked and appropriate sensitivity to both increases and decreases in levels 
of endogenous dopamine.  
PET Imaging with [11C]PHNO in Monkeys at Yale University PET Center 
We have successfully prepared [11C]PHNO with high radiochemical purity and high specific 
activity at the Yale University PET Center radiochemistry laboratory.  One test scan and two 
control-preblock studies were performed in three different rhesus monkeys using [11C]PHNO 
(Table 1). For the control-preblock studies, injections of [11C]PHNO were delivered as a 2-
minute bolus followed by 118 minutes of constant infusion. In the preblock studies, 2 mg/kg of 
SB-277011, a DA D3 receptor antagonist, was infused for a total of 3 hours, starting 1 hour 
before the second scan. Images for the baseline and preblock studies are shown in Fig 1. Changes 
in radiotracer concentration following the preblock were measured in cerebellum, basal ganglia, 
and thalamus brain regions.  Equilibrium was reached by about 60 minutes post-injection and 
BPND was calculated by the equilibrium ratio of regions to cerebellum averaged from 60 to 90 
minutes (Table 1). These results indicate that [11C]PHNO is a DA D3 receptor-preferring 
radiotracer. Respiratory and cardiovascular functions were closely monitored throughout the 
PET scans, and no noticeable effects were observed on the respiratory or cardiovascular 
functions of the animals,
Table 1. Binding potential results from control-preblock experiments of  [11C]PHNO 
Study
Region
BPcontrol*
BPpreblock*
% 
Blockade
[11C]PHNO 
#1
Caudate
Putamen
Pallidum
Thalamus
4.03 
4.35 
3.68 
1.02 
3.18 
4.43 
2.32 
0.39 
20%
-2%
35%
65%
[11C]PHNO 
#2
Caudate
Putamen
Pallidum
Thalamus
3.91 
3.92 
4.12 
0.79 
2.72 
3.35 
1.79 
0.26 
30%
14%
56%
68%
*BP values are calculated from 60 to 90 minutes post-injection
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A. 
 
B.
 
 
C.   
   
Figure 1.  [11C]PHNO uptake summed from 60 to 90 minutes post-injection (A) at baseline and 
(B) after preblock with the D3 receptor antagonist SB277011;(C) Corresponding MRI slices are 
displayed for anatomical reference.  The left panels show slices at the level of the striatum and 
globus pallidus in coronal orientation, while the right panels show the striatum and globus 
pallidus in transverse orientation.  Brain regions with the highest concentrations of [11C]PHNO 
are shown in red, with progressively lower concentrations displayed in yellow, green, and blue, 
respectively. 
2.2.Dosimetry Studies of [11C]PHNO in Monkeys at Yale PET Center
Radiation dosimetry for [11C]PHNO has been estimated (using the standard MIRD absorption 
fractions program MIRDOSE 3.1) based on PET scans with a tracer dose of [11C]PHNO on four 
Rhesus monkeys (2 females and 2 males) to obtain organ concentration and residence time. The 
percentage of the injected dose per cm3 was calculated from ROIs drawn on the PET images, and 
average organ weights for female and male Rhesus monkeys were used to derive the amount of 
activity per organ as a function of time.  The scans were carried out to 120 minutes and the 
residence times calculated from a trapezoidal approximation of the integral.
Based on these numbers we estimated that the maximum allowable injection dose for 
[11C]PHNO is 55 mCi per single injection. We tabulated the dosimetry below (all entries refer to 
scan data from monkeys).
 
Absorbed Dose (rad/mCi)
 
 
RDRC MAX DOSE
 
mean
SD
 
max rad/study
 
Brain
1.84E-02
2.08E-03
 
5
2.72E+02
 
Breasts
6.64E-03
4.08E-04
 
5
7.53E+02
 
Gallbladder Wall
4.63E-02
2.66E-02
 
5
1.08E+02
 
LLI Wall
9.72E-03
4.12E-04
 
5
5.14E+02
 
Small Intestine
2.95E-02
4.77E-03
 
5
1.70E+02
 
Stomach Wall
1.32E-02
2.45E-03
 
5
3.80E+02
 
ULI Wall
1.20E-02
4.11E-04
 
5
4.18E+02
 
Heart Wall
1.43E-02
1.26E-03
 
5
3.50E+02
 
Kidneys
9.07E-02
2.83E-02
 
5
5.51E+01
 
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Liver
5.56E-02
9.17E-03
 
5
9.00E+01
 
Lungs
2.86E-02
7.17E-03
 
5
1.75E+02
 
Muscle
7.79E-03
4.49E-04
 
5
6.42E+02
 
Ovaries
1.06E-02
4.24E-04
 
5
4.73E+02
 
Pancreas
3.21E-02
7.45E-03
 
3
9.34E+01
 
Red Marrow
1.10E-02
5.45E-04
 
5
4.57E+02
 
Osteogenic Cells
1.23E-02
6.85E-04
 
3
2.43E+02
 
Skin
5.89E-03
5.34E-04
 
5
8.49E+02
 
Spleen
1.71E-02
3.81E-03
 
5
2.92E+02
 
Testes
7.03E-03
6.25E-04
 
5
7.11E+02
 
Thymus
7.77E-03
4.70E-04
 
3
3.86E+02
 
Thyroid
6.90E-03
7.79E-04
 
5
7.25E+02
 
Urinary Bladder Wall
7.01E-02
1.88E-02
 
5
7.13E+01
 
Uterus
1.37E-02
2.32E-03
 
5
3.65E+02
 
Total Body
1.04E-02
0.00E+00
 
5
4.81E+02
 
 
 
 
 
 
 
 
Effective Dose Equivalent
2.69E-02
4.72E-03
 
 
 
 
Effective Dose (rem/mCi)
1.92E-02
2.57E-03
 
Critical organ
Max Dose (mCi)
ED with Stom Adjust
2.08E-02
2.81E-03
 
Kidneys
5.51E+01
 
 
 
 
 
 
 
 
The maximum allowable dose for a single injection is 3000 mR to the whole body, active blood-
forming organs, lens of the eye and gonads. The dose to any other organ cannot exceed 5000 
mR.
The maximum allowable dose for one year is 5000 mR to the whole body, active blood-forming 
organs, lens of the eye and gonads. The dose to any other organ cannot exceed 15000 mR.
Critical Organ(s): kidneys
For comparison, the average person in the United States receives a radiation exposure of 0.3 rem 
(or 300 mrem) per year from natural background sources, such as from the sun, outer space, and 
from radioactive materials that are found naturally in the earth’s air and soil. The dose that a 
subject will receive from participation in this research study would be less than that obtained in 
one year from natural sources.
2.3. Clinical Characterization and Safety profile of [11C]PHNO
[11C]PHNO has been used in several PET centers.  It was initially developed at the PET Center, 
University of Toronto, and the safety of its use in humans has been demonstrated. 
It states that oral or intravenous administration of therapeutic doses of PHNO (0.2 up to 60 mg) 
produces side effects commonly seen with other dopamine agonists.  In addition to orthostasis, 
the most frequently reported side effects were dystonia and nausea. Drop-outs in these studies 
were most commonly due to somnolence or orthostatic reactions.  None of the studies reported 
significant drug- related hematological, biochemical, or electrocardiographic changes.  In the 
first 18 healthy subjects studied with tracer doses of [11C]PHNO, there were no changes in vital 
signs, EKG, or biochemical markers. 
The most common adverse event reported has been self-limiting transient nausea shortly after 
injection (well before the peak brain uptake). To date, they have carried out 331 PET scans with 
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[11C]PHNO (≤ 20 mCi for each injection) in healthy controls and in patients with schizophrenia 
at the Toronto CAMH PET Centre. Of those, 12% experienced nausea, 1% emesis and 3% 
reported other effects such as dizziness, headache or a warm sensation.  The occurrence of these 
side effects had no relation with the injected mass (2.3 ± 0.4 μg/kg; range 1.0–5.6  μg/kg), 
subject's sex, body mass or age. 
 
No serious adverse effects are expected from tracer doses of [11C]PHNO, with a recommended 
maximum mass less 0.5 nmoles/kg body weight, which is one thousand fold less than the 
pharmacological dose used during the therapeutic trials of PHNO. 
[11C]PHNO has been used in humans at the Yale University PET center in several studies 
including HIC 0910005822.
Previous studies involving IV physostigmine in humans 
At least 2 human studies employing same paradigm as described above have been conducted in 
human subjects without adverse side effects from the low dose of IV physostigmine 
administration. 
1. Koeppe et al (1999) conducted a PET brain imaging study in 23 healthy volunteers 
to evaluate a new radiotracer. A subgroup of these individuals received 
physostigmine at a dose of 1.5mg/hr for one hour. They were pre-treated with 
glycopyrrolate. No unexpected adverse effects were reported. One subject was 
reported to feel nauseated but was able to complete the study.
2. Esterlis et al (2013) conducted a SPECT imaging study in 7 healthy volunteers to 
evaluate whether [123I]5IA radioligand is susceptible to increases in endogenous 
ACh. Subjects underwent the same physostigmine paradigm as described in #1 
above and in the current protocol. One subject vomited as consequence of 
physostigmine administration. No serious or unexpected side effects were noted.
d-Amphetamine:
Amphetamine is administered to measure changes in [11C]PHNO binding due to dopamine release. 
This dose was chosen because it is expected to produce a quantifiable displacement of the 
radiotracer. The risks are outlined in Section VI.  Oral and IV d-amphetamine administration to 
healthy humans and individuals with psychiatric disorders has been safely used in many PET 
imaging studies. We will use the oral route due to the greater safety and ease of administration.
3. Source: a) Identify the source of the drug or biologic to be used. 
[18F]NCFHEB and [11C]PHNO will be synthesized at the Yale University PET Center 
radiochemistry Laboratory.  Physostigmine, glycopyrrolate, and d-amphetamine will be provided 
by the YNHH pharmacy.
b) Is the drug provided free of charge to subjects? 
 Yes  
 No
    If yes, by whom? PET center
APPROVED BY THE YALE UNIVERSITY IRB 3/10/2021

A P P R O V E D B Y T H E Y A L E U NI V E R SI T Y I R B 3/ 1 0/ 2 0 2 1
Pa ge 5 1 of 6 8
4. St or a ge,  Pre p ar ati o n a n d Use:  Descri be t he met h o d of st ora ge, pre parati o n, sta bilit y i nf or mati o n, 
a n d f or pare nteral pr o d ucts, met h o d of sterilizati o n a n d met h o d of testi n g sterilit y a n d 
p yr o ge nicit y.
[1 8F] N C F H E B
Pre parati o n of [ 1 8F] -(-)-N C F H E B will be carrie d o ut i n acc or da nce wit h pr oce d ures a n d q ualit y 
s pecificati o ns c o ntai ne d i n l ocal Dr u g Master File. Briefl y, [ 1 8F] -(-)-N C F H E B is s y nt hesize d b y 
reacti n g [1 8F]fl u ori de wit h t he ra di ola beli n g prec urs or i n a n a p pr o priate reacti o n s ol ve nt f oll o we d 
b y de pr otecti o n of t he B oc pr otecti n g gr o u p.  P urificati o n of t he ra di ola bele d pr o d uct is perf or me d 
b y se mi- pre parati ve H P L C, f oll o we d b y re m o val of t he H P L C el ue nt via s oli d- p hase e xtracti o n, 
f or m ulati o n i n sterile sali ne c o ntai ni n g et ha n ol a n d sterile filtrati o n t o yiel d t he rea d y-f or-i njecti o n 
ra di otracer [1 8F] -(-)-N C F H E B i n sali ne s ol uti o n c o ntai ni n g < 1 0 % et ha n ol.
T he ra di oacti ve pr o d uct is st ore d at r o o m te m perat ure a n d is sta ble f or at least 8 h o urs after 
pre parati o n. P yr o ge n test is perf or me d f or eac h batc h of pr o d uct. Sterilit y is ac hie ve d b y passi n g 
t he pr o d uct t hr o u g h a me m bra ne filter f or ter mi nal sterilizati o n as t he last ste p i n t he pre parati o n 
pr ocess, a n d c o nfir me d b y sterilit y test perf or me d after a d mi nistrati o n.
[1 1C] P H N O  
T he starti n g material f or t he pr o d ucti o n of [1 1C] P H N O, des- N- pr o p yl- P H N O, is p urc hase d fr o m 
A B X A d va nce d Bi oc he micals. We ha ve s uccessf ull y pre pare d [ 1 1 C] P H N O wit h hi g h 
ra di oc he mical p urit y a n d hi g h s pecific acti vit y at t he Yale U ni versit y P E T Ce nter ra di oc he mistr y 
la b orat or y.  We ha ve als o c o n d ucte d se veral P E T st u dies i n n o n- h u ma n pri mates (see t he 
preli mi nar y data secti o n).  Ra di os y nt hesis of [ 1 1 C] P H N O was perf or me d at t he Yale U ni versit y 
P E T Ce nter ra di oc he mistr y la b usi n g a f o ur-ste p s y nt hetic se q ue nce starti n g fr o m [ 1 1 C] C O 2.  A 
fe w mi n utes bef ore t he e n d of b o m bar d me nt ( E O B), Gri g nar d s ol uti o n ( pre pare d b y diss ol vi n g 1 
M Et M g Br wit h a n a p pr o priate a m o u nt of a n h y dr o us et h yl et her) was i ntr o d uce d i nt o a vessel 
pre vi o usl y fl us he d wit h ar g o n.  P urifie d [ 1 1 C] C O 2 was s we pt i nt o t he Gri g nar d vessel u n der strea m 
of ar g o n u ntil acti vit y pea ke d i n t he vessel.  T he n p ht hal o yl dic hl ori de was i ntr o d uce d a n d t he 
mi xt ure was all o we d t o sta n d f or ~ 2 0 sec o n ds. 2, 6- di- tert- b ut yl p yri di ne was t he n a d de d a n d t he 
res ulti n g s ol uti o n was heate d u n der a strea m of ar g o n t o distill 1 1 C- pr o pi o n yl c hl ori de i nt o ice 
c ol d s ol uti o n of t he prec urs or a n d N, N - diis o pr o p ylet h yla mi ne u ntil acti vit y pea ke d.  T he res ulti n g 
s ol uti o n was heate d at ~ 8 0° C f or a fe w mi n utes. After c o oli n g i n ice bat h,  L A H s ol uti o n i n T H F 
was a d de d t o t he reacti o n vial a n d t he mi xt ure was heate d f or ~ 5 mi n utes at ~ 1 0 0° C u n der a 
strea m of ar g o n t o re m o ve T H F. After c o oli n g i n ice bat h, dil ute d H Cl(a q) was i ntr o d uce d a n d t he 
s ol uti o n was heate d f or ~ 4 mi n utes at ~ 1 0 0° C. T he res ulti n g s ol uti o n was c o ole d, dil ute d wit h 
dei o nize d water, t he n p urifie d b y se mi pre parati ve H P L C. T he pr o d uct fracti o n was c ollecte d a n d 
dil ute d wit h ~ 5 0 m L dei o nize d water, t he n passe d t hr o u g h a C 1 8 Se p- Pa k ( Waters). T he Se p- Pa k 
was was he d wit h 1 0 m L of dil ute d H Cl(a q) a n d el ute d wit h 1 m L et ha n ol f oll o we d b y 3 m L of 
sali ne. T he c o m bi ne d et ha n ol a n d sali ne s ol uti o n was t he n passe d t hr o u g h a sterile 0. 2 m
A P P R O V E D B Y T H E Y A L E U NI V E R SI T Y I R B 3/ 1 0/ 2 0 2 1
 filter 
a n d c ollecte d i nt o a ve nte d sterile vial c o ntai ni n g 7 m L sali ne. P urit y a n d s pecific acti vit y were 
deter mi ne d b y a nal ytical H P L C.
 
T he ra di oc he mical yiel d was 1 9. 0 ± 4. 2 m Ci.  T he a vera ge s y nt hesis ti me was 6 6 mi n.  T he 
s pecific acti vit y was 7 0 5 ± 2 0 5 Ci/ m m ol at e n d of s y nt hesis ( E O S).  T he fi nal f or m ulate d pr o d uct 
was > 9 9 % p ure ra di oc he micall y. T he ra di oli ga n d is pr o d uce d acc or di n g t o t he l ocal Sta n dar d 
Ma n ufact uri n g Pr oce d ure a n d t o l ocal q ualit y c o ntr ol pr oce d ures i n effect at t he Yale U ni versit y 
P E T Ce nter. T his tracer has bee n vali date d at Yale U ni versit y P E T Ce nter Ra di oc he mistr y La b a n d 
has bee n a p pr o ve d f or h u ma n st u dies.

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Physostigmine, glycopyrrolate, and amphetamine will be stored and prepared at Yale New 
Haven Hospital pharmacy.
Check applicable Investigational Drug Service utilized:
 
 YNHH IDS
 Yale Cancer Center 
 CMHC Pharmacy
 West Haven VA
 PET Center    
 None
      
 Other:
Note: If the YNHH IDS (or comparable service at CMHC or WHVA) will not be utilized, explain in 
detail how the PI will oversee these aspects of drug accountability, storage, and preparation.  
  
5. Use of Placebo: 
 Not applicable to this research project
If use of a placebo is planned, provide a justification which addresses the following:
a.
Describe the safety and efficacy of other available therapies. If there are no other
available therapies, state this.
b.   State the maximum total length of time a participant may receive placebo while on the study. 
c.   Address the greatest potential harm that may come to a participant as a result of  receiving    
  
   placebo. 
d.   Describe the procedures that are in place to safeguard participants receiving placebo. 
6. Use of Controlled Substances:
Will this research project involve the use of controlled substances in human subjects?
 Yes  
 No See HIC Application Instructions to view controlled substance listings. 
If yes, is the use of the controlled substance considered:
 Therapeutic: The use of the controlled substance, within the context of the research, has the 
potential to benefit the research participant.
 Non-Therapeutic: Note, the use of a controlled substance in a non-therapeutic research study 
involving human subjects may require that the investigator obtain a Laboratory Research License. 
Examples include controlled substances used for basic imaging, observation or biochemical 
studies or other non-therapeutic purposes. See Instructions for further information.
Dextro-amphetamine (0.5 mg/kg) will be given by mouth to each subject 3 hours prior to the second 
[11C]PHNO PET scan in Aim 3.
7. Continuation of Drug Therapy After Study Closure   
 Not applicable to this project
Are subjects provided the opportunity to continue to receive the study drug(s) after the study has 
ended?
 
 Yes   If yes, describe the conditions under which continued access to study drug(s) may apply 
as well as conditions for termination of such access. 
 
 No    If no, explain why this is acceptable.
SECTION IV: RECRUITMENT/CONSENT AND ASSENT PROCEDURES 
1.  Targeted Enrollment: Give the number of subjects
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a.
  targeted for enrollment at Yale for this protocol 150
b.    If this is a multi-site study, give the total number of subjects targeted across all 
sites___ 
5. Indicate recruitment methods below.  Attach copies of any recruitment materials that will 
be used.
 Flyers
 Internet/Web Postings
 Radio
 Posters
 Mass E-mail Solicitation
 Telephone
 Letter
 
 Departmental/Center Website
 Television
 Medical Record Review
 Departmental/Center Research Boards
 Newspaper
 Departmental/Center Newsletters
 Web-Based Clinical Trial Registries
 YCCI Recruitment Database
 Clinicaltrials.gov Registry (do not send materials to HIC)
 Other (describe):
6.  Recruitment Procedures:
a.
Describe how potential subjects will be identified.  
b. Describe how potential subjects are contacted.  
c.
Who is recruiting potential subjects?   
Subjects will be recruited through flyers, public advertisement (newspaper, radio, internet 
posting), by word of mouth, contact with community service groups, and clinics and local 
treatment facilities (the VA Hospital, West Haven, CMHC, the Yale Psychiatric Hospital, Mood 
Disorders Research Program, the Yale Depression Research Program).  The subjects will be 
asked to call us if they are interested in participating in the research study.  The PI, in 
collaboration with study investigators, is responsible for subject recruitment.
7. Screening Procedures
a. Will email or telephone correspondence be used to screen potential subjects for eligibility 
prior to the potential subject coming to the research office? 
 Yes  
 No
b. If yes, identify any health information and check off any of the following HIPAA 
identifiers to be collected and retained by the research team during this screening process. 
HEALTH INFORMATION TO BE COLLECTED:
HIPAA identifiers: 
 Names 
 All geographic subdivisions smaller than a State, including: street address, city, county, precinct, zip codes and their 
equivalent geocodes, except for the initial three digits of a zip code if, according to the current publicly-available data from 
the Bureau of the Census: (1) the geographic unit formed by combining all zip codes with the same three initial digits contains 
more than 20,000 people, and (2) the initial three digits of a zip code for all such geographic units containing 20,000 or fewer 
people is changed to 000. 
  Telephone numbers
 Fax numbers 
 E-mail addresses
 Social Security numbers 
 Medical record numbers
 Health plan beneficiary numbers 
 Account numbers 
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  All elements of dates (except year) for dates related to an individual, including: birth date, admission date, discharge 
date, date of death, all ages over 89 and all elements of dates (including year) indicative of such age, except that such ages 
and elements may be aggregated into a single category of age 90 or older 
 Certificate/license numbers 
 Vehicle identifiers and serial numbers, including license plate numbers 
 Device identifiers and serial numbers 
 Web Universal Resource Locators (URLs) 
 Internet Protocol (IP) address numbers 
 Biometric identifiers, including finger and voice prints 
 Full face photographic images and any comparable images 
 Any other unique identifying numbers, characteristics, or codes 
8. Assessment of Current Health Provider Relationship for HIPAA Consideration:
Does the Investigator or any member of the research team have a direct existing clinical 
relationship with any potential subject? 
 Yes, all subjects
 Yes, some of the subjects
 No
If yes, describe the nature of this relationship.
9. Request for waiver of HIPAA authorization: (When requesting a waiver of HIPAA 
Authorization for either the entire study, or for recruitment purposes only.  Note: if you are 
collecting PHI as part of a phone or email screen, you must request a HIPAA waiver for 
recruitment purposes.)
Choose one: For entire study: ______ For recruitment purposes only: ___X___
i. Describe why it would be impracticable to obtain the subject’s authorization for 
use/disclosure of this data;
ii. If requesting a waiver of signed authorization, describe why it would be 
impracticable to obtain the subject’s signed authorization for use/disclosure of this 
data;
By signing this protocol application, the investigator assures that the protected 
health information for which a Waiver of Authorization has been requested will not 
be reused or disclosed to any person or entity other than those listed in this 
application, except as required by law, for authorized oversight of this research 
study, or as specifically approved for use in another study by an IRB.
Researchers are reminded that unauthorized disclosures of PHI to individuals outside of the Yale 
HIPAA-Covered entity must be accounted for in the “accounting for disclosures log”, by subject 
name, purpose, date, recipients, and a description of information provided.  Logs are to be 
forwarded to the Deputy HIPAA Privacy Officer.
 
10. Required HIPAA Authorization: If the research involves the creation, use or disclosure of  
protected health information (PHI), separate subject authorization is required under the 
HIPAA Privacy Rule. Indicate which of the following forms are being provided:
 Compound Consent and Authorization form
 HIPAA Research Authorization Form
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11. Consent Personnel: List the names of all members of the research team who will be 
obtaining consent/assent:
Kelly Cosgrove, Ph.D., Irina Esterlis, Ph.D., Stephen Baldassari, M.D., Nicole DellaGioia, Jon 
Mikael Anderson, Ansel Hillmer, Ph.D., Shivani Bhatt, Sophie Holmes, Ph.D., Sarah O’Grady, 
Yasmin Zakiniaeiz, Halle Thurnauer, Kim Bielen, Grai Bluez, Gina Creatura, Michael 
Kleinberg, and Emma Deaso
12. Process of Consent/Assent: Describe the setting and conditions under which consent/assent    
    will be obtained, including parental permission or surrogate permission and the steps taken to 
   ensure subjects’ independent decision-making. 
The consent process is a multistep process, whereby information about the risks and 
benefits of the study will be provided to potential subjects across several sessions. The number of 
sessions over which this information will be provided will depend on how well the subject 
understands and retains the information. The process begins with the subject initiating contact 
via telephone. The research staff will provide a brief description of the study following which the 
subject is screened by a member of the research team. Thereafter, potentially eligible candidates 
are scheduled for a face-to-face interview. The study procedures will be described as a research 
tool with potential to enhance our knowledge about the brain. Subjects will also be informed of 
all potential risks of participation. Subjects will be required to read the informed consent form 
and the investigator will additionally describes the risks and discomforts. 
To ensure that the study subject understands the study, the subject will be asked questions 
about the study procedures and the risks associated with participation. If any concern arises that 
the study subject did not fully understand the study, the principal investigator (PI) may decide 
that the subject is not suitable for participation. This process generally takes about one hour. If 
the subject is still interested after all questions have been answered, the PI or staff member 
consenting, will ask the subject to sign the informed consent form. Any subject who appears 
incapable of providing informed consent will be excluded. Subjects will be informed that they 
can decline to participate in the study without penalty and given the opportunity to withdraw 
from the study prior to analysis of their data. Following the resolution of any questions, the 
subjects will be asked to sign the consent form if he/she agrees to participate.
 The decision not to participate will not affect an individual’s eligibility to participate in future 
studies, to receive treatment at Yale-New Haven Hospital, or to receive treatment on a private 
basis from a referring clinician.  A copy of the signed consent form will be provided to all 
participating subjects.  For subjects who are not eligible, all PHI will be destroyed.
13. Evaluation of Subject(s) Capacity to Provide Informed Consent/Assent: Indicate how the 
personnel obtaining consent will assess the potential subject’s ability and capacity to consent 
to the research being proposed. 
In cases in which capacity is in doubt, the PI will assess the subject’s understanding of the study 
and the subject’s capacity to decide to participate.
14.   Documentation of Consent/Assent: Specify the documents that will be used during the 
   consent/assent process. Copies of all documents should be appended to the protocol, in the 
   same format that they will be given to subjects. 
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Compound Authorization Form
15. Non-English Speaking Subjects: Explain provisions in place to ensure comprehension for 
   research involving non-English speaking subjects. Translated copies of all consent materials 
   must be submitted for approval prior to use. 
Non-English speaking subjects will not be invited to participate in the studies.  All of our 
materials are in English only, and staff members are fluent in English.  Furthermore, cognitive 
testing is validated in English-speaking subjects only.
16. Consent Waiver: In certain circumstances, the HIC may grant a waiver of signed 
consent, or a full waiver of consent, depending on the study. If you will request either a 
waiver of consent, or a waiver of signed consent for this study, complete the appropriate 
section below.  
 
 Not Requesting a consent waiver 
 
 Requesting a waiver of signed consent
       
 Requesting a full waiver of consent
   
A. Waiver of signed consent: (Verbal consent from subjects will be obtained. If PHI is 
collected, information in this section must match Section IV, Question 6)
 Requesting a waiver of signed consent for Recruitment/Screening only 
If requesting a waiver of signed consent, please address the following:
a. Would the signed consent form be the only record linking the subject and the research? 
 Yes  
 No
b. Does a breach of confidentiality constitute the principal risk to subjects? 
 Yes  
 No
OR
c. Does the research activity pose greater than minimal risk? 
 Yes If you answered yes, stop. A waiver cannot be granted.  Please note: 
Recruitment/screening is generally a minimal risk research activity  
 No 
AND
d. Does the research include any activities that would require signed consent in a non-
research context? 
 Yes  
 No
 Requesting a waiver of signed consent for the Entire Study (Note that an information 
sheet may be required.)
If requesting a waiver of signed consent, please address the following:
a. Would the signed consent form be the only record linking the subject and the research? 
 Yes  
 No
b. Does a breach of confidentiality constitute the principal risk to subjects? 
 Yes  
 No
OR
c. Does the research pose greater than minimal risk? 
 Yes  If you answered yes, stop. 
A waiver cannot be granted.  
 No 
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AND
d. Does the research include any activities that would require signed consent in a non-
research context? 
 Yes  
 No
B. Full waiver of consent: (No consent from subjects will be obtained for the activity.) 
 Requesting a waiver of consent for Recruitment/Screening only 
a. Does the research activity pose greater than minimal risk to subjects?  
 Yes  If you answered yes, stop. A waiver cannot be granted. Please note: 
Recruitment/screening is generally a minimal risk research activity 
 No
b. Will the waiver adversely affect subjects’ rights and welfare? 
 Yes  
 No
c. Why would the research be impracticable to conduct without the waiver? 
d. Where appropriate, how will pertinent information be returned to, or shared with 
subjects at a later date? 
 Requesting a full waiver of consent for the Entire Study (Note: If PHI is 
collected, information here must match Section IV, question 6.)
If requesting a full waiver of consent, please address the following:
a. Does the research pose greater than minimal risk to subjects?  
 Yes If you answered 
yes, stop. A waiver cannot be granted.   
 No
b. Will the waiver adversely affect subjects’ rights and welfare? 
 Yes  
 No
c. Why would the research be impracticable to conduct without the waiver? 
d. Where appropriate, how will pertinent information be returned to, or shared with 
subjects at a later date?  
SECTION V: PROTECTION OF RESEARCH SUBJECTS
  Confidentiality & Security of Data:
a.    
What protected health information (medical information along with the HIPAA 
identifiers) about subjects will be collected and used for the research?   
       
Required private identifiable information about individuals, such as their medical history, current 
medications, psychiatric problems, and family history, will be collected by research staff and be 
used for research purposes and charting after consent is obtained.
b.    How will the research data be collected, recorded and stored? 
The data are collected and recorded by trained research personnel. The data will be recorded on 
Excel spreadsheets that will be saved onto a server or will be in the form of questionnaires that 
are filled out by the subject or the researcher. These paper research materials containing 
confidential information are stored in locked filing cabinets.  Additional brain data is collected 
during the brain imaging scans by trained technologists and is stored on password-protected and 
encrypted computers with identifying information carefully in compliance with HIPAA 
regulations. 
c.    How will the digital data be stored? 
 CD  
 DVD  
 Flash Drive  
 Portable Hard 
Drive  
 Secured Server  
 Laptop Computer  
 Desktop Computer  
 Other
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d.  What methods and procedures will be used to safeguard the confidentiality and security of 
the identifiable study data and the storage media indicated above during and after the subject’s 
participation in the study?
Do all portable devices contain encryption software? 
 Yes   
 No
      
   If no, see http://hipaa.yale.edu/guidance/policy.html
 
All staff members that come into contact with the data are fully trained to the current HIPAA 
regulations and are informed as to the proper use of all data.
Identifiable paper information is kept in locked file drawers and password protected computer 
files.  Results are published as group data without the use of characteristics that would identify 
individual subjects.  We quote information only by number in conference discussions, scientific 
reports, or publications, in order to maintain anonymity.
Identifiable research data, including recruitment and screening information and code keys, are 
stored on a secure database located on the internal PET Center Network.  The PET network is 
protected by a Cisco PIX firewall operated by ITS.  All research data are backed up nightly to a 
Dell PV-136T library wit 4 IBM Ultrium-TD2 tape drives using the backup software Legato 
Networker 7.3 from EMC.  Human subjects enrolled in the study are assigned a subject-specific 
random identifier.   Subject identifiers and the means to link the subject names and codes with the 
research data are stored in separate locations within the database. The software of the database 
limits the ability to connect the random identifier to the actual subject identification information 
to research team members only.  Access to the database is password protected and each research 
team member is required to have a unique ID and password to gain access to the database.  
Authorized users employ their netid and authentication is performed using Yale’s central 
authentication server.  Users always access research data through the random identifier only. Direct 
identifiers belonging to subjects who withdraw from the study, will be stripped from the key.
e.
What will be done with the data when the research is completed? Are there plans to destroy 
the identifiable data? If yes, describe how, by whom and when identifiers will be destroyed. If 
no, describe how the data and/or identifiers will be secured.
The data will be stored in locked filing cabinets and on the password-protected secure database on 
the internal Yale University PET Center Network for at least 7 years, accessed only by authorized 
personnel.
f.  Who will have access to the protected health information (such as the research sponsor, the  
investigator, the research staff, all research monitors, FDA, Yale Cancer Center Data and 
Safety Monitoring Committee (DSMC), SSC, etc.)? (please distinguish between PHI and de-
identified data) 
The investigator and research staff (e.g., PET center nuclear technologists, recruiters) will have 
access to the PHI only on as needed to know basis.  The FDA may also have access to the PHI.
g.   If appropriate, has a Certificate of Confidentiality been obtained?
NA
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h.   Are any of the study procedures likely to yield information subject to mandatory reporting 
requirements? (e.g. HIV testing – reporting of communicable diseases; parent interview -
incidents of child abuse, elderly abuse, etc.). Please verify to whom such instances will need 
to be reported. 
No.
SECTION VI: POTENTIAL BENEFITS
Potential Benefits: Identify any benefits that may be reasonably expected to result from the 
research, either to the subject(s) or to society at large. (Note: Payment of subjects is not 
considered a benefit in this context of the risk-benefit assessment.) 
There are no direct benefits to the nonsmoking subjects for participating in this study.  Smoking 
subjects receive smoking cessation support to abstain from tobacco smoking. This research will 
benefit scientific knowledge by contributing to the understanding of the use of PET imaging in 
tobacco addiction and schizophrenia.  This may have clinical application in the future.
         SECTION VII: RESEARCH ALTERNATIVES AND ECONOMIC CONSIDERATIONS
       1.   Alternatives: What alternatives are available to the study subjects outside of the
              research?
The alternative to participation in this research protocol is to not participate.  Subjects will be 
informed that they are free to choose not to participate and, if they do agree to become a subject, 
they will be free to withdraw from the study at any time during its course.  They will also be 
informed that if they choose not to participate or if they withdraw, it will not adversely affect 
their relationship with their doctors or the hospital (see attached Consent Form).
       2.   Payments for Participation (Economic Considerations): Describe any payments that will
             be made to subjects and the conditions for receiving this compensation. 
The subjects will be compensated for their time commitment and inconveniences necessary for 
completing the study. Subjects will have no financial responsibilities for any portion of the study. 
For all Aims compensation may be $550 for each NCFHEB PET scan, $350 for each PHNO 
PET scan, $50 for each arterial line placement and $50 for each MRI scan and $40 for cognitive 
testing at baseline.  Subjects who participate in the Probabilistic Reward Task may also be 
compensated for the amount that they “win” during the task, up to $60.  Subjects may also 
receive an extra $10 for each Cold Pressor Task that they participate in.  Subjects who complete 
the Conditioned Hallucinations Task (Hearing Task in the Consent) will receive $75. Subjects 
will be paid either by check, and are advised to allow 4-6 weeks for receipt of payment, or they 
will be given a credit card or cash.  In addition, subjects will be provided with a light meal, at the 
end of the PET imaging day.  Reasonable transportation costs will be reimbursed.  Receipts must 
be submitted. If participation in the PET Scan has already begun, then compensation will be 
based on involvement in the study, and will be up to the discretion of the PI.
Smokers will also receive $10 for each contingency management appointment at which their 
carbon monoxide and urine cotinine levels show that they have not smoked.  Subjects can decide 
if they want to receive this amount at each appointment in cash, or receive it all at the end of the 
study in the form of a credit card or check or cash.  Smokers will receive an additional $100 
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Page 60 of 68
bonus for completing the study.  Aim 6 subjects will receive an additional $100 on PET day 
because of the longer scan day they will encounter as a result of the bolus to infusion scan.
Subjects with schizophrenia: Because smoking subjects with schizophrenia will be asked to 
participate in the smoking cessation part of the study as inpatient, a different payment schedule 
was devised (as in HIC 27532). Subjects will be paid $25 for first day of smoking cessation, $50 
for second, $75 for third, etc (increments of $25 for each successful day, for up to $525). Thus, 
smokers with schizophrenia may earn up to $1325 for smoking cessation, PET and MRI scans. 
Nonsmokers with schizophrenia may earn up to $800. 
Cancellations:  If a PET scan should get cancelled for a reason outside of the subject's control 
(i.e. radiotracer synthesis failure) the subject will be paid $50 minimum, or a higher amount not 
to exceed the payment for a full scan day.  The amount of the payment for cancellation will be 
based on the subject's length of participation on that scan day prior to the cancellation, and will 
be up to the discretion of the PI.
       3.   Costs for Participation (Economic Considerations): Clearly describe the subject’s costs
             associated with participation in the research, and the interventions or procedures of the study
             that will be provided at no cost to subjects.   
There will be no costs to subjects related to participation in this research intervention.
  
4.   In Case of Injury: This section is required for any research involving more than minimal 
      risk.
a.     Will medical treatment be available if research-related injury occurs? 
b.     Where and from whom may treatment be obtained? 
c.     Are there any limits to the treatment being provided? 
d.     Who will pay for this treatment? 
e.     How will the medical treatment be accessed by subjects? 
Medical treatment will be offered to the subjects for any physical injuries that they receive as a 
result of participating in this research.  However, the subject or his/her insurance company is 
responsible for the cost.  Federal regulations require that subjects be told that if they are 
physically injured, no additional financial compensation is available.
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 Other Locations, Specify: 
 International Research Site 
(Specify location(s)):
2. Probable Duration of Project: State the expected duration of the project, including all 
follow-up and data analysis activities. 
5 years
      
3. Research Type/Phase: (Check all that apply)
a. Study Type
    
 Single Center Study
    
 Multi-Center Study
Does the Yale PI serve as the PI of the multi-site study? Yes 
No 
  
 
 Coordinating Center/Data Management
   
 
 Other: 
b. Study Phase
 N/A
    
 Pilot
 Phase I
 Phase II
 Phase III
 Phase IV
4.    Is this study a clinical trial? Yes 
No 
NOTE the current ICMJE (International Committee of Medical Journal Editors) definition of a 
clinical trial: “any research study that prospectively assigns human participants or groups of 
humans to one or more health-related interventions to evaluate the effects on health outcomes.” 
Health-related interventions include any intervention used to modify a biomedical or health-
related outcome (for example, drugs, surgical procedures, devices, behavioral treatments, 
dietary interventions, and process-of-care changes). Health outcomes include any biomedical or 
health-related measures obtained in patients or participants, including pharmacokinetic 
measures and adverse events”
If yes, where is it registered?
Clinical Trials.gov registry 
Other (Specify) 
Registration of clinical trials at their initiation is required by the FDA, NIH and by the ICMJE.
If this study is registered on clinicaltrials.gov, there is new language in the consent form and 
compound authorization that should be used.
For more information on registering clinical trials, including whether your trial must be 
registered, see the YCCI webpage, http://ycci.yale.edu/researchers/ors/registerstudy.aspx  or 
 contact YCCI at 203.785.3482)
5. Will this study have a billable service as defined by the Billable Service Definition?
Yes 
 No
If you answered "yes", this study will need to be set up in Patient Protocol Manager (PPM)
http://medicine.yale.edu/ymg/systems/ppm/index.aspx 
6. Are there any procedures involved in this protocol that will be performed at YNHH or one of 
its affiliated entities?  Yes _ __ No X  If Yes, please answer questions a through c and note 
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Page 4 of 38
instructions below.  If No, proceed to Section II.
a. Does your YNHH privilege delineation currently include the specific procedure that 
you will perform?
 
b. Will you be using any new equipment or equipment that you have not used in the past 
for this procedure?
 
c. Will a novel approach using existing equipment be applied?
 
If you answered “no” to question 6a, or "yes" to question 6b or c, please contact the YNHH 
Department of Physician Services (688-2615) for prior approval before commencing with your 
research protocol.
SECTION II: RESEARCH PLAN
1. Statement of Purpose: State the scientific aim(s) of the study, or the hypotheses to be tested.
Tobacco smoking is one of the only leading causes of death that is 100% preventable. While 
most smokers express a desire to quit smoking, only ~6% achieve abstinence in a given year and 
the majority relapse in the first 2 weeks. This highlights the acute withdrawal period as the 
critical window in maintaining abstinence. Nicotine is the primary addictive chemical in tobacco 
smoke and exerts its initial reinforcing effects through the mesolimbic dopamine (DA) 
system(1). Specifically, nicotine binds to beta2 subunit-containing nicotinic acetylcholine 
receptors (beta2*-nAChRs) located throughout the brain and this leads to widespread changes in 
neurotransmitter levels including dopamine (DA), and upregulation, i.e., increase in number, of 
beta2*-nAChRs. beta2*-nAChRs are also responsible for controlling the dynamic range of DA 
release, with chronic nicotine administration or deletion of the beta2*-nAChRs resulting in 
reduced DA release(2). This suggests that chronic smokers may have blunted DA release 
compared to nonsmokers and that changes in DA may be tied to other neurochemical alterations 
in the brains of smokers. However, the basic dopaminergic mechanisms involved in withdrawal 
and relapse in tobacco smokers remain unknown.
We have a radiotracer at the Yale PET center that will allow us to measure dopaminergic 
neurotransmission in the brains of smokers. The radiotracer is called [11C]PHNO and it has 
advantages over other dopamine D2 receptor ligands because it is an agonist and measures the 
high affinity, functionally active D2 receptors and not the low affinity D2 receptors. This has 
been recently shown to produce an increased sensitivity over other PET ligands to measure 
changes in synaptic dopamine levels and thus provides a novel paradigm to investigate drug-
induced dopamine release, such as amphetamine-induced dopamine release. We have 
preliminary data suggesting tobacco smokers at approximately 2 weeks of abstinence have 
blunted DA release compared to nonsmokers. The overall goal of the study is to measure 
amphetamine-induced DA release during early and prolonged withdrawal in smokers (vs. 
nonsmokers) and to examine relationships to relapse and other clinical correlates of tobacco 
smoking. 
Aim 1.  To determine amphetamine-induced DA release in nonsmokers and in tobacco 
smokers during acute and prolonged withdrawal. We will scan 50 Healthy Smokers and 50 
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Non Smoker healthy controls. Each tobacco smoking subject will participate in up to 4 
[11C]PHNO PET scans, one pair of scans during the first 2 weeks of smoking abstinence and, for 
those who remain abstinent, the option to have another pair of scans at 2-8 weeks abstinence. We 
aim to have at least one week in between sets of scans. Ideally, two PET scans will be carried out 
in the same day. We first obtain a baseline scan, then approximately three hours before the 
second scan, amphetamine (0.5 mg/kg, PO) will be administered. Nonsmokers will only be asked 
to participate in one set of scans. We hypothesize that smokers at 1-2 weeks of withdrawal will 
have amphetamine-induced DA release that is blunted compared to healthy nonsmokers. We also 
hypothesize that the smokers who are scanned again at 2-8 weeks withdrawal will exhibit some 
‘normalization’ of the dopamine system and will have a larger amphetamine-induced DA release 
compared to their first scan. We hypothesize that smokers with amphetamine-induced DA 
release similar to nonsmokers, or that increases over time, will have better abstinence outcomes. 
                                                           
2.   Background: Describe the background information that led to the plan for this project.   
      Provide references to support the expectation of obtaining useful scientific data. 
Tobacco Smoking: There are enormous costs to society and to individuals from tobacco 
smoking addiction and related disease, e.g., ~80% of smokers will die from a smoking related 
disease. Smokers are clearly aware of the negative consequences; but 20% of the adult American 
population continues to smoke. Most smokers want to quit smoking and make yearly quit 
attempts, but only 6% are successful in a given year and the vast majority cannot quit for more 
than 2 weeks, even with all of the currently available treatments. We do not know how to help 
smokers remain abstinent. The behavioral manifestations of withdrawal symptoms are clear – 
cognitive dysfunction, intense craving, irritability, anxiety, and bad mood – but because tobacco 
smoking is also a brain-based addiction, it is imperative that we uncover the molecular 
mechanisms that drive these symptoms during acute and prolonged withdrawal. Understanding 
the time course of the molecular changes that occur over withdrawal and determining 
relationships between neurochemistry, clinical symptoms, and their predictive relationship to 
relapse will achieve several goals: 1) provide to the public a greater understanding of the 
neurobiological basis of tobacco smoking withdrawal, e.g., it is not just a “bad habit”; 2) provide 
the public and clinicians neurochemical data on how long the brain needs to recover from the 
addiction; 3) inform the field about directions we should take in terms of targeting new drug 
development and in creating more easily obtained biomarkers or “proxies” of dysfunctional 
neurotransmission over the first few months of withdrawal, to personalize treatment selection 
consistent with a smoker’s individual neurochemistry.
How does the dopaminergic system regulate tobacco smoking and withdrawal? A 
substantial literature demonstrates that nAChRs and the cholinergic system dynamically control 
the mesolimbic DA system by enhancing, inhibiting and filtering striatal DA release(3, 4). 
Nicotine releases DA by binding to β2*-nAChRs located on the mesolimbic DA neurons in the 
ventral tegmental area, resulting in neuronal firing and DA release in the nucleus accumbens and 
dorsal caudate (5). β2*-nAChRs are also critical for the reinforcing(6) and motivational effects of 
nicotine, e.g., tying cues to drug consumption(7). Nicotine also “filters” DA release by 
modifying the sensitivity of DA synapses through its desensitization of the nAChRs. As 
reviewed in Exley and Cragg (2008)(3), nicotine promotes DA release through both burst and 
tonic activity. Nicotine functions as an agonist and also as an antagonist via its 
desensitization/blocking actions and both may ultimately enhance DA neurotransmission. Thus, 
β2*-nAChRs are responsible for controlling the dynamic range of DA release. A recent 
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preclinical study reported that chronic nicotine administration or deletion of the β2*-nAChRs 
(i.e., β2* knockout) reduced acute DA release(2). This suggests that chronic smokers may have a 
blunted stimulus-induced DA release compared to nonsmokers; however, this has not been 
examined. Several PET studies have demonstrated blunted amphetamine-induced DA release in 
the ventral striatum in both cocaine and alcohol dependent populations at approximately 2 weeks 
of withdrawal vs. controls(8-10). In the cocaine-dependent subjects, those with a more blunted 
drug-induced DA release responded less well to a behavioral treatment that incorporated positive 
reinforcement(10). Their findings suggest that individuals with dysfunctional DA transmission 
are not able to “switch” well from drug-reinforced behavior to more natural alternative 
rewards(8, 10), and this is supported by a preclinical study in which rats with a lesioned nucleus 
accumbens displayed an inability to choose greater magnitude delayed rewards vs. immediate 
rewards of lesser value(11). We have preliminary data suggesting tobacco smokers at 
approximately 2 weeks of abstinence have blunted DA release compared to nonsmokers. 
 
Imaging amphetamine-induced DA release in smokers vs. nonsmokers measured with 
[11C]PHNO and PET. Amphetamine administration results in a reliable and robust increase in 
extracellular DA and has been widely used in brain imaging studies as a marker of DA function 
in psychiatric and healthy populations(8, 9, 12-16). We have chosen oral amphetamine based on 
its robust and reliable DA signal and because it is routinely used in PET studies(17-23) and by 
our group to probe DA neurotransmission. The amphetamine robustly increases synaptic levels 
of DA. The increased DA competes with the radiotracer to bind at the DA receptor, thus an 
increase in DA results in a decrease in radiotracer binding compared to baseline. This allows 
calculation of the “occupancy” of the receptors by DA or a change in binding potential (BP), and 
is an indirect measure of DA release based on the “occupancy model”(13). We have obtained 
preliminary data that demonstrates blunted DA release in smokers at 2-3 weeks of abstinence vs. 
controls and is similar to what has previously been found in cocaine- and alcohol- dependent 
individuals(8, 9). While the previous studies(8, 9) used [11C]raclopride which measures striatal 
D2/3 receptor binding, we propose to use [11C]PHNO which measures striatal and extrastriatal 
(substantia nigra and globus pallidum) D2/3 binding(24). Importantly, blunted DA release in 
cocaine-dependent individuals was associated with the choice to self-administer cocaine over 
money(8) and with treatment failure(10). In the current study, we will determine whether 
smokers have blunted DA release compared to nonsmokers and if the degree of DA release in 
smokers is associated with time to relapse. 
3. Research Plan: Summarize the study design and research procedures using non-technical 
language that can be readily understood by someone outside the discipline. Be sure to distinguish 
between standard of care vs. research procedures when applicable, and include any flowcharts of 
visits specifying their individual times and lengths. 
. 
3.1 Overall plan for Aim 1: To determine amphetamine-induced DA release in nonsmokers 
and tobacco smokers during acute and prolonged nicotine withdrawal. We will image up to 
50 smokers and 40 nonsmokers.
Smoker-subjects:  Subjects that qualify for the study will be asked to participate in the 
following: 
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a) One MRI scan, which will take approximately 30-60 minutes. This will take place at the 
Anlyan Center at 300 Cedar St.
b)  Up to 8 weeks of smoking abstinence- meet with staff multiple times a week to ensure 
abstinence. For the first week the subject will meet with staff every day (barring an unforeseen 
circumstance that prevents the subject from meeting). Meetings will occur either at 2 Church St 
South, the Yale PET center, or a meeting point midway between the subject and Yale due to 
distance. As the weeks proceed, meetings will taper down.
c) Two sets of two PET scans with [11C]PHNO approximately 1-2 weeks after quitting smoking 
and again at 2-8 weeks with at least a week break in between scan sets. Both scans of each set 
will take about 2 hours each and should be done on the same day.  Starting up to 3 hours prior to 
the second [11C]PHNO scan of the set, subjects will be asked to take a drug called amphetamine 
by mouth. These will take place at the Yale PET Center. 
Sequence of procedures
Order
Study procedure
How Long
When
1.
MRI scan/Day 0 appointment: This includes, 
mri, computer testing, cold pressor task, and 
withdrawal guidelines. 
2 hours
After screening
2.
Smoking abstinence for about 2-8 weeks w/ 
daily appts for the first week tapering down as 
the weeks proceed..
2-8 weeks
After MRI scan
3.
Set of 2 PHNO PET scans with amphetamine 
challenge (with computer task and cold 
pressor task)
All Day
At ~1-2 weeks of 
smoking abstinence
4.
MRI scan(with possible computer task and 
Cold pressor task)
1 hour
At ~ 2-8 weeks of 
smoking abstinence
5.
Set of 2 PHNO PET scans with amphetamine 
challenge (with computer task and cold 
pressor task)
All Day
At ~2-8 weeks of 
smoking abstinence
6.
May be called followed up with Monthly  to 
check in on smoking status
15 mins
monthly
Non-Smoker subjects: 
Subjects that qualify for this study will be asked to participate in the following: 
a) One MRI scan, which will take approximately 30-60 minutes. This will take place at the 
Anlyan Center at 300 Cedar St.
b) One set of two PET scans with [11C]PHNO. Both scans of the set will take about 2 hours each 
and should be done on the same day. Starting up to 3 hours prior to the second [11C]PHNO scan 
of the set, subjects will be asked to take a drug called amphetamine by mouth. This will take 
place at the Yale PET Center. 
Sequence of procedures
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Order
Study procedure
How Long
When
1.
MRI scan/Day 0 appointment(with computer 
task and cold pressor task)
2 hours
After screening
2.
Set of 2 PHNO PET scans with amphetamine 
challenge(with computer task and cold 
pressor task)
All Day
After screening
3.2 Subject recruitment. 
We have an established program to recruit healthy controls. For the present study, subjects will 
be recruited through our program as well as flyers, public advertisement (newspaper, radio, 
internet postings), and word of mouth. 
3.3 Screening for eligibility. 
After completing the informed consent process, subjects will have a physical and neurological 
examination. The following lab tests will be performed at screening to exclude medical illnesses: 
complete blood count (CBC) and differential, chemistries, kidney function tests (creatinine, 
BUN, urinalysis), liver function tests, and TSH.  A urine drug screen and a pregnancy test (for 
women) will be done at screening and before radiotracer administration on each PET scan day. 
The psychiatric assessment will include a psychiatric history, a structured clinical interview 
(SCID), and assessment of subsyndromal depression with the Center for Epidemiological Studies 
Depression Scan – CES-D and subsyndromal anxiety with Speilberger State-Trait Inventory 
(STAI). 
Aim 1 Healthy nonsmokers

smoked < 100 cigarettes in lifetime

urinary cotinine levels 0-30 ng/mL both at intake evaluation and on scan day
Aims 1 Healthy Smokers 

have been smoking cigarettes on a daily basis for at least 1 year 

carbon monoxide levels > 8 ppm during intake evaluation

plasma nicotine levels > 10 ng/mL during intake evaluation

plasma cotinine levels of > 50 ng/mL during intake evaluation

wish or willing to quit smoking for at least 1 week and up to 8 weeks 
3.4 Assessments:
All participants will be screened initially using a telephone screen that will include questions to 
evaluate medical history, personal and familial psychiatric and smoking history.  
3.4a. General Intake Assessments 
1.
Demographic Questionnaire This questionnaire will obtain: (1) basic demographic 
information including age, gender, marital status, employment status, occupation, (2) 
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alcohol/drug history, (3) family history of alcohol/drug use, depression, anxiety, and smoking 
history.
2.
Medical History This questionnaire will obtain a basic medical history (personal and 
family) including past or current conditions such as neurological, endocrine, cardiovascular, 
renal, liver, and thyroid pathology. Current body weight and current medications will also be 
assessed. 
3.
Medical Assessments will include a physical exam by a state licensed physician 
(overseen by Dr. David Matuskey), a complete blood count, blood urea nitrogen, creatinine, 
fasting blood sugar, electrolytes, liver function tests, thyroid function tests (including T3, T4, 
T3RU, estimated free T4), thyroid stimulating hormone levels, urine toxicology, EKG, and 
urinalysis. Female subjects will have serum pregnancy tests. All EKGs are read by a state 
licensed cardiologist and all abnormal MRIs will be reviewed by a state licensed 
neuroradiologist.
4.
Illicit Drug/Pregnancy Screen A urine sample will be collected to determine current illicit 
drug use (for all potential subjects), positive results other than THC may be cause for exclusion 
from the study, however positive results will not be kept on file due to the sensitive nature of this 
information. In addition, urine samples will be collected for the intake visit and on each MR and 
PET scan day to confirm that the subject is not pregnant.  Note: the urine pregnancy test will not 
be required prior to the MRI if the serum pregnancy test was done within 1 week prior to the MR 
imaging session.
5.
Structured Clinical Interview for DSM-IV Axis I Disorders The psychotic screening and 
depression sections of the Structured Clinical Interview for DSM-IV Axis I Disorders (SCID) 
will be used to determine whether subjects meet exclusion criteria for diagnosis of psychotic 
disorders and major depression (25).
3.4b.  Mood Measures 
We may of mood and anxiety at intake and also on the PET scan day including the following.
1. 
Center for Epidemiological Studies Depression Scale (CES-D) The CES-D (26) is a 20-
item self-report instrument, which has been extensively used in both clinical and nonclinical 
populations to measure the frequency and severity of depressive symptoms over the past week. 
The CES-D, which has been used to document the severity of depressive symptoms in adults 
(27) and has been shown to be a sensitive measure of negative affect in smokers (28), will be 
used in the proposed studies to exclude for presence of major depression, and to measure level of 
mild depressive symptoms commonly noted in tobacco smokers.
2. 
Anxiety: The State-Trait Anxiety Inventory (29) is a 40-item, self-report measure, 
comprised of two subscales. The State-Anxiety scale is 20 items and assesses transitory states 
characterized by feelings of tension, apprehension, and heightened autonomic reactivity. The 
Trait-Anxiety scale is 20 items and assesses stable individual differences in anxiety proneness.
3.
Impulsivity: Barratt Impulsiveness Scale (BIS; (30)) is a 30 item self-report instrument 
designed to assess the personality/behavioral construct of impulsiveness. 
 
3.4c. Smoking Measures
We will obtain smoking measures at intake and also on the PET scan day, which may include the 
following. 
1.Fagerstrom Test for Nicotine Dependence (FTND).(31) This will be used to measure the 
severity of nicotine dependence.  It is a 6-item scale with an internal consistency of .61 and its 
total score is closely related to biochemical measures of intensity of smoking.  
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2. Smoking History. This questionnaire will assess basic smoking status and history such as 
number of years smoked, number and length of quit attempts, reasons for quitting, and second 
hand smoke exposure.  
3. Nicotine Withdrawal Checklist.(32) This measures the severity of eight withdrawal symptoms 
on 5-point Likert scales.  
4. Tiffany Questionnaire of Smoking Urges (QSU).(33) The QSU-brief is a 10-item 
questionnaire that evaluates the structure and function of smoking urges.  Subjects indicate on a 
likert-type scale how strongly they agree or disagree with each statement with a score of 1 
(strongly agree) to 7 (strongly disagree).  This characterizes ‘urges to smoke’ into a negative 
affect related to relief of withdrawal symptoms and positive affect related to expectancy of 
reinforcement.   
3.4d Cognitive Measures
We will obtain these measures at baseline and up to two times on the PET days.
1. Cogstate Battery: This computerized test battery will assess memory and cognition.  The 
tasks may include:
a. International Shopping List Task – a computerized task to assess verbal learning and memory.
b.  Groton Maze Learning Task – a computerized task to assess executive function and spatial 
problem solving.
c.  Detection Task – a computerized task to assess psychomotor function and speed of 
processing.
d.  Identification Task – a computerized task to assess visual attention and vigilance.
e.  One Card Learning Task – a computerized task to assess visual learning and memory.
f.  One Back Task – a computerized task to assess attention and working memory
2. Probabilistic Reward Task: The PRT has been successfully used to assess reward 
responsiveness (166-168). In each trial, subjects choose which of two difficult-to-differentiate 
stimuli was presented. Stimuli consist of simple cartoon faces (diameter: 25 mm; eyes: 7 mm) 
presented in the center of the monitor. At the beginning of the trial, the face has no mouth. After 
a given delay, either a straight mouth of 11.5 mm (“short mouth”) or 13 mm (“long mouth”) is 
presented for 100 ms. Subjects are instructed to press an appropriate button to decide whether a 
long or small mouth had been presented.  Unbeknownst to subjects, correct identification of one 
stimulus (“rich stimulus”) is rewarded three times more frequently (“Correct! You won 20 
cents”) than the other (“lean”) stimulus. In healthy controls, this reinforcement schedule leads to 
a response bias (i.e., a preference for the more frequently rewarded stimulus). The degree of 
response bias toward the more frequently reinforced alternative will be used for operationalizing 
sensitivity to reward.
3. Cold Pressor Task: Subjects may be asked to participate in the Cold Pressor Task.  The cold 
pressor task (CPT) is a stress task used to measure pain sensitivity and pain tolerance.  This task 
will be used to determine alterations in pain thresholds as a result of nicotine use.  Participants 
will immerse their hand (up to the wrist) for up to 3 minutes in the experimental (ice-cold 
temperature 0-4°C)) and control (room temperature (20°C)) conditions.  Physiological measure 
(heart rate, blood pressure and subject responses (stress, mood) will be collected 5 minutes 
before, 1 minute into, and immediately after the CPT.
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3.5 Experimental Methods
MRI.
Within approximately 6 months of the PET study, anatomical MRIs will be acquired at the Yale 
University MRI Center. Subjects will be taken through a ferromagnetic metal detector before 
entering the scan room. The purpose of the MRI scan is to direct the region of interest placement 
on the lower resolution PET images.  The T1 weighted images will be acquired on a 3 Tesla 
Siemens Scanner.  There will also be an additional resting state scan with subjects in the scanner, 
eyes open, fixating on a cross.  Smokers who participate in the 2-8 week PET scan set may return 
to have a second MRI with resting state at 2-8 weeks withdrawal A member of the research team 
will accompany the subject and remain for the duration of the scan. 
Prisma Scan sequence: 64 Channel
Series 1: 3 plane localize 
Series 2: Sag 3d tfl; 256fov; 1mm thick slices; 176 slices total; TE 2.77; TR 2530; TI 1100; FA 
7; 256X256 1 average. 
Series 3: Resting state: Ep2d bold; 210fov; 2.5mm thick slices; TE 30; TR 3400; FA 85; 84x84 
(Run twice). 
Series 4: Diffusion Tensor Imaging (DTI) sequence
MR images provide a matching anatomical atlas for creating individualized region-of-interest 
templates for each subject. We will also examine functional connectivity at rest.
Amphetamine challenge
Dextroamphetamine sulfate is the dextro isomer of the compound d,l-amphetamine sulfate, a
sympathomimetic amine of the amphetamine group. It is an FDA approved drug available for the
treatment of narcolepsy and attention deficit hyperactivity disorder (maximum approved total
daily dose of 5-60 mg). After the first [11C]PHNO scan, dextro-amphetamine (0.5 mg/kg, PO) 
will be given in increments of 5 or 10 mg, as it is available in 5 mg and 10 mg tablets, and will 
be rounded down to approximate 0.5 mg/kg total dose without exceeding it. Total dextro-
amphetamine dose will not exceed 50 mg per scan. A second transmission and emission scan 
will be acquired approximately 2.5-3 hours post amphetamine identical to the methods outlined 
previously. The timing of the second [11C]PHNO administration and subsequent PET scanning 
(i.e., 2.5-3 hours) corresponds to the time of maximum plasma concentration of amphetamine as 
stated in the respective FDA-approved product labeling. EKG and frequent BP monitoring will 
occur throughout the study and until the vital signs are within normal limits. Supplemental 
oxygen will be provided via nasal cannula if necessary.  If the systolic BP reaches or exceeds 
200 mmHg for more than 5 minutes, the study doctor will take the appropriate clinical measures 
in order to lower the BP, which may include phentolamine administration (5 mg IV over 10 min) 
or other appropriate measures.
Following the post-amphetamine PET scan, subjects will be assessed (EKG and vital signs) by 
one of the research nurses.  Subjects will be discharged when vital signs are within normal limits 
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and when behavioral changes (if any) are found to be not clinically significant by the MD 
attending to the subject at the PET Center. If subjects experience any adverse events, they will be 
treated until they become asymptomatic, prior to discharge.
Behavioral response to amphetamine will be measured by self-ratings with a simplified version 
of the Amphetamine Interview Rating Scale (van Kammen and Murphy, 1975).  Four items will 
be investigated: euphoria ("feel good"), alertness ("feel energetic"), restlessness ("feel like 
moving") and anxiety ("feel anxious").  Self-ratings will be obtained by analog scales at the 
following times relative to the d-amphetamine administration: -5 minutes, 0, and hourly 
thereafter until end of the second scan. 
PET
PET scans may be performed on the High Resolution Research Tomograph (HRRT, 2-3 mm 
resolution) or another similar camera.  Venous catheters will be used for i.v. administration of 
the radiotracers, venous blood sampling of activity, and for venous blood sampling of 
amphetamine plasma levels and hormone levels. At the beginning of scan, the subject’s head will 
be immobilized and a transmission scan will be obtained for attenuation correction. PET scans 
will be acquired using bolus or bolus to infusion administration of up to 10 mCi of [11C]PHNO 
and subjects will be scanned for up to 2 hours. Dynamic images of radioactivity 
concentration are reconstructed with corrections for attenuation, normalization, random events, 
scatter, and deadtime. Subject motion is corrected automatically on an event-by-event basis with 
the Vicra motion tracking system.  Vital signs (blood pressure, pulse and respiration) are 
collected prior to and during each PET scan. Urine pregnancy test will be again administered on 
the PET scan day prior to the initiation of any imaging procedures. Smoking abstinence, when 
appropriate, will also be confirmed for smoking subjects prior to PET scanning. 
PET scanning will then proceed as following for each aim: 
Aim 1. Smoking subjects will be asked to come in for two [11C]PHNO scans with amphetamine 
administration. The first scan will be during the first 2 weeks of withdrawal with the option to 
come back again at 2-8 weeks of withdrawal. Nonsmoking subjects will be asked to come in for 
one set of two [11C]PHNO scans with amphetamine administration. Up to 10 mCi of PHNO will 
be administered per scan. After the first scan, subjects will take amphetamine (0.5mg/kg) by 
mouth, and then will be imaged again with PHNO.  If technical difficulties arise, the second PET 
scan of the set may have to be re-scheduled and will be scheduled as soon as possible. In that 
case, subjects may be asked to re-take amphetamine on the day of the rescheduled PHNO scan, 
approximately 3 hrs prior to the scan. Amphetamine dose will not exceed 50mg per scan.  
Subjects will not be allowed to drive home.
In the rare event that a scan would fail post injection, subjects may repeat each scan set up to a 
total of 8 PHNO scans for smokers and 4 PHNO scans for nonsmokers.  
3.6 Image analysis.
The primary outcome measure is the binding potential (BPND), which in turn is proportional to 
the available receptor concentration (Bavail), given that there is no change in affinity (KD) and that 
nondisplaceable (nonspecific and free) uptake does not differ between subjects and studies.  We 
will examine the regions-of-interest listed below with the cerebellum used as a reference region 
because it is devoid of D2/3 receptors.
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We obtain an MRI (as previously described) to use as a guide to place our regions of interest.  
This is because we can define anatomical regions of interest on the MRI, which provides 
structural information and then we can apply these regions of interest to the PET scans.  The PET 
scans alone are not sufficient to identify brain regions. The PET image sets are aligned and 
resliced to yield images in the same planes and spatial system as the MRI images using AAL 
template. Primary ROIs will be caudate, putamen, ventral striatum, globus pallidus, and 
substantia nigra. Cerebellum will be the reference region. 
3.7 Statistical analysis. 
Demographics between smokers and nonsmokers will be compared using t tests or Chi-Square 
tests, as appropriate. Scores on psychological tests such as depressive symptoms at baseline will 
be compared between groups and may be used as covariates in subsequent analyses. All 
outcomes will be summarized descriptively and assessed for normality prior to analysis using 
normal probability plots and Kolmogorov test statistics. Transformations or nonparametric 
analyses will be performed as necessary. All tests will be two-sided and considered statistically 
significant at alpha = 0.05. Post-hoc comparisons will be conducted as necessary with 
significance levels adjusted for multiple comparisons basing the adjustment on the number of 
conceptually related statistical tests within each hypothesis. All statistical analyses will be 
conducted using SAS version 9.3 (Cary, NC).
Aim 1. To determine if amphetamine-induced DA release is blunted during abstinence in 
tobacco smokers compared to nonsmokers. Hypothesis 1. We hypothesize that amphetamine-
induced striatal DA release will be blunted in smokers compared to nonsmokers. Power 
Calculations for Hypothesis 1: Our preliminary data revealed blunted amphetamine-induced DA 
release (d=0.91) in the ventral striatum among smokers (-21%±12.0) compared to nonsmokers (-
29%±6.3). Assuming a two-sided t-test with α=0.05, 40 nonsmokers and 50 smokers assessed 
after 14-21 days of abstinence will provide 80% statistical power to detect effect sizes as small as 
d=0.63, which compares favorably with the effects observed above. 
Regional [11C]PHNO uptake for each subject will be quantified as BPND described above for 
each ROI and will represent the primary outcome measure for the proposed experiments.  
Repeated measures ANOVA will be used to evaluate the change between scans (deltaBP) for 
brain measures for the scans.  Delta BP will be computed as [(BPbaseline-BPcondition)/BPbaseline X 
100]. Analysis will be conducted using SAS version 9.1 (Cary, NC). N=20 subjects per group is 
necessary to detect differences between smokers and nonsmokers. Potential associations, e.g., 
relationship between magnitude of dopamine release and time to relapse, will be examined using 
correlation analysis with Type I error corrected for multiple comparisons. Exploratory analyses 
of the other cognitive and behavioral measures and their relationship to relapse will also be 
performed. 
3.8 Contingency Reinforcement for Smoking Cessation (for healthy smokers)
We plan to image smokers during the first 8 weeks of abstinence. Contingency management 
techniques have been successfully used to help smokers quit smoking by a number of investigators 
including our group for many years (34-36). 
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We will help the subjects set a quit date and prior to the quit date they will meet with the 
research staff who will provide them with brief advice on quitting smoking based on AHCPR 
guidelines (The Smoking Cessation and Clinical Practice Guideline Panel and Staff, 1996). They 
will be advised about the risks and benefits of quitting smoking and told that they will be monitored 
daily to ensure abstinence.  Subjects will be informed of payment schedules (see below) for CO 
levels indicating abstinence and also given information on how quit rates in the first week of 
smoking cessation predicted sustained abstinence.  We use CO levels < 11 ppm to define abstinence 
from cigarettes.  During the first week of abstinence we will obtain CO levels during contingency 
management meetings from subjects up to twice daily. In addition, we will obtain urine samples 
once daily to measure cotinine levels (a nicotine metabolite). These meeting will occur either in the 
research office, the Yale PET center, or in a midway point if subjects travel a far distance. 
For each contingency management appointment, subjects will get $10 if their CO levels are less 
than 11ppm and urine cotinine levels are less than 100 ng/ml.  
         4.  Genetic Testing    N/A
5. Subject Population Provide a detailed description of the targeted population of human    
       subjects for this research project.
Target numbers for participation are 40 Healthy controls and 50 Healthy Smokers. Healthy 
controls and healthy smokers will be recruited from the community through advertisements as 
approved by the Yale University Human Investigations Committee (HIC).  Interested individuals 
contacting the clinic by phone in response to advertisements are told that the information they 
give over the phone is written down and discussed by the research team.  They are advised that if 
they do not enroll in research with the clinic the information is destroyed, and that if they do, it 
becomes part of their research chart.  A phone screen is completed after they give verbal 
authorization.  If an individual appears to meet enrollment criteria and is interested in 
participating, a face-to-face interview is conducted.  A release of information is obtained for 
review of any available historical and clinical data.  Written authorization is also obtained from 
each subject, permitting the research team to use, create, or disclose the subject's PHI for 
research purposes.  The nature of the project, procedures, relative risks and benefits, and 
alternatives to participation in the project are discussed with the individual.  Following this 
discussion, the individual is given a copy of the consent form to review, and any questions are 
answered. We will obtain written consent from all participants.
6. Subject Classifications: Will subjects who may require additional safeguards or other 
   considerations be enrolled in the study? If so, identify the population of subjects requiring 
   special safeguards and provide a justification for their involvement.
   a. Is this research proposal designed to enroll children who are wards of the state as potential 
   subjects? 
 Yes  
 No (If yes, see Instructions section VII #4 for further requirements) 
7. Inclusion/Exclusion Criteria: What are the criteria used to determine subject inclusion 
or    exclusion?
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General inclusion criteria: 

men and women, aged 18-55 years

who are able to read and write 

who are able to give voluntary written informed consent

have no current uncontrolled medical condition such as neurological, cardiovascular, 
endocrine, renal, liver, or thyroid pathology 

drink less than <21 drinks/week for women and less than <35 drinks per week for men 

If female, not pregnant or breast feeding

If female of childbearing age, must use an acceptable method of birth control, as 
determined by the principal investigator

do not suffer from claustrophobia or any MR contradictions

willing to be followed up monthly after study participation via phone or email contact
General exclusion criteria:

Presence of acute or unstable medical or neurological illness. Subjects will be excluded 
from the study if they present with any history of serious medical or neurological illness 
or if they show signs of a major medical or neurological illness on examination or lab 
testing including history of seizures, head injury, brain tumor, heart, liver or kidney 
disease, eating disorder, diabetes.

Presence of an Axis I diagnosis other than nicotine dependence 

Regular use of any psychotropic drugs including anxiolytics and antidepressants and 
other over-the-counter medications and herbal products in the past six months other than 
THC, per the PI’s discretion. The PI will take a number of factors into consideration on a 
case-by-case basis including type of psychotropic drug used, frequency, and dose.

Pregnancy/Breast feeding

Subjects with a pacemaker or other ferromagnetic material in body.

Subjects with a sitting pulse rate >100 bpm will be excluded

Subjects with hypertension defined as sitting systolic blood pressure of >160 mmHg 
and/or sitting diastolic blood pressure of >100 mmHg will be excluded.  Those 
individuals with hypertension that is well controlled by medication (e.g., within the above 
mentioned range) are not excluded

Specifically, we will exclude subjects who have any active clinically significant deviation 
from the normal range in their electrocardiogram (EKG).  However, subjects who have 
abnormalities in their EKG but the condition has been present for a while and the study 
cardiologist has evaluated and feels comfortable with the condition, would not be 
excluded on the basis of their cardiac condition.  Examples of conditions that may meet 
these criteria (e.g., condition has been present for a while) include but are not limited to 
T-wave abnormalities, atrial fibrillation, prolonged PR interval, and right bundle branch 
block. 

Subjects with history of prior radiation exposure for research purposes within the past 
year such that participation in this study would place them over FDA limits for annual 
radiation exposure. This guideline is an effective dose of 5 rem received per year.

Subjects with current, past or anticipated exposure to radiation in the work place

Blood donation within eight weeks of the start of the study.
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
History of a bleeding disorder or are currently taking anticoagulants (such as Coumadin, 
Heparin, Pradaxa, Xarelto).
8. How will eligibility be determined, and by whom? 
Eligibility to participate will be determined by the PI of this study after completion of the 
medical and psychiatric evaluation of the potential participant.
9. Risks: Describe the reasonably foreseeable risks, including risks to subject privacy, 
     discomforts, or inconveniences associated with subjects’ participation in the research. 
Risks from this study include 1) risks associated with radiation exposure, 2) risks associated with 
MRI, 3) intravenous lines and blood drawing, 4) nicotine withdrawal symptoms, 5) amphetamine
1. Risks Associated with Radiation 
The Yale New Haven  Radiation Safety Committee and the FDA will review the use of radiation 
in this research study as the radiotracer [11C]PHNO PET will be under IND # 134138, This 
research study involves exposure to radiation from [11C]PHNO PET scanning. This radiation 
exposure is not necessary for medical care and is for research purposes only. 
For each individual [11C]PHNO PET scan, subjects will receive up to ≤ 10 mCi of [11C]PHNO, 
plus transmission scans. This is equal to an effective dose equivalent of 0.2146 rem per injection.
The maximum amount of radiation per year an individual subject will receive in this study is 
from up to 8 injections of ≤ 10mCi each of [11C]PHNO, plus transmission scans. The intended 
amount is from up to 4 injections of ≤ 10mCi each of [11C]PHNO for smokers and up to 2 
injections of  ≤ 10mCi each of [11C]PHNO in nonsmokers, however in case of scan failures post 
injection we have included up to 8 injections if scan sets need to be repeated within the study 
time frame (i.e. 1-2 wks withdrawal and 2-8 wks withdrawal). Nonsmokers maximum number of 
injections including re-scans would be 4 scans total. Should the second scan of the set fail, we 
will make every effort to reschedule the scan for repeat within 1 wk, however if scheduling does 
not permit this, we may ask the subject to repeat the whole set due to timing related data. 
Although each organ will receive a different dose, the maximum intended amount of radiation 
exposure subjects will receive per year from this study is equal to an effective dose equivalent of 
0.858400001 rem for a total of up to 40 mCi of [11C] PHNO in 4 injections of ≤ 10mCi each for 
smokers and 0.4292 rem for a total of up to 20 mCi  [11C] PHNO in 2 injections of ≤ 10mCi each 
for non smoker. This calculated value is used to relate the dose received by each organ to a 
single value.
However, if scans should need to be repeated the maximum dose for smokers would be equal to 
an effective dose of 1.716800002 rem from up to 8 injections and for non smokers would be 
0.858400001 rem from up to 4 injections. We have every intention to limiting radiation exposure 
to subjects and avoiding re-scans if possible. 
2 injections PHNO
0.4292 rem
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4 injections PHNO
0.8584 rem
8 injections PHNO
1.7168 rem
The amount of radiation subjects will receive in this study is below the dose guidelines established 
by the FDA and monitored by the Yale University Radioactive Drug Research Committee for 
research subjects. This guideline sets an effective dose limit of 5 rem per year.
2.  MRI
MR carries a risk for subjects who have pacemakers, metal pieces, aneurysm clips, or other 
contraindications for MR.
Magnetic resonance (MR) is a technique that uses magnetism and radio waves, not x-rays, to 
take pictures and measure chemicals of various parts of the body.  The United States Food and 
Drug Administration (FDA) has set guidelines for magnet strength and exposure to radio waves, 
and we carefully observe those guidelines. 
 
Subjects will be watched closely throughout the MR study.  Some people may feel 
uncomfortable or anxious.  If this happens, the subject may ask to stop the study at any time and 
we will take them out of the MR scanner.  On rare occasions, some people might feel dizzy, get 
an upset stomach, have a metallic taste or feel tingling sensations or muscle twitches.  These 
sensations usually go away quickly but we will ask subjects to tell the research staff if they have 
them.  
There are some risks with an MR study for certain people.  If subjects have a pacemaker or some 
metal objects inside their body, they may not be in this study because the strong magnets in the 
MR scanner might harm them.  Another risk is the possibility of metal objects being pulled into 
the magnet and hitting a subject.  To reduce this risk we require that all people involved with the 
study remove all metal from their clothing and all metal objects from their pockets.  We also ask 
all people involved with the study to walk through a detector designed to detect metal objects.  It 
is important to know that no metal can be brought into the magnet room at any time.  Also, once 
subjects are in the magnet, the door to the room will be closed so that no one from outside 
accidentally goes near the magnet.
We want subjects to read and answer very carefully the questions on the MR Safety 
Questionnaire related to their personal safety. We will be sure that subjects have read the MR 
Safety Questionnaire and tell us any information they think might be important.  
This MR study is for research purposes only and is not in any way a clinical examination.  The 
scans performed in this study are not designed to find abnormalities.  The primary investigator, 
the lab, the MR technologist, and the Magnetic Resonance Research Center are not qualified to 
interpret the MR scans and are not responsible for providing a diagnostic evaluation of the 
images.  If a worrisome finding is seen on a subject’s scan, a radiologist or another physician 
will be asked to review the relevant images.  Based on his or her recommendation (if any), the 
primary investigator or consulting physician will contact the subject, inform them of the finding, 
and recommend that they seek medical advice as a precautionary measure.  The decision for 
additional examination or treatment would lie solely with the subject and their physician.  The 
investigators, the consulting physician, the Magnetic Resonance Research Center, and Yale 
University are not responsible for any examination or treatment that a subject receives based on 
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these findings.  The images collected in this study are not a clinical MR exam and for that 
reason, they will not be made available for diagnostic purposes.
3. Blood Drawing and IV line Insertion
Drawing blood and inserting an intravenous line (IV) into an arm vein are safe and standard 
medical procedures. Sometimes a bruise will occur at the puncture site and rarely a blood clot or 
infection will occur in the vein. Certain individuals may feel light-headed during venipuncture. 
The volume of blood collected during this study, may include screening laboratories, MRI and 
PET scans, will be approximately 32 tablespoons. This is not expected to have any serious 
negative effects on a study participant. 
4.  Nicotine Withdrawal
Smokers that quit smoking may experience symptoms of nicotine withdrawal such as craving 
cigarettes, mild anxiety, restlessness, irritability, difficulty concentrating, loss of energy, and 
excessive hunger. These are typical symptoms that people experience when they stop smoking 
and they can be uncomfortable but they are not life threatening. 
5.  Risks of oral d-amphetamine 
Risks of amphetamine administration include both medical and psychiatric risks.
The frequent somatic side effects of d-amphetamine administration are cardiovascular 
(hypertension, palpitations, tachycardia, bradycardia, orthostasis). General effects such as 
sweating, feeling warm or cold, nausea, diarrhea, muscle and abdominal cramping, have been 
reported frequently. Behavioral effects in this dose range are increased level of alertness, 
talkativeness, restlessness, agitation, mood changes (usually euphoria) and anxiety. In our 
experience, these effects are generally transient and well tolerated. This dose of amphetamine 
has not been reported to induce psychotic symptoms in non-schizophrenic subjects. Infrequently 
blurred vision, headaches and chest tightness, and changes in EKG have been reported. There is 
a rare risk of permanent neurological damage and death as a result of cardiac arrest or stroke.
Psychiatric or behavioral side effects:  General behavioral effects of amphetamine in this dose 
range are increased level of alertness, talkativeness, restlessness, agitation, mood changes 
(usually euphoria) and anxiety.  In our experience, these effects are generally transient and well 
tolerated.  This dose of amphetamine has not been reported to induce psychotic symptoms in non 
schizophrenic subjects and we confirm this observation. 
10. Minimizing Risks: Describe the manner in which the above-mentioned risks will be  
    minimized. 
1.  The dose of radiation will be submitted for approval to the Yale New Haven Hopsital 
Radiation Safety Committee. All scans will be done in the presence of medical supervision and 
trained staff in an institution specifically designed to support imaging studies. In the event of 
serious medical complications, the PET scan facilities have immediate access to or consultation 
with specialized medical units at the Yale-New Haven Hospital. Preparation of 
radiopharmaceuticals and performance of PET scans will be by radiochemists, physicians, and 
technologists of the Department of Diagnostic Radiology, Yale University School of Medicine. 
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These professionals are qualified by training and experience in the safe use and handling of 
radiopharmaceuticals. Subjects will be asked about their previous radiation exposure and those 
who have had research exposure within the past year will be excluded if their cumulative annual 
exposure (including the present study) exceeds FDA limits.   The information on the previous 
radiation exposure of study subjects will be notified to the study doctor. 
No PET studies will be performed on pregnant or potentially pregnant women, as confirmed by 
pregnancy testing during evaluation and on each scan day before initiation of any scan 
procedures. If subjects are breastfeeding they will not be able to participate in this research 
study. 
2. Minimizing risks with MRI: All subjects will be screened for any metallic objects other MR 
contraindications that they may be holding or have implanted in their bodies using a 
questionnaire and all potential subjects with contraindications for MR will be excluded. This 
questionnaire will be repeated immediately before each measurement to insure that no metallic 
materials are brought into close proximity of the magnet, where they might be pulled toward the 
magnet.  For additional security, subjects will be taken through a ferromagnetic metal detector 
immediately before going to the scan room.
3. Minimizing risks with blood draws: The risks of bruising, clotting, and infection will be 
minimized by having venipuncture performed by trained and experienced personnel using 
aseptic technique. To avoid injury due to fainting, the venous catheters will be inserted when the 
subjects are recumbent. The blood draws during PET scanning sessions will be obtained from the 
already inserted catheter, to minimize discomfort.
4. Minimizing risks with nicotine withdrawal: Subjects will meet with research staff and be 
counseled prior to the quit attempt to provide them with skills to help ease the withdrawal 
symptoms. 
5. Minimizing risks with oral d-amphetamine administration: Medical side effects: Subjects 
will be carefully screened for absence of significant medical history and current medical 
conditions with a complete medical history, physical examination, routine blood tests, urine 
toxicology and EKG. Inclusion in the study will be limited to individuals who are between the 
ages of 18-55. Patients will be excluded if they have any h/o severe medical or neurological 
illness, any clinically significant brain abnormality, insulin dependent diabetes, a history of 
cardiovascular disease, or hypertension. Administration of oral d-amphetamine will take place at 
the PET center by a research nurse, with a physician on site.  The research nurse will report vital 
signs to the physician prior to administering the amphetamine.
 If several of the subject’s blood pressure readings are recorded at >100 or <60 for diastolic BP 
or >160 or <90 for systolic BP while at rest, they will be evaluated by the MD.  The study may 
be cancelled at the discretion of the MD after evaluation.  Any automated blood pressure results 
that are abnormal will be repeated manually. The manual reading will be the official reading. 
Constant EKG and frequent BP monitoring will occur until the vital signs are within normal 
limits. If the systolic BP reaches or exceeds 200 mmHg for more than 5 minutes, an infusion of 
phentolamine (5 mg IV, over 10 min) or other appropriate measures may be initiated to control 
the blood pressure response.  The study physician will be notified if those parameters are reached 
and he/she will supervise the treatment.  
In case of chest pain, chest tightness or other symptoms suggestive of cardiac ischemia, the 
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experiment may be cancelled and an EKG will be obtained to rule out angina (ST segment 
elevation or depression as compared to the baseline EKG). Appropriate treatment will be 
initiated.
Upon discharge, patients will be given the phone numbers of the study physicians and will not be 
allowed to drive home.  They will either arrange a ride or a taxi.
For Data and Safety Monitoring Plan templates, see 
http://www.yale.edu/hrpp/forms-templates/biomedical.html 
Data Safety Monitoring Plan:
1. Personnel responsible for the safety review and its frequency:
The principal investigator will be responsible for monitoring the data, assuring protocol 
compliance, and conducting the safety reviews at the specified frequency which must be 
conducted at a minimum of every 6 months (including when re-approval of the protocol is 
sought).  During the review process, the principal investigator (monitor) will evaluate whether 
the study should continue unchanged, require modification/amendment, or close to enrollment. 
Either the principal investigator, the IRB or Safety Monitoring Committee (DSMC) have the 
authority to stop or suspend the study or require modifications.
2. The risks associated with the current study are deemed moderate for the following 
reasons: 
1. We do not view the risks associated with the radiotracer [11C]PHNO as minimal.
2. We do not view the risks associated with the combined use of ________and_________ as 
minimal.
3. Given the now established safety and validity of the current _________ in our prior work, 
we do not view the proposed studies as high risk.
4. Given our experience with the combined co-administration_________, we do not view 
the proposed studies as high risk.
Although we have assessed the proposed study as one of moderate risk, the potential exists for 
anticipated and/or unanticipated adverse events, serious or otherwise, to occur since it is not 
possible to predict with certainty the absolute risk in any given individual or in advance of first-
hand experience with the proposed study methods. Therefore, we provide a plan for monitoring 
the data and safety of the proposed study as follows:
3. Attribution of Adverse Events:
Adverse events will be monitored for each subject participating in the study and attributed to the 
study procedures / design by the principal investigator Kelly Cosgrove, Ph.D. according to the 
following categories:
a.) Definite: Adverse event is clearly related to investigational procedures(s)/agent(s).
b.) Probable: Adverse event is likely related to investigational procedures(s)/agent(s).
c.) Possible: Adverse event may be related to investigational procedures(s)/agent(s).
d.) Unlikely: Adverse event is likely not to be related to the investigational 
procedures(s)/agent(s).
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e.) Unrelated: Adverse event is clearly not related to investigational procedures(s)/agent(s).
4. Plan for Grading Adverse Events:
The following scale will be used in grading the severity of adverse events noted during the study:
1. Mild adverse event
2. Moderate adverse event
3. Severe 
5. Plan for Determining Seriousness of Adverse Events:
Serious Adverse Events:
In addition to grading the adverse event, the PI will determine whether the adverse event meets 
the criteria for a Serious Adverse Event (SAE).  An adverse event is considered serious if it:
1. is life-threatening OR
2. results in in-patient hospitalization or prolongation of existing hospitalization OR
3. results in persistent or significant disability or incapacity OR
4. results in a congenital anomaly or birth defect OR
5. results in death OR
6. based upon appropriate medical judgment, may jeopardize the subject’s health and may 
require medical or surgical intervention to prevent one of the other outcomes listed in 
this definition, OR
7. adversely affects the risk/benefit ratio of the study
An adverse event may be graded as severe but still not meet the criteria for a Serious Adverse 
Event.  Similarly, an adverse event may be graded as moderate but still meet the criteria for an 
SAE.  It is important for the PI to consider the grade of the event as well as its “seriousness” 
when determining whether reporting to the HIIRB is necessary.
6. Plan for reporting serious AND unanticipated AND related adverse events, anticipated 
adverse events occurring at a greater frequency than expected, and other unanticipated 
problems involving risks to subjects or others to the IRB
The investigator will report the following types of adverse events to the IRB: a) serious AND 
unanticipated AND possibly, probably or definitely related events; b) anticipated adverse events 
occurring with a greater frequency than expected; and c) other unanticipated problems involving 
risks to subjects or others.
These adverse events or unanticipated problems involving risks to subjects or others will be 
reported to the IRB within 48 hours of it becoming known to the investigator, using the 
appropriate forms found on the website.
7. Plan for reporting adverse events to co-investigators on the study, as appropriate the 
protocol’s research monitor(s), e.g., industrial sponsor, Yale Cancer Center Data and 
Safety Monitoring Committee (DSMC), Protocol Review Committee (PRC), DSMBs, study 
sponsors, funding and regulatory agencies, and regulatory and decision-making bodies.
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For the current study, the following individuals, funding, and/or regulatory
agencies will be notified:
□ All Co-Investigators listed on the protocol.
□ Safety Monitoring Committee (DSMC)
□ National Institutes of Health
The principal investigator Kelly Cosgrove, Ph.D. will conduct a review of all adverse events 
upon completion of every study subject. The principal investigator will evaluate the frequency 
and severity of the adverse events and determine if modifications to the protocol or consent form 
are required.
11. Statistical Considerations: Describe the targeted number of subjects and the statistical 
     analyses that support the study design. 
Demographics between smokers and nonsmokers will be compared using t tests or Chi-Square 
tests, as appropriate. Scores on psychological tests such as depressive symptoms at baseline will 
be compared between groups and may be used as covariates in subsequent analyses. All 
outcomes will be summarized descriptively and assessed for normality prior to analysis using 
normal probability plots and Kolmogorov test statistics. Transformations or nonparametric 
analyses will be performed as necessary. All tests will be two-sided and considered statistically 
significant at alpha = 0.05. Post-hoc comparisons will be conducted as necessary with 
significance levels adjusted for multiple comparisons basing the adjustment on the number of 
conceptually related statistical tests within each hypothesis. All statistical analyses will be 
conducted using SAS version 9.3 (Cary, NC).
Aim 1. To determine if amphetamine-induced DA release is blunted during abstinence in 
tobacco smokers compared to nonsmokers. Hypothesis 1. We hypothesize that amphetamine-
induced striatal DA release will be blunted in smokers compared to nonsmokers. Power 
Calculations for Hypothesis 1: Our preliminary data revealed blunted amphetamine-induced DA 
release (d=0.91) in the ventral striatum among smokers (-21%±12.0) compared to nonsmokers (-
29%±6.3). Assuming a two-sided t-test with α=0.05, 40 nonsmokers and 50 smokers assessed 
after 14-21 days of abstinence will provide 80% statistical power to detect effect sizes as small as 
d=0.63, which compares favorably with the effects observed above. 
Regional [11C]PHNO uptake for each subject will be quantified as BPND described above for 
each ROI and will represent the primary outcome measure for the proposed experiments.  
Repeated measures ANOVA will be used to evaluate the change between scans (deltaBP) for 
brain measures for the scans.  Delta BP will be computed as [(BPbaseline-BPcondition)/BPbaseline X 
100]. Analysis will be conducted using SAS version 9.1 (Cary, NC). N=20 subjects per group is 
necessary to detect differences between smokers and nonsmokers. Potential associations, e.g., 
relationship between magnitude of dopamine release and time to relapse, will be examined using 
correlation analysis with Type I error corrected for multiple comparisons. Exploratory analyses 
of the other cognitive and behavioral measures and their relationship to relapse will also be 
performed. 
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SECTION III: RESEARCH INVOLVING DRUGS, BIOLOGICS, RADIOTRACERS, PLACEBOS AND 
DEVICES
           If this section (or one of its parts, A or B) is not applicable, state N/A and delete the rest of the 
section.
DRUGS, BIOLOGICS and RADIOTRACERS
12. Identification of Drug, Biologic or Radiotracer: What is (are) the name(s) of the drug(s),
biologic(s) or radiotracer(s) being used? Identify whether FDA approval has been granted and for what 
indication(s). 
[11C]PHNO, IV, radioactivity dose of no more than 10 millicuries for one injection. [11C]PHNO has 
been used in humans and has been shown to be safe and well tolerated after its administration to healthy 
subjects or patients.  No serious adverse effects are expected from tracer doses, which is one thousand 
fold ≤ the pharmacological used during the therapeutic trials of PHNO.  This will be used under the PET 
Center’s approved IND#134138 for the radiotracer [11C]PHNO. 
d-Amphetamine, dose 0.5 mg/kg, PO by study physician to participants so no IND necessary per 21 
CFR 312.2(b).
All protocols which utilize a drug, biologic or radiotracer not approved by, but regulated by, the FDA 
must provide the following information:  
   
a.   What is the Investigational New Drug (IND) number assigned by the FDA? 134138
b.  Who holds the IND? Yale PET Center
c. All protocols which utilize a radiotracer not approved by, but regulated by the FDA must 
provide the IND number:
Alternatively, use of the investigational radiotracer may be under RDRC/RSC oversight: (check 
if appropriate)_____________
For all investigational radiotracers, attach a copy of the RDRC/RSC application ( for 
radioisotopes used in the PET Center, PET Center personnel may complete this step)
Go to http://rsc.med.yale.edu/login.asp?url=myApps.asp.  When you have logged in, complete 
the application and attach a copy to this submission.     
Alternatively, an exemption from IND filing requirements may be sought for a clinical
 investigation of a drug product that is lawfully marketed in the United States. If there is no IND and
 an exemption is being sought, review the following categories and complete the category that applies
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 (and delete the inapplicable categories):
Exempt Category 1 (d-Amphetamine)
The clinical investigation of a drug product that is lawfully marketed in the United States can be
exempt from IND regulations if all of the following are yes:
i.   The intention of the investigation is NOT to report to the FDA as a well-controlled study in support
     of a new indication for use or to be used to support any other significant change in the labeling for
     the drug.  
 Yes  
 No
ii. The drug that is undergoing investigation is lawfully marketed as a prescription drug product, and
      the intention of the investigation is NOT to support a significant change in the advertising for the
      product. 
 Yes  
 No
iii.  The investigation does NOT involve a route of administration or dosage level or use in populations
      or other factor that significantly increases the risks (or decreases the acceptability of the risks)   
      associated with the use of the drug product. 
 Yes  
 No
iv. The investigation will be conducted in compliance with the requirements for institutional (HIC)   
      review and with the requirements for informed consent of the FDA regulations (21 CFR Part 50  
and 21 CFR Part 56). 
 Yes  
 No
v.   The investigation will be conducted in compliance with the requirements regarding promotion and 
charging for investigational drugs. 
 Yes  
 No
Exempt Category 2 (all items i, ii, and iii must be checked to grant a category 2 exemption)
 i. The clinical investigation is for an in vitro diagnostic biological product that involves one or
more of the following (check all that apply):
 Blood grouping serum
 Reagent red blood cells 
 Anti-human globulin
 ii. The diagnostic test is intended to be used in a diagnostic procedure that confirms the
diagnosis made by another, medically established, diagnostic product or procedure; and
 iii. The diagnostic test is shipped in compliance with 21 CFR §312.160.
Exempt Category 3
 The drug is intended solely for tests in vitro or in laboratory research animals if shipped in 
accordance with 21 CFR 312.60
Exempt Category 4
 A clinical investigation involving use of a placebo if the investigation does not otherwise 
require submission of an IND.
1. Background Information: Provide a description of previous human use, known risks, and 
data addressing dosage(s), interval(s), route(s) of administration, and any other factors that 
might influence risks. If this is the first time this drug is being administered to humans, 
include relevant data on animal models. 
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Previous human use of [11C]PHNO
The use of [11C]PHNO in PET imaging in human was first reported in 2006 (37). No relevant 
abnormalities in blood pressure, heart rate, or ECG were reported at any time in the study. There 
also were no relevant findings in physical or neurological exams or in routine blood and urine 
analyses during the study. However nausea was reported approximately 2–3 min after tracer 
injection, which subsided rapidly 2–3 min later. This effect was attributed to the transient high 
[11C]PHNO concentration resulting from the bolus injection may have been responsible for the 
reported transient self-limiting nausea (37).
[11C]PHNO has been used extensively used in many clinical trials. A survey of the 
ClinicalTrials.gov website shows 12 completed trials, 5 recruiting trials, and 1 active clinical 
trial with [11C]PHNO investigating a variety of behaviors and disorders: alcohol drinking, 
addictive behavior, behavioral symptoms, cocaine-related disorders, compulsive behavior, 
depression, depressive disorder, drinking behavior, impulsive behavior, mental disorders, mood 
disorders, obsessive-compulsive disorder, psychotic disorders, schizophrenia studies, 
schizophrenia spectrum and other psychotic disorders, psychological stress, substance-related 
disorders, and tobacco use disorder. 
[11C]PHNO has been used in humans at the Yale University PET center in several studies 
including HIC 0910005822. To date, more than 250 human [11C]PHNO scans have been 
conducted at Yale PET Center. All injected doses were limited to ≤ 0.03 µg/kg. These studies 
included smokers, cocaine users, pathological gamblers, schizophrenia subjects, and healthy 
controls. Adverse events were observed in studies which involved administration of 
amphetamine in addition to [11C]PHNO. The typical protocol involved a morning [11C]PHNO 
administration, followed by oral amphetamine, followed by a second PHNO injection, starting 3 
hours post-amphetamine. Because nausea is attributed to the transient high [11C]PHNO 
concentration 2–3 min after the bolus injection of the tracer (37), the Yale PET Center 
administers [11C]PHNO as a bolus over a period of 5 minutes, i.e., at a PHNO dose rate of 7 
ng/min for the average 70 kg subject. Administration of [11C]PHNO dose at this rate is about 5 
times slower than lowest rate reported in the clinical study involving i.v. administration of 
PHNO (38).
Proposed single study dose limit
[11C]PHNO will be administered intravenously.
The proposed radioactivity dose of 10 mCi per single administration is below the 21 CFR 361.1 
estimated dose limit of 75.5 mCi for a 70 kg Hermaphrodite Male (77.7 mCi for female). The 
estimated dose limit calculations are based upon the liver as the critical organ; 5 rem per single 
study limit: 0.0662 rem per mCi to the combined male and female livers. 
The maximum allowable dose for a single injection is 3000 mR to the whole body, active blood-
forming organs, lens of the eye and gonads. The dose to any other organ cannot exceed 5000 
mR.
The maximum allowable dose for one year is 5000 mR to the whole body, active blood-forming 
organs, lens of the eye and gonads. The dose to any other organ cannot exceed 15000 mR.
For comparison, the average person in the United States receives a radiation exposure of 0.3 rem 
(or 300 mrem) per year from natural background sources, such as from the sun, outer space, and 
from radioactive materials that are found naturally in the earth’s air and soil. The dose that a 
subject will receive from participation in this research study would be less than that obtained in 
one year from natural sources.
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As previously mentioned, the maximum amount of radiation per year an individual subject will 
receive in this study is from up to 8 injections of ≤ 10mCi each of [11C]PHNO, plus transmission 
scans. The intended amount is from up to 4 injections of ≤ 10mCi each of [11C]PHNO for 
smokers and up to 2 injections of  ≤ 10mCi each of [11C]PHNO in nonsmokers, however in case 
of scan failures post injection we have included up to 8 injections if scan sets need to be repeated 
within the study time frame (i.e. 1-2 wks withdrawal and 2-8 wks withdrawal). Nonsmokers 
maximum number of injections including re-scans would be 4 scans total. Should the second 
scan of the set fail, we will make every effort to reschedule the scan for repeat within 1 wk, 
however if scheduling does not permit this, we may ask the subject to repeat the whole set due to 
timing related data. 
2 injections PHNO
0.4292 rem
4 injections PHNO
0.8584 rem
8 injections PHNO
1.7168 rem
d-Amphetamine:
Amphetamine is administered to measure changes in [11C]PHNO binding due to dopamine release. 
This dose was chosen because it is expected to produce a quantifiable displacement of the 
radiotracer. The risks are outlined in Section VI.  Oral and IV d-amphetamine administration to 
healthy humans and individuals with psychiatric disorders has been safely used in many PET 
imaging studies. We will use the oral route due to the greater safety and ease of administration.
3. Source: a) Identify the source of the drug or biologic to be used. 
 [11C]PHNO will be synthesized at the Yale University PET Center radiochemistry Laboratory 
under the supervision of Drs. Henry Huang & Nabeel Nabulsi.
d-amphetamine will be provided by the YNHH pharmacy.
b) Is the drug provided free of charge to subjects? 
 Yes  
 No
    If yes, by whom? PET center
4. Storage, Preparation and Use: Describe the method of storage, preparation, stability information, 
and for parenteral products, method of sterilization and method of testing sterility and 
pyrogenicity.
[11C]PHNO
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Due to the short half-life, PET drugs are prepared and formulated immediately before 
administration, and therefore there are no issues with storage or stability. PET drug products are 
stored at room temperature and are stable for at least 60 min after preparation.
The preparation of sterile PET drug products is validated prior to human use. Sterility is achieved 
by passing the PET drug product through a 0.22 micron membrane filter during the last step of the 
formulation process. Prior to release for administration, a bubble point test is performed on the 
membrane filter used for terminal sterilization in order to validate and verify its integrity during 
the filtration process. Due to the short half-life, a sample of the PET drug product is tested for 
sterility after administration for further confirmation.
The level of endotoxin in each batch of the final PET drug product is determined quantitatively 
prior to release for administration using the FDA approved Charles River Laboratory’s Portable 
Testing System (Endosafe®-PTS).
[11C]PHNO will be prepared at the Yale PET Center in accordance with local Chemistry 
Manufacturing & Control (CMC) procedures and quality specifications described in
local FDA-approved Drug Master File (IND No. 134138). Briefly, [11C]propionyl chloride is 
prepared by reaction of [11C]CO2 with ethylmagnesium bromide, followed by treatment with 
phthaloyl dichloride. [11C]propionyl chloride then reacts with 9-hydroxynaphthoxazine to generate 
a [11C]-amide, which is subsequently reduced by lithium aluminium hydride to yield [11C]PHNO. 
The resulting PET drug product is purified first by semi-preparative HPLC, followed by solid-
phase extraction to remove the HPLC buffer mixture. Finally [11C]PHNO is formulated in <10% 
ethanolic saline solution (USP), and the resulting PET drug product is then passed through a 0.22 
micron sterile membrane filter for terminal sterilization and collected in a sterile pyrogen free 
collection vial to afford a formulated I.V. solution ready for dispensing and administration.
Amphetamine 
Amphetamine will be stored and prepared at Yale New Haven Hospital pharmacy.
Check applicable Investigational Drug Service utilized:
 
 YNHH IDS
 Yale Cancer Center 
 CMHC Pharmacy
 West Haven VA
 PET Center    
 None
      
 Other:
Note: If the YNHH IDS (or comparable service at CMHC or WHVA) will not be utilized, explain in 
detail how the PI will oversee these aspects of drug accountability, storage, and preparation.  
  
5. Use of Placebo: 
 Not applicable to this research project
If use of a placebo is planned, provide a justification which addresses the following:
a.
Describe the safety and efficacy of other available therapies. If there are no other
available therapies, state this.
b.   State the maximum total length of time a participant may receive placebo while on the study. 
c.   Address the greatest potential harm that may come to a participant as a result of  receiving    
  
   placebo. 
d.   Describe the procedures that are in place to safeguard participants receiving placebo. 
6. Use of Controlled Substances:
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Will this research project involve the use of controlled substances in human subjects?
 Yes  
 No See HIC Application Instructions to view controlled substance listings. 
If yes, is the use of the controlled substance considered:
 Therapeutic: The use of the controlled substance, within the context of the research, has the 
potential to benefit the research participant.
 Non-Therapeutic: Note, the use of a controlled substance in a non-therapeutic research study 
involving human subjects may require that the investigator obtain a Laboratory Research License. 
Examples include controlled substances used for basic imaging, observation or biochemical 
studies or other non-therapeutic purposes. See Instructions for further information.
Dextro-amphetamine (0.5 mg/kg) will be given by mouth to each subject 3 hours prior to the second 
[11C]PHNO PET scan.
7. Continuation of Drug Therapy After Study Closure   
 Not applicable to this project
Are subjects provided the opportunity to continue to receive the study drug(s) after the study has 
ended?
 
 Yes   If yes, describe the conditions under which continued access to study drug(s) may apply 
as well as conditions for termination of such access. 
 
 No    If no, explain why this is acceptable.
SECTION IV: RECRUITMENT/CONSENT AND ASSENT PROCEDURES 
1.  Targeted Enrollment: Give the number of subjects
a.
  targeted for enrollment at Yale for this protocol: 100
b.    If this is a multi-site study, give the total number of subjects targeted across all 
sites___ 
2. Indicate recruitment methods below.  Attach copies of any recruitment materials that will 
be used.
 Flyers
 Internet/Web Postings
 Radio
 Posters
 Mass E-mail Solicitation
 Telephone
 Letter
 
 Departmental/Center Website
 Television
 Medical Record Review
 Departmental/Center Research Boards
 Newspaper
 Departmental/Center Newsletters
 Web-Based Clinical Trial Registries
 YCCI Recruitment Database
 Clinicaltrials.gov Registry (do not send materials to HIC)
 Other (describe):
3.  Recruitment Procedures:
a.
Describe how potential subjects will be identified.  
b. Describe how potential subjects are contacted.  
c.
Who is recruiting potential subjects?   
Subjects will be recruited through flyers, public advertisement (newspaper, radio, internet 
posting), by word of mouth, contact with community service groups, and clinics and local 
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treatment facilities (the VA Hospital, West Haven, CMHC, the Yale Psychiatric Hospital, Mood 
Disorders Research Program, the Yale Depression Research Program).  The subjects will be 
asked to call us if they are interested in participating in the research study.  The PI, in 
collaboration with study investigators, is responsible for subject recruitment.
4. Screening Procedures
a. Will email or telephone correspondence be used to screen potential subjects for eligibility 
prior to the potential subject coming to the research office? 
 Yes  
 No
b. If yes, identify any health information and check off any of the following HIPAA 
identifiers to be collected and retained by the research team during this screening process. 
HEALTH INFORMATION TO BE COLLECTED: We will ask about general health, neurological disorders, past 
surgeries, past injuries especially to the head, psychological history, immediate family psychological history. 
HIPAA identifiers: 
 Names 
 All geographic subdivisions smaller than a State, including: street address, city, county, precinct, zip codes and their 
equivalent geocodes, except for the initial three digits of a zip code if, according to the current publicly-available data from 
the Bureau of the Census: (1) the geographic unit formed by combining all zip codes with the same three initial digits contains 
more than 20,000 people, and (2) the initial three digits of a zip code for all such geographic units containing 20,000 or fewer 
people is changed to 000. 
  Telephone numbers
 Fax numbers 
 E-mail addresses
 Social Security numbers 
 Medical record numbers
 Health plan beneficiary numbers 
 Account numbers 
  All elements of dates (except year) for dates related to an individual, including: birth date, admission date, discharge 
date, date of death, all ages over 89 and all elements of dates (including year) indicative of such age, except that such ages 
and elements may be aggregated into a single category of age 90 or older 
 Certificate/license numbers 
 Vehicle identifiers and serial numbers, including license plate numbers 
 Device identifiers and serial numbers 
 Web Universal Resource Locators (URLs) 
 Internet Protocol (IP) address numbers 
 Biometric identifiers, including finger and voice prints 
 Full face photographic images and any comparable images 
 Any other unique identifying numbers, characteristics, or codes 
5. Assessment of Current Health Provider Relationship for HIPAA Consideration:
Does the Investigator or any member of the research team have a direct existing clinical 
relationship with any potential subject? 
 Yes, all subjects
 Yes, some of the subjects
 No
If yes, describe the nature of this relationship.
6. Request for waiver of HIPAA authorization: (When requesting a waiver of HIPAA 
Authorization for either the entire study, or for recruitment purposes only.  Note: if you are 
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collecting PHI as part of a phone or email screen, you must request a HIPAA waiver for 
recruitment purposes.)
Choose one: For entire study: ______ For recruitment purposes only: ___X___
i. Describe why it would be impracticable to obtain the subject’s authorization for 
use/disclosure of this data;
ii. If requesting a waiver of signed authorization, describe why it would be 
impracticable to obtain the subject’s signed authorization for use/disclosure of this 
data;
By signing this protocol application, the investigator assures that the protected 
health information for which a Waiver of Authorization has been requested will not 
be reused or disclosed to any person or entity other than those listed in this 
application, except as required by law, for authorized oversight of this research 
study, or as specifically approved for use in another study by an IRB.
Researchers are reminded that unauthorized disclosures of PHI to individuals outside of the Yale 
HIPAA-Covered entity must be accounted for in the “accounting for disclosures log”, by subject 
name, purpose, date, recipients, and a description of information provided.  Logs are to be 
forwarded to the Deputy HIPAA Privacy Officer.
 
7. Required HIPAA Authorization: If the research involves the creation, use or disclosure of  
protected health information (PHI), separate subject authorization is required under the 
HIPAA Privacy Rule. Indicate which of the following forms are being provided:
 Compound Consent and Authorization form
 HIPAA Research Authorization Form
8. Consent Personnel: List the names of all members of the research team who will be 
obtaining consent/assent:
Located in IRES
9. Process of Consent/Assent: Describe the setting and conditions under which consent/assent    
    will be obtained, including parental permission or surrogate permission and the steps taken to 
   ensure subjects’ independent decision-making. 
The consent process is a multistep process, whereby information about the risks and 
benefits of the study will be provided to potential subjects across several sessions. The number of 
sessions over which this information will be provided will depend on how well the subject 
understands and retains the information. The process begins with the subject initiating contact 
via telephone. The research staff will provide a brief description of the study following which the 
subject is screened by a member of the research team. Thereafter, potentially eligible candidates 
are scheduled for a face-to-face interview. The study procedures will be described as a research 
tool with potential to enhance our knowledge about the brain. Subjects will also be informed of 
all potential risks of participation. Subjects will be required to read the informed consent form 
and the investigator will additionally describe the risks and discomforts. 
To ensure that the study subject understands the study, the subject will be asked questions 
about the study procedures and the risks associated with participation. If any concern arises that 
the study subject did not fully understand the study, the principal investigator (PI) may decide 
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that the subject is not suitable for participation. This process generally takes about one hour. If 
the subject is still interested after all questions have been answered, the PI or staff member 
consenting, will ask the subject to sign the informed consent form. Any subject who appears 
incapable of providing informed consent will be excluded. Subjects will be informed that they 
can decline to participate in the study without penalty and given the opportunity to withdraw 
from the study prior to analysis of their data. Following the resolution of any questions, the 
subjects will be asked to sign the consent form if he/she agrees to participate.
 The decision not to participate will not affect an individual’s eligibility to participate in future 
studies, to receive treatment at Yale-New Haven Hospital, or to receive treatment on a private 
basis from a referring clinician.  A copy of the signed consent form will be provided to all 
participating subjects.  For subjects who are not eligible, all PHI will be destroyed.
10. Evaluation of Subject(s) Capacity to Provide Informed Consent/Assent: Indicate how the 
personnel obtaining consent will assess the potential subject’s ability and capacity to consent 
to the research being proposed. 
In cases in which capacity is in doubt, the PI will assess the subject’s understanding of the study 
and the subject’s capacity to decide to participate.
11.   Documentation of Consent/Assent: Specify the documents that will be used during the 
   consent/assent process. Copies of all documents should be appended to the protocol, in the 
   same format that they will be given to subjects. 
Compound Authorization Form
12. Non-English Speaking Subjects: Explain provisions in place to ensure comprehension for 
   research involving non-English speaking subjects. Translated copies of all consent materials 
   must be submitted for approval prior to use. 
Non-English speaking subjects will not be invited to participate in the studies.  All of our 
materials are in English only, and staff members are fluent in English.  Furthermore, cognitive 
testing is validated in English-speaking subjects only.
13. Consent Waiver: In certain circumstances, the HIC may grant a waiver of signed 
consent, or a full waiver of consent, depending on the study. If you will request either a 
waiver of consent, or a waiver of signed consent for this study, complete the appropriate 
section below.  
 
 Not Requesting a consent waiver 
 
 Requesting a waiver of signed consent
       
 Requesting a full waiver of consent
   
A. Waiver of signed consent: (Verbal consent from subjects will be obtained. If PHI is 
collected, information in this section must match Section IV, Question 6)
 Requesting a waiver of signed consent for Recruitment/Screening only 
If requesting a waiver of signed consent, please address the following:
a. Would the signed consent form be the only record linking the subject and the research? 
 Yes  
 No
b. Does a breach of confidentiality constitute the principal risk to subjects? 
 Yes  
 No
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OR
c. Does the research activity pose greater than minimal risk? 
 Yes If you answered yes, stop. A waiver cannot be granted.  Please note: 
Recruitment/screening is generally a minimal risk research activity  
 No 
AND
d. Does the research include any activities that would require signed consent in a non-
research context? 
 Yes  
 No
 Requesting a waiver of signed consent for the Entire Study (Note that an information 
sheet may be required.)
If requesting a waiver of signed consent, please address the following:
a. Would the signed consent form be the only record linking the subject and the research? 
 Yes  
 No
b. Does a breach of confidentiality constitute the principal risk to subjects? 
 Yes  
 No
OR
c. Does the research pose greater than minimal risk? 
 Yes  If you answered yes, stop. 
A waiver cannot be granted.  
 No 
AND
d. Does the research include any activities that would require signed consent in a non-
research context? 
 Yes  
 No
B. Full waiver of consent: (No consent from subjects will be obtained for the activity.) 
 Requesting a waiver of consent for Recruitment/Screening only 
a. Does the research activity pose greater than minimal risk to subjects?  
 Yes  If you answered yes, stop. A waiver cannot be granted. Please note: 
Recruitment/screening is generally a minimal risk research activity 
 No
b. Will the waiver adversely affect subjects’ rights and welfare? 
 Yes  
 No
c. Why would the research be impracticable to conduct without the waiver? 
d. Where appropriate, how will pertinent information be returned to, or shared with 
subjects at a later date? 
 Requesting a full waiver of consent for the Entire Study (Note: If PHI is 
collected, information here must match Section IV, question 6.)
If requesting a full waiver of consent, please address the following:
a. Does the research pose greater than minimal risk to subjects?  
 Yes If you answered 
yes, stop. A waiver cannot be granted.   
 No
b. Will the waiver adversely affect subjects’ rights and welfare? 
 Yes  
 No
c. Why would the research be impracticable to conduct without the waiver? 
d. Where appropriate, how will pertinent information be returned to, or shared with 
subjects at a later date?  
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SECTION V: PROTECTION OF RESEARCH SUBJECTS
  Confidentiality & Security of Data:
a.    
What protected health information (medical information along with the HIPAA 
identifiers) about subjects will be collected and used for the research?   
       
Required private identifiable information about individuals, such as their medical history, current 
medications, psychiatric problems, and family history, will be collected by research staff and be 
used for research purposes and charting after consent is obtained. Identifiers collected may 
include name, birthdate, and social security number. 
b.    How will the research data be collected, recorded and stored? 
The data are collected and recorded by trained research personnel. The data will be recorded on 
Excel spreadsheets that will be saved onto a server or will be in the form of questionnaires that 
are filled out by the subject or the researcher. These paper research materials containing 
confidential information are stored in locked filing cabinets.  Additional brain data is collected 
during the brain imaging scans by trained technologists and is stored on password-protected and 
encrypted computers with identifying information carefully in compliance with HIPAA 
regulations. 
c.    How will the digital data be stored? 
 CD  
 DVD  
 Flash Drive  
 Portable Hard 
Drive  
 Secured Server  
 Laptop Computer  
 Desktop Computer  
 Other
d.  What methods and procedures will be used to safeguard the confidentiality and security of 
the identifiable study data and the storage media indicated above during and after the subject’s 
participation in the study?
Do all portable devices contain encryption software? 
 Yes   
 No
      
   If no, see http://hipaa.yale.edu/guidance/policy.html
 
All staff members that come into contact with the data are fully trained to the current HIPAA 
regulations and are informed as to the proper use of all data.
Identifiable paper information is kept in locked file drawers and password protected computer 
files.  Results are published as group data without the use of characteristics that would identify 
individual subjects.  We quote information only by number in conference discussions, scientific 
reports, or publications, in order to maintain anonymity.
Identifiable research data, including recruitment and screening information and code keys, are 
stored on a secure database located on the internal PET Center Network.  The PET network is 
protected by a Cisco PIX firewall operated by ITS.  All research data are backed up nightly to a 
Dell PV-136T library wit 4 IBM Ultrium-TD2 tape drives using the backup software Legato 
Networker 7.3 from EMC.  Human subjects enrolled in the study are assigned a subject-specific 
random identifier.   Subject identifiers and the means to link the subject names and codes with 
the research data are stored in separate locations within the database. The software of the 
database limits the ability to connect the random identifier to the actual subject identification 
information to research team members only.  Access to the database is password protected and 
each research team member is required to have a unique ID and password to gain access to the 
database.  Authorized users employ their netid and authentication is performed using Yale’s 
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central authentication server.  Users always access research data through the random identifier 
only. Direct identifiers belonging to subjects who withdraw from the study, will be stripped from 
the key.
e.
What will be done with the data when the research is completed? Are there plans to destroy 
the identifiable data? If yes, describe how, by whom and when identifiers will be destroyed. If 
no, describe how the data and/or identifiers will be secured.
The data will be stored in locked filing cabinets and on the password-protected secure database 
on the internal Yale University PET Center Network for at least 7 years, accessed only by 
authorized personnel.
f.  Who will have access to the protected health information (such as the research sponsor, the 
investigator, the research staff, all research monitors, FDA, Yale Cancer Center Data and 
Safety Monitoring Committee (DSMC), SSC, etc.)? (please distinguish between PHI and de-
identified data) 
The investigator and research staff (e.g., PET center nuclear technologists, recruiters) will have 
access to the PHI only on as needed to know basis.  The FDA may also have access to the PHI.
g.   If appropriate, has a Certificate of Confidentiality been obtained?
This protocol is funded by NIH. As such, according to the NIH policy issues in October 2017, 
the information collected from subjects is automatically protected by a Certificate of 
Confidentiality (CoC). 
h.   Are any of the study procedures likely to yield information subject to mandatory reporting 
requirements? (e.g. HIV testing – reporting of communicable diseases; parent interview -
incidents of child abuse, elderly abuse, etc.). Please verify to whom such instances will need 
to be reported. 
No.
SECTION VI: POTENTIAL BENEFITS
Potential Benefits: Identify any benefits that may be reasonably expected to result from the 
research, either to the subject(s) or to society at large. (Note: Payment of subjects is not 
considered a benefit in this context of the risk-benefit assessment.) 
There are no direct benefits to the nonsmoking subjects for participating in this study.  Smoking 
subjects receive smoking cessation support to abstain from tobacco smoking. This research will 
benefit scientific knowledge by contributing to the understanding of the neurochemical changes 
that occur during the recovery from tobacco smoking.  This may have clinical application in the 
future.
         SECTION VII: RESEARCH ALTERNATIVES AND ECONOMIC CONSIDERATIONS
       1.   Alternatives: What alternatives are available to the study subjects outside of the
              research?
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The alternative to participation in this research protocol is to not participate.  Subjects will be 
informed that they are free to choose not to participate and, if they do agree to become a subject, 
they will be free to withdraw from the study at any time during its course.  They will also be 
informed that if they choose not to participate or if they withdraw, it will not adversely affect 
their relationship with their doctors or the hospital (see attached Consent Form).
       2.   Payments for Participation (Economic Considerations): Describe any payments that will
             be made to subjects and the conditions for receiving this compensation. 
The subjects will be compensated for their time commitment and inconveniences necessary for 
completing the study. Subjects will have no financial responsibilities for any portion of the study. 
For Aim 1 payment would be $350 for each PHNO PET scan, $50 for each MRI scan, and $40 
for cognitive testing at baseline.  Subjects who participate in the Probabilistic Reward Task may 
also be compensated for the amount that they “win” during the task, up to $50. Subjects may also 
receive an extra $10 for each Cold Pressor Task that they participate in. Subjects will be paid 
either by check, and are advised to allow 4-6 weeks for receipt of payment, or they will be given 
a credit card or cash.  In addition, subjects will be provided with a light meal, at the end of the 
PET imaging day.  Reasonable transportation costs will be reimbursed.  Receipts must be 
submitted. If participation in the PET Scan has already begun, then compensation will be based 
on involvement in the study, and will be up to the discretion of the PI.
Smokers will also receive $10 for each contingency management appointment at which their 
carbon monoxide and urine cotinine levels show that they have not smoked.  Subjects can decide 
if they want to receive this amount at each appointment in cash, or receive it all at the end of the 
study in the form of a credit card or check or cash.  Smokers will receive an additional $100 
bonus for completing the study. 
Cancellations:  If a PET scan should get cancelled for a reason outside of the subject's control 
(i.e. radiotracer synthesis failure) the subject will be paid $50 minimum, or a higher amount not 
to exceed the payment for a full scan day.  The amount of the payment for cancellation will be 
based on the subject's length of participation on that scan day prior to the cancellation, and will 
be up to the discretion of the PI.
       3.   Costs for Participation (Economic Considerations): Clearly describe the subject’s costs
             associated with participation in the research, and the interventions or procedures of the study
             that will be provided at no cost to subjects.   
There will be no costs to subjects related to participation in this research intervention.
  
4.   In Case of Injury: This section is required for any research involving more than minimal 
      risk.
a.     Will medical treatment be available if research-related injury occurs? 
b.     Where and from whom may treatment be obtained? 
c.     Are there any limits to the treatment being provided? 
d.     Who will pay for this treatment? 
e.     How will the medical treatment be accessed by subjects? 
Medical treatment will be offered to the subjects for any physical injuries that they receive as a 
result of participating in this research.  However, the subject or his/her insurance company is 
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responsible for the cost.  Federal regulations require that subjects be told that if they are 
physically injured, no additional financial compensation is available.
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