Brain Networks and Addiction Susceptibility

Objective. Identification of genetic risk factors predisposing to nicotine abuse and
dependence, and elucidation of their neurobiological mechanisms of action, is critical to
individualized treatments and prevention of nicotine addiction. Nicotine exerts its effects
on the brain through nicotinic acetylcholine receptors (nAChRs). The non-synonymous single
nucleotide polymorphism (SNP) rs16969968 in the CHRNA5 gene encoding the 5 subunit of nAChRs
has been unequivocally associated with smoking behavior and nicotine dependence in
genome-wide association studies (GWAS). At the brain level, the salience network (SN) or the
neural circuitry connecting the anterior insula (AI), dorsal anterior cingulate cortex
(dACC), the ventral striatum (VS), and extended amygdala has been shown to be crucially
involved in nicotine addiction. The SN detects salient events and initiates a rapid switch
between large-scale brain networks, the default-mode network (DMN) and executive control
network (ECN), in control of behavior. Genetic influences on the SN may therefore explain
some of the individual differences in susceptibility to addiction. We have previously shown
that resting-state connectivity of the SN is decreased in smokers and non-smokers with the
rs16969968 risk allele. But the underlying neurobiological processes are still unknown. Given
a well-established role of dopamine (DA) in addiction, and the presence of nAChRs on DA
neurons, one plausible mechanism involves cholinergic modulation of DA transmission.
Consequently, we will employ an integrative imaging pharmacogenetics approach to test for DA
mediation of the rs16969968 effects on the SN in healthy non-smoking participants, with the
goal of elucidating the neurobiological mechanism underlying the association between this SNP
and susceptibility to nicotine dependence without the confound of chronic smoking.

Study Population. Sixty pre-screened participants will be classified into two equal groups (n
= 30 per group) based on their rs16969968 genotype: 1) rs16969968 risk allele homozygotes, or
A/A genotype ( Risk Group ); and 2) rs16969968 non-risk allele homozygotes, or G/G genotype (
Non-Risk Group ). Participants will be healthy, right-handed males and females, aged 18-55,
non-smokers and free of lifetime substance dependence.

Design. A double-blind, placebo-controlled crossover design will be used. Each participant
will complete a screening session (under the Screening Protocol 06-DA-N415); an orientation
session, which will include a nicotine patch tolerance test; and 4 imaging visits, each with
a different pharmacological pre-treatment prior to scanning: 1) placebo pill + placebo patch;
2) 20 mg oral methylphenidate + placebo patch; 3) 2 mg oral haloperidol + placebo patch; and
4) placebo pill + 7 mg nicotine patch.

Outcome Measures. The study will use neuroimaging (fMRI) to assess the impact of rs16969968
genotype and drug condition (MPH vs. haloperidol vs. nicotine vs. placebo) on the SN, ECN,
and DMN function at rest and during task performance. The primary outcome measures will be:
1) network coherence, as indexed by resting-state and task-related functional connectivity
(FC); 2) dynamic task-related interactions; and 3) behavioral task performance. We will also
test for genetic effects on 4) self-report measures of impulsivity and other traits
associated with addiction susceptibility.

- INCLUSION CRITERIA:

The inclusion criteria are as follows:

1. Participants must be between 18-55 years of age. Justification: Many neural processes
change with age, including the attributes of, and interactions between, the three
brain networks assessed in the study. In addition, the risk of difficult-to-detect
medical abnormalities such as silent cerebral infarcts increases with age. Assessment
tool(s): driver s license, birth certificate, or other government-issued forms of
identification.

2. Participants must be right-handed. Justification: Some of the neural processes
assessed in this protocol may be lateralized in the brain. In order to reduce
potential variance, participants will be required to be right-handed. Assessment
tool(s): Edinburgh Handedness Inventory.

3. Participants must be in good health. Justification: Many illnesses may alter fMRI
signals as well as neural functioning. Assessment tool(s): Participants will provide a
brief health history during phone screening, and undergo a medical history and
physical examination with an IRP clinician.

4. Participants must be free of lifetime substance dependence, and free of substance
abuse in the last 2 years, for any substance, including nicotine, alcohol,
prescription drugs, and illicit drugs, according to DSM-IV diagnostic criteria. With
respect to tobacco use, participants must not be current smokers and must never have
been daily smokers for more than 1 month. In addition, the MAI and/or PI will use
their medical/scientific judgment on a case-by-case basis to disqualify potential
participants from participation at lower levels of use. Justification: Abuse or
dependence on drugs or alcohol may result in unique CNS deficits that could confound
results. This eligibility criterion is particularly important in the current study,
which examines the neurobiological processes underlying susceptibility to drug
dependence in the absence of, and preceding, chronic use and the development of such
dependence. Assessment tool(s): The computerized SCID and clinical substance
abuse/dependence assessment. While recreational/intermittent use of alcohol and/or
marijuana will be tolerated, individuals will be excluded from participation if they
meet lifetime DSM-IV diagnosis of abuse or dependence.

5. Both male and female participants will be enrolled in the study.

6. While we will target the A/A homozygotes and G/G homozygotes at the rs16969968 locus,
we will also allow A/G heterozygotes to

become enrolled in the study. The enrollment will be open to all racial and ethnic groups,
including Caucasians, African Americans, and Asians, as well as Hispanic and non-Hispanics,
and we will make an effort to include all racial and ethnic groups as long as all
participants meet the rs16969968 genotype criteria. Justification: The allele frequencies
at the rs16969968 locus vary greatly between racial and ethnic groups, making it extremely
difficult to ensure a balance of ethnicities across the genotype groups. In particular, the
minor A allele of rs16969968 (the Risk allele in the current study), associated with
increased risk of heavy smoking and nicotine dependence, has a frequency of 0.42 in
Caucasians, but is very rare in Asians (0.03) and African Americans (0.07) (Saccone et al.,
2010b). Therefore, assuming Hardy-Weinberg Equilibrium, we will need to recruit
approximately 275 Caucasian subjects in order to

meet the enrollment target of n = 40 per genotype group (i.e., 40 A/A and 40 G/G), with the
A/A homozygotes being the limiting factor. For comparison, because the A/A is very rare in
African Americans (0.0049; or 49 in 10,000), we would need to recruit over 8,000 African
American subjects to meet our enrollment target of n = 40 for that ethnic group; if we
aimed at only 20 African American subjects in our sample (closer to the distribution in the
Baltimore area), we would still have to recruit over 4,000 African American subjects. The
frequency of the A/A homozygotes in Asian population is even more daunting (0.0009; or 9 in
10,000). Importantly, because the association between the rs16969968 locus and nicotine
addiction severity has been demonstrated in all three ethnic groups in a recent
meta-analysis (Chen et al., 2012), we argue that the racial and ethnic groups not enrolled
in the current study will still benefit from basic, mechanistic knowledge gained from this
study if translated to clinical treatment and prevention of nicotine abuse and dependence
in the future. The estimated number of potential participants to be screened is 300.
Assessment tools: Genotype group membership will be determined by genotyping. Racial and
ethnic group membership will be initially determined by self-report. Additionally, we will
examine ancestry information markers to verify and extend the self-reported ethnic
background and to test for possible modulatory effects of specific genetic-ancestry groups
with respect to the rs16969968 effects in our data.

EXCLUSION CRITERIA:

Participants will be excluded if they:

1. are not suitable to undergo an fMRI experiment due to certain implanted devices
(cardiac pacemaker or neurostimulator, some artificial joints, metal pins, surgical
clips or other implanted metal parts), body morphology, or claustrophobia.
Justification: MR scanning is one of the primary measurement tools used in the
protocol. Assessment tool(s): Prospective participants will fill out an MRI screening
questionnaire and undergo an interview with an MR technologist. Questions concerning
suitability for scanning will be referred to the MR Medical Safety Officer.
Prospective participants will be questioned about symptoms of claustrophobia and
placed in the mock scanner during their first visit to assess for possible difficulty
tolerating the confinement of the scanner and for ability to fit into the scanner.

2. have coagulopathies, history of, current superficial, or deep vein thrombosis,
musculoskeletal abnormalities restricting an individual s ability to lie flat for
extended periods of time. Justification: MR scanning sessions require participants to
lie flat on their backs and remain perfectly still for approximately two hours.
Therefore, conditions that would make that difficult (e.g. chronic back pain,
significant scoliosis) or dangerous (e.g. familial hypercoagulability syndrome,
history of thrombosis) will be exclusionary. Assessment tool(s): History and physical
examination by an IRP clinician, supplemented with a trial of lying in the mock
scanner to assess comfort issues.

3. have HIV or Syphilis. Justification: HIV and Syphilis both can have central nervous
system (CNS) sequelae, thus introducing unnecessary variability into the data.
Assessment tool(s): Oral HIV followed by blood test if oral test is + and RPR+ (>1:8
without history of adequate treatment).

4. regularly use some prescriptions (e.g., antidepressants, benzodiazepines,
antipsychotics, anticonvulsants, barbiturates), over-the-counter (e.g., cold medicine)
or herbal medications (e.g., Kava, Gingko biloba, St. John s wort) that may alter CNS
function, cardiovascular function, or neuronal-vascular coupling. Justification: The
use of these substances may alter the fMRI signal and/or neural functions of interest
in the current study. Assessment tool(s): History and comprehensive urine drug
screening to detect antidepressants, benzodiazepines, antipsychotics, anticonvulsants,
and barbiturates. The MAI/PI will use their medical/scientific judgment on a
case-by-case basis.

5. currently use moderate or strong CYP3A4 or 2D6 inhibitors not already covered by other
exclusion criteria. Justification: Inhibitors of CYP3A4 or 2D6 may cause mild to
moderately increased haloperidol concentrations when taken concomitantly, thereby
increasing the possibility and/or duration of side effects associated with haloperidol
administration. Assessment tool: All medications reported by participants will be
reviewed for their effects on CYP3A4 and 2D6. Additionally, we will instruct
participants to not consume grapefruit juice (2D6 inhibitor) on study days.

6. have any current, or a history of, neurological illnesses including, but not limited
to, seizure disorders, frequent migraines or on prophylaxis medications, multiple
sclerosis, movement disorders, history of significant head trauma, or CNS tumor.
Justification: Neurological diseases alter CNS function and, possibly, the
neuronal-vascular coupling that forms the basis of the fMRI signal. Assessment
tool(s): History and physical examination by a qualified IRP clinician, urine drug
screening for anticonvulsants not disclosed by history. History of head trauma with
loss of consciousness of more than 30 minutes or with post-concussive sequelae lasting
more than two days, regardless of loss of consciousness, will be exclusionary.

7. have any current, or a history of, major psychiatric disorders, including major
depressive disorder (single past episode with at least three years symptom-free off
medication will be allowed), schizophrenia, bipolar disorder or
attention-deficit/hyperactivity disorder (ADHD), or are currently under antidepressant
or antipsychotic medication treatment. Justification: Psychiatric disorders may be
accompanied by alternations in brain structure and/or function. Assessment tool(s):
Computerized SCID, Beck Depression Inventory, Beck Anxiety Inventory, Adult ADHD
Self-Report Scales and clinical interview confirmation by clinician.

8. are cognitively impaired or learning disabled. Justification: Cognitive impairment and
learning disabilities may be associated with altered brain functioning in regions
recruited during laboratory task performance. Cognitive impairment may affect one s
ability to give informed consent. Assessment tool(s): History examination and Wechsler
Abbreviated Scale of Intelligence (WASI). IQ estimate must be greater than or equal to
85.

9. have significant cardiovascular or cerebrovascular conditions. Justification: Such
conditions may alter blood flow, the fMRI signal and other autonomic signals, and
increase risks associated with nicotine patch use. Assessment tool(s): History and
physical exam, including EKG.

10. have QTc greater than 450. Justification: Haloperidol may increases the risk of
Torsades de Pointes and QTc prolongation. Assessment tool: EKG.

11. have any other major medical condition that in the view of the investigators would
compromise the safety of an individual during participation. Justification: Many
illness not explicitly covered here may increase risk or alter important outcome
measures. Assessment tool(s): History and physical examination by an IRP clinician and
CBC, urinalysis, NIDA chemistry panel (liver function tests, electrolytes, kidney
function). The following lab values will result in exclusion from the study:

1. Hemoglobin < 10 g/dl

2. White Blood Cell Count < 2400/microL

3. Liver Function Tests > 3 times normal

4. Serum glucose > 200 mg/dl

5. Urine protein > 2+

6. Estimated glomerular filtration rate <60ml/min

12. pregnant, planning to become pregnant, or breastfeeding. Justification: Study
procedures and drugs used in the current protocol may complicate pregnancy or be
transferred to nursing children. Assessment tool/s: Urine pregnancy tests will be
conducted at the beginning of each scanning visit.

The MAI will retain discretion to exclude based on less extreme lab results. After the
screening process has been completed, the MAI will take into account all data collected in
order to decide if there is an existing medical illness that would compromise participation
in this research.