Acetylcholine, Tobacco Smoking, Genes and Nicotinic Receptors

- Magnetic resonance image. Within approximately two weeks 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.

- Physostigmine challenge 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 On PHNO PET days, subjects will have a baseline PHNO scan and then
will receive amphetamine by mouth (0.5mg/kg). Approximately 2.5 hours after amphetamine
administration, subjects will be scanned with PHNO again.

- Positron emission tomography 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 radiotracer, venous blood sampling of AChE activity,
and for the administration of glycopyrrolate and physostigmine. A radial artery catheter
will be inserted by an experienced physician before the PET scan 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 10
millicuries of NCFHEB or PHNO. 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.

Aim 2. Subjects will participate in one PET scan day. Aim 3. Subjects from Aim 2 who are able
to continue smoking abstinence will be asked to come back for another PET scan after about
6-8 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).

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).

Control subjects may participate in more than 1 aim. For example, nonsmoking subjects may
complete Aim 1 and if chose, participate in Aim 4. Thus, subjects may participate in up to 4
PET scans for this protocol.

Aim 7. Subjects from Aim 4 will be asked to participate in 2 PHNO PET scans and amphetamine
administration.

Inclusion Criteria:

- 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).

- 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

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 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.

- Regular use of antidepressants or psychotropic over-the-counter medications and herbal
products within the last year

- 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.