Molecular and Genetic Bases of Nicotine DependenceNoboru Hiroi, Ph.D.

Department of Psychiatry and Behavioral SciencesDepartment of Neuroscience

Albert Einstein College of Medicine

Smoking as DependenceConfession of a smoker

- He smoked 22-40 cigars per day.

- "To cease smoking is the easiest thing I ever did. I ought to know because I've done it a thousand times."

- “As an example to others, and not that I care for moderation myself, it has always been my rule never to smoke when asleep* and never to refrain when awake.” --70th birthday speech

- * “He always went to bed with a cigar in his mouth, and sometimes, mindful of my fire insurance, I went up and took it away, still burning, after he had fallen asleep." William Dean Howells.

Samuel Langhorne Clemens

-

“Nicotine is not addictive”

Nicotine as an addictive substance

- Smokers prefer nicotine-containing cigarettes to de- nicotinized cigarettes.

- Smokers experience withdrawal when switching to light cigarettes.

- Nicotine replacement alleviates withdrawal symptoms.

How easily would you develop dependence?

Nicotine

Heroin

Cocaine

Alcohol

Stimulants other than cocaine (d-amphetamine and methamphetamine)

Cannabis (marijuana, hashish, or both)

Anxiolytics/sedative and hypnotic drugs (secobarbital, diazepam, flurazepam, alprazolam, and triazolam)

Analgesics (morphine, propoxyphene, and codeine)

% of individuals with dependence among extra-medical users.

n=8,098, 15-54 years old. (Anthony et al., 1994)

32%

23%

17%

15%

11%

9%

9%

8%

http://www.bbc.co.uk/bbcfour/documentaries/features/dna.shtml

How to define substance dependence (by features)Physiological Dependence• Tolerance • Withdrawal

Behavioral Dependence (Addiction)• Substance taken in larger amounts or over a longer period than

intended.• Persistent desire or unsuccessful attempts to control or

decrease use.• Great deal of time spent in activities to obtain substance.• Substance use significantly or completely takes place of social,

occupational, or recreational activities.• Continued use despite adverse consequences.(Three or more of the above within a 12 month period) DSM-IV-TR

Problems• All or none• Not mechanism based• Heterogeneous nature ignored• Not much translational value

Affiliative attachment Automaticity Behavioral choice-melioration Loss of control Cognitive enhancement Craving Cue exposure-associative processes Negative reinforcement Positive reinforcement Social and environmental goads Taste and sensory properties Tolerance

WISDM: Wisconsin Inventory of Smoking Dependence MotivesPiper et al (2004)

How to define substance dependence (by reasons)

Subjective reasons for smoking

Affiliative attachment

Automaticity

Behavioral choice-melioration

Loss of control

Cognitive enhancement

Craving

Cue exposure-associative processes

Negative reinforcement

Positive reinforcement Social and environmental goads

Taste and sensory properties

Tolerance

How to model elements of dependence in experimental animals

?

?

?

?

Memory tasks

?

Cue reactivity

Withdrawal and cue reactivity

Self-administration and cue control

?

?

?

Do you have an urge to smoke?

Drug-associated cues

Droungas, et al., 1995

Neutral cuesSmoking-related cuesUnpleasant cues

Nicotine

Rodent model of cue reactivity

CONDITIONING DAY: MORNING

Saline

CONDITIONING DAY: AFTERNOON

TEST DAY

Affiliative attachment

Automaticity

Behavioral choice-melioration

Loss of control

Cognitive enhancement

Craving

Cue exposure-associative processes

Negative reinforcement

Positive reinforcement Social and environmental goads

Taste and sensory properties

Tolerance

How to model elements of dependence in experimental animals

?

?

?

?

Memory tasks

?

Cue reactivity

Withdrawal and cue reactivity

Self-administration and cue control

?

?

?

Nicotine

Withdrawal Cue Aversion

Chronic nicotine intake

Mecamylamine

CONDITIONING DAY: MORNING

Precipitated withdrawal

Saline

No withdrawal

TEST DAY

http://www.bbc.co.uk/bbcfour/documentaries/features/dna.shtml

N. Accumbens

DA

VTA

Nicotine Acts on the Mesolimbic Dopamine Pathway

Nicotinic receptorNicotine

Mesolimbic Dopamine Pathway

Caudate-Putamen

N. Accumbens

Frontal/cingulate cortex

VTA

Hiroi (2005)

Double Dissociation of Nicotine Cue Preference and Withdrawal-Cue Aversion

CPP

Pretest Test 1 Test 2 Test 3 Test 4 Test 5 Test 6 Test 7 Test 8 Test 9 Test 10Tim

e d

iffer

enc

e (

Nic

otin

e-p

aire

d -

sa

line

-pa

ired,

sec

)

-300

-200

-100

0

100

200

300

400 InsulaOrbitofrontal CortexSham Cortical control

Pretest Test 1 Test 2 Test 3 Test 4 Test 5 Test 6 Test 7 Test 8 Test 9 Test 10

Tim

e d

iffe

ren

ce (

Me

cam

yla

min

e-p

aire

d -

sa

line

-pa

ired

, se

c)

-400

-300

-200

-100

0

100

200

300

400InsulaOrbitofrontal CortexSham Cortical control

Scott and Hiroi (in preparation)

Nicotine Cue Apporach Withdrawal-Cue Aversion

http://www.bbc.co.uk/bbcfour/documentaries/features/dna.shtml

N. Accumbens

DA

VTA

Nicotine Acts on the Mesolimbic Dopamine Pathway

Nicotinic receptorNicotine

Transcription Factor

Downstream genes

Second Messenger

Nicotine Dependence?

Dopamine

Hypothetical Intracellular Cascades of Addiction

?

Saline Nicotine Sal Nic Sal Nic Sal Nic

0.5 1 6 0.5 0.5 1 6 0.5 0.5 1 6 0.5 0.5 1 6 0.5

WT KO WT KO WT KO WT KO

Accumbens Caudate-Putamen Ventral tegmental area Substantia Nigra

FosB

DFosB

β-actin

Time (hr)

FosB/DFosB induction by nicotine along the mesolimbic dopamine pathway

N. Accumbens

Caudate-Putamen

VTASN

Zhu et al (2007) Human Molecular Genetics

Strategy

Mouse with deletion of single genes

Characterize behavioral phenotypes in rodentmodels of nicotine dependence

FosB KO mouse• Backcrossed to C57BL/6J mice for 7 generations

• Reduced locomotor habituation (Zhu et al., 2007)• Basally low blood corticosterone level (Zhu et al., 2007)

Normal in:• neurological functions (Gruda et al., 1996)• olfactory discrimination (Brown et al., 1996)• learning (Brown et al., 1996)• anatomy (Hiroi et al., 1997)• regulation of other Fos family proteins (Hiroi et al., 1999)• ethanol preference (Korkosz et al., 2004)• nicotine metabolism (Zhu et al., 2007)• novelty approach (Zhu et al., 2007)• anxiety-related behavior (Zhu et al., 2007)

Cue-induced Approach

Time difference = Time in nicotine side - time in saline side Zhu et al (2007) Human Molecular Genetics

Nicotine (base mg/kg)0.025 0.05 0.2 0.6 0.8 2

Tim

e di

ffere

nce

(sec

)

-400

-200

0

200

400 WT

FosB KO

*

Preference

Aversion

Transcription Factor

Downstream genes

cAMP

Nicotine Cue Approach

Dopamine

FosB

PKA

GC (soluble guanylate cyclase)

Nicotine Dependence?

Nitric Oxide (NO)

cGMP (cyclic guanosine monophosphate)

PKG (cGMP-dependent protein kinase)

PThr34P-Thr75

DARPP-32

Nicotine

Allen Brain Atlas (2009)

PKGII mRNA

Region-specific activation by nicotine of PKG activity

PFC NA CA AMY VTA SN

Per

cent

of c

ontr

ol

60

70

80

90

100

110

120

130

140

150

160

170

180SalineNicotine (0.2 mg/kg)

**

**

**

Scott et al., 2009

PKG-II KO mice

• Congenic C57BL/6 background• Reduced bone length• Defective intestinal water secretion• Enhanced anxiety• Increased ethanol consumption• Altered light-induced circadian clock

resetting

Maintenance of Nicotine Cue Approach

Pretest 1 2 3 4 5 6 7 8 9 10Tim

e di

ffere

nce

(sec

, nic

otin

e-pa

ired

- sa

line-

paire

d)

-400

-300

-200

-100

0

100

200

300

WTHTKO

**

** **

**

**

++++

+ +

#

PKGII KO

PKGII WT

Scott et al., 2009

Pretest Test 1 Test 2 Test 3 Test 4 Test 5 Test 6 Test 7 Test 8 Test 9 Test 10

Tim

e d

iffe

ren

ce (

me

cam

yla

min

e-p

aire

d -

sa

line

-pai

red,

se

c)

-500

-400

-300

-200

-100

0

100

200

WT HT KO

Withdrawal-Cue Aversion

Scott et al., 2009

GC

Long-term Nicotine Cue ApproachNot withdrawal-cue aversion

Nitric Oxide (NO)

cGMP

PKG

Nicotine

Nicotine

Long-term retention ofNicotine cue approachPKG-II

Acute Nicotine cue approachFosB

Translational Approach

Nicotine cue approachWithdrawal cue aversion

InsularOrbitofrtontal cortexStriatumAmygdala

FosBPKGDARPP-32MAOA

fosB 19q13.32Prkg2 4q13.1-q21.1DARPP-32 17q12Maoa Xp11.3

Albert Einstein College of MedicineLaboratory of Molecular PsychobiologyDepartment of Psychiatry and Behavioral SciencesDepartment of NeuroscienceNoboru Hiroi, Ph.D.Takehito Sawamura, M.D. Hongwen Zhu, M.D. , Ph.D. Soh Agatsuma, M.D., Ph.D.Go Suzuki, M.D.Takeshi Hiramoto, Ph.D.Kathryn Harper, M.Sc.Daniel Scott, M.Sc.Gina Kang, B.Sc.

Franz Hofmann, Ph.D. Jens Schlossmann, Ph.D. Robert Feil, Ph.D. Suzanne Feil, Ph.D.

Supported by: NIDA (R01DA13232 and R01DA024330), NARSAD, Transgenic Core/ Albert Einstein College of Medicine

The Rockefeller UniversityPaul Greengard, Ph.D.

Children’s Hospital/Harvard Medical SchoolMichael E. Greenberg, Ph.D.

University of Southern CaliforniaJean C. Shih, Ph.D.Kevin Chen, Ph.D.

University of Paris-SudIsabelle Seif

Technical University of Munich

ACKNOWLEDGEMENTS

,