Nestler (2001) Nature Reviews Neuroscience 2, 110-128.

1. Molecular and Genetic Bases of Nicotine Dependence Noboru Hiroi, Ph.D. Department of Psychiatry and Behavioral Sciences Department of Neuroscience Albert Einstein College of Medicine Nestler (2001) Nature Reviews Neuroscience 2, 110-128.

2. - 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." Smoking as DependenceConfession of a smoker - “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

3. “Nicotine is not addictive” -

4. 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.

5. How easily would you develop dependence? 32% 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) 23% 17% 15% 11% 9% 9% 8% % of individuals with dependence among extra-medical users. n=8,098, 15-54 years old.(Anthony et al., 1994)

7. 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

8. 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 WISDM: Wisconsin Inventory of Smoking Dependence Motives Piper et al (2004)

9. How to model elements of dependence in experimental animals ? ? ? ? Memory tasks ? Cue reactivity Withdrawal and cue reactivity Self-administration and cue control ? ? ? • 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

10. Do you have an urge to smoke?

11. Drug-associated cues Neutral cues Smoking-related cues Unpleasant cues Droungas, et al., 1995

12. Rodent model of cue reactivity CONDITIONING DAY: MORNING Nicotine

13. CONDITIONING DAY: AFTERNOON Saline

14. TEST DAY

15. How to model elements of dependence in experimental animals ? ? ? ? Memory tasks ? Cue reactivity Withdrawal and cue reactivity Self-administration and cue control ? ? ? • 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

16. Withdrawal Cue Aversion Chronic nicotine intake Nicotine

17. Precipitated withdrawal CONDITIONING DAY: MORNING Mecamylamine

18. No withdrawal Saline

19. TEST DAY

21. Nicotine Acts on the Mesolimbic Dopamine Pathway DA N. Accumbens VTA Nicotinic receptor Nicotine

22. Mesolimbic Dopamine Pathway Caudate-Putamen Frontal/cingulate cortex VTA N. Accumbens Hiroi (2005)

23. Double Dissociation of Nicotine Cue Preference and Withdrawal-Cue Aversion Nicotine Cue Apporach Withdrawal-Cue Aversion Scott and Hiroi (in preparation)

25. Nicotine Acts on the Mesolimbic Dopamine Pathway DA N. Accumbens VTA Nicotinic receptor Nicotine

26. Hypothetical Intracellular Cascades of Addiction Dopamine Second Messenger Transcription Factor  ? Downstream genes Nicotine Dependence?

27. FosB/DFosB induction by nicotine along the mesolimbic dopamine pathway Caudate-Putamen SN VTA N. Accumbens Accumbens Caudate-Putamen Ventral tegmental area Substantia Nigra WT KO WT KO WT KO WT KO Saline Nicotine Sal Nic Sal Nic Sal Nic Time (hr) 0.5 1 6 0.5 0.5 1 6 0.5 0.5 1 6 0.5 0.5 1 6 0.5 FosB DFosB β-actin Zhu et al (2007) Human Molecular Genetics

28. Strategy Mouse with deletion of single genes Characterize behavioral phenotypes in rodent models of nicotine dependence

29. 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)

30. Cue-induced Approach WT 400 Preference FosB KO 200 * 0 Time difference (sec) -200 Aversion -400 0.025 0.05 0.2 0.6 0.8 2 Nicotine (base mg/kg) Time difference = Time in nicotine side - time in saline side Zhu et al (2007) Human Molecular Genetics

31. Dopamine cAMP PKA Transcription Factor  FosB Downstream genes Nicotine Cue Approach

32. Nicotine Nitric Oxide (NO) GC (soluble guanylate cyclase) cGMP (cyclic guanosine monophosphate) PKG (cGMP-dependent protein kinase) PThr34 P-Thr75 DARPP-32 Nicotine Dependence?

33. PKGII mRNA Allen Brain Atlas (2009)

34. Region-specific activation by nicotine of PKG activity ** ** ** Scott et al., 2009

35. 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

36. Maintenance of Nicotine Cue Approach PKGII WT ++ ++ ** ** + + ** * * * * # PKGII KO Scott et al., 2009

37. Withdrawal-Cue Aversion Scott et al., 2009

38. Nicotine Nitric Oxide (NO) GC cGMP PKG Long-term Nicotine Cue Approach Not withdrawal-cue aversion

39. Long-term retention of Nicotine cue approach PKG-II Nicotine Acute Nicotine cue approach FosB

40. Translational Approach Insular Orbitofrtontal cortex Striatum Amygdala FosB PKG DARPP-32 MAOA fosB 19q13.32 Prkg2 4q13.1-q21.1 DARPP-32 17q12 Maoa Xp11.3 Nicotine cue approach Withdrawal cue aversion

41. ACKNOWLEDGEMENTS Albert Einstein College of Medicine Laboratory of Molecular Psychobiology Department of Psychiatry and Behavioral Sciences Department of Neuroscience Noboru 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 University Paul Greengard, Ph.D. Children’s Hospital/Harvard Medical School Michael E. Greenberg, Ph.D. University of Southern California Jean C. Shih, Ph.D. Kevin Chen, Ph.D. University of Paris-Sud Isabelle Seif Technical University of Munich ,