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Three responses to chronic nicotine exposure: Studies on

Three responses to chronic nicotine exposure: Studies on genes, proteins, drugs, cells, circuits, and behavior. Henry Lester. November, 2007. 1. How does one explain nicotine addiction?. Does it matter? Won’t everyone stop smoking soon? Smokeless tobacco?.

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Three responses to chronic nicotine exposure: Studies on

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  1. Three responses to chronic nicotine exposure: Studies on genes, proteins, drugs, cells, circuits, and behavior Henry Lester November, 2007

  2. 1. How does one explain nicotine addiction? Does it matter? Won’t everyone stop smoking soon? Smokeless tobacco? 2. Nicotine as an imperfect therapeutic drug Best example: Parkinson’s disease 3. Cellular / molecular approaches to better therapies

  3. United States, 1997–2006 30 25 20 percent 15 10 5 (National Health Interview Survey) 0 1996 1998 2000 2002 2004 2006 year Progress on smoking cessation is very slow Prevalence of current smoking among adults aged 18 years and over “Self-medication” may be the reason In 2002, individuals with a current psychiatric disorder comprised 7% of the US population, but they smoked 34-46% of all cigarettes in the US.

  4. receptors become “bored” 1 million channels nicotine 20 seconds The nicotine video Produced for Pfizer to explain varenicline (Chantix) to physicians This summarizes knowledge in ~ 2004. “ligand” is a molecule that binds to another. “physical” addiction vs “psychological” addiction. “Desensitization“ and “Upregulation” Some abbreviations on future slides: ACh, acetylcholine nAChR, nicotinic acetylcholine receptor DA, dopamine

  5. What are the mechanisms? Focus on a4β2 receptors Conclusions from knockout andhypersensitive mice (2005): Activation of a4b2-containing (a4b2*) receptors by nicotine Is necessary and sufficient for sensitization, tolerance, reward, (but withdrawal?)

  6. Chronic nicotine exposure causes tolerance of dopamine release • The “yoked self-administration” experiment 4.0 Yoked saline 3.5 Yoked nicotine 3.0 2.5 Dialysate DA (nM) 2.0 1.5 1.0 0.5 Saline Nicotine 0.0 Yoked animal 0 40 80 120 160 -40 Master animal Time (min) Rahman, Zhang, Engleman, & Corrigall, 2004

  7. 2. Chronic nicotine exposure causes cognitive sensitization In the human context, cognitive sensitization is epitomized by smokers’ reports that they think better when they smoke; this anecdotal observation is confirmed by data that smokers who smoke nicotine cigarettes (but not nicotine-free cigarettes) display several cognitive enhancements. In the rodent context, rats show more contextual fear conditioning if, one day after withdrawal from chronic nicotine, they receive an acute nicotine dose; also chronic nicotine produces better spatial working memory performance in the radial arm maze.

  8. 3. Inverse correlation between long-term tobacco smoking and Parkinson’s disease In identical twins discordant for both Parkinson’s disease & smoking, the unaffected twin smoked at a significantly higher rate. In those twins where one or both smoked, The unaffected twin smoked 12 pack-years more. There are good indications that nicotine itself is a protective agent. Clinical trials of nicotine patches have given mixed results because of side effects Beneficial results of short-term nicotine exposure: Pain reduction. Increased concentration: ADHD, Schizophrenia. Alzheimer (Aricept = donepezil, a cholinesterase inhibitor; Reminyl = galantamine) Decreased inflammation. Antidepressant actions.

  9. receptor G protein i q s t kinase effector channel enzyme intracellular messenger cAMP Ca2+ phosphorylated protein Possible mechanism 1: The “Molecular Relay Race”: Signal transduction triggered by a ligand-gated channel nAChRs are highly permeable to Ca2+ as well as to Na+.

  10. neuroprotection? If the upregulated receptors are Desensitized (“bored”), this might cause decreased synaptic transmission and excitation, leading to tolerance. But this does not explain cognitive sensitization. If the upregulated receptors are active, excitotoxicity might exacerbate Parkinson’s disease. If the upregulated receptors are desensitized, this might be neuroprotective. • The “Bored Receptor” (desensitized) • versus • b. The “Exuberant Receptor” (upregulated) Possible Mechanisms 2a, 2b: Chronic exposure to nicotine induces more nicotinic receptors The “Receptor Dilemma”: How (if at all) do changed receptors contribute to . . . nicotine addiction? If the upregulated receptors are Active (“exuberant”), upregulation might cause better synaptic transmission and excitation, leading to cognitive sensitization. But this does not explain tolerance.

  11. Strategy to choose between the “bored” or “exuberant” receptors in the response to chronic nicotine exposure 1. Generate mice with fully functional, fluorescent a4* receptors. (Why mice?) 2. Chronically expose the mice to nicotine (2 weeks). 3. Find the brain regions and cell types with changed fluorescence. 4. Perform experiments on these regions and cells to decide whether the new receptors are “bored” or “exuberant”. 5. Model the cellular and circuit changes

  12. Functional studies show: the new receptors are “exuberant”, not “bored” V Chronic nicotine increases a4 fluorescence ~ 2-fold in hippocampus --a brain area that provides a good model for cognition. Alveus Py Or Rad LMol 200 mm Temperoammonic Path Medial Perforant Path

  13. Midbrain dopaminergic cells (tyrosine hydroxylase stain) Substantia nigra pars compacta (SNc, controls motion); Ventral tegmental area (VTA, controls reward) Substantia nigra pars reticulata (SNr, GABAergic)

  14. a4-YFP knock-in: substantia nigra pars compacta neurons Spectrally unmixed background autofluorescence Spectrally unmixed a4YFP 10 mm 10 mm a 4YFP 1500 Background 1000 YFP Intensity 500 0 500 520 540 560 580 600 Wavelength (nm)

  15. Substantia nigra data also support the “exuberant receptor” idea Chronic nicotine does not change a4 levels in dopaminergic neurons . . . Substantia Nigra Pars Compacta α4 intensity per TH+ neuron . . . but does upregulate a4 levels in GABAergic inhibitory neurons. Substantia Nigra Pars Reticulata α4 intensity per GAD+ neuron

  16. Chronic Nicotine Tolerance Upregulated a4* nAChRs Endogenous ACh 2A Craving Reward Endogenous ACh Decreased Reward Plus Acute Nicotine (1st expsoure) 2B 1B Plus Acute Nicotine (repeated exposure) + acute nicotine Chronic nicotine cell-specifically upregulates a4* receptors: Basis for circuit-based tolerance in midbrain via “exuberant inhibition” Chronic Saline Endogenous ACh VTA NAc LDT 1A DAergic Cholinergic GABAergic 4.0 Yoked saline 3.5 Yoked nicotine 1B 3.0 2.5 1A Dialysate DA (nM) 2.0 1.5 1.0 2B 2A 0.5 Saline Nicotine 0.0 0 20 40 60 80 120 140 160 180 -40 -20 100 Time (min) Rahman et al, 2004

  17. Striatum SNc DAergic PPTg Thalamus, superior colliculus Cholinergic GABAergic SNr Endogenous ACh . . . As produced by “deep brain stimulation” in subthalamic nucleus Hypothesis: Circuit-based neuroprotection by chronic nicotine in substantia nigra via Cholinergic, Dopaminergic, and GABAergic neurons in Hindbrain & Midbrain Upregulated a4* nAChRs GABAergic neurons may have increased or more regular firing in chronic nicotine. . .

  18. Conclusions from hypersensitive & fluorescent mice When a4* nicotinic receptors are repeatedly occupied/activated these receptors become “exuberant” in specific neurons. This produces improved cognition via forebrain synapses, but tolerance occurs via changes in a GABA-dopamine circuit. How do we develop better therapeutics based on these ideas?

  19. The nicotinic receptor’s interfacial “aromatic box” occupied by nicotine Showing the cation-p interaction with unnatural amino acids aY198 C2 aW149 B aY93 A non-aW55 D aY190 C1 Collaboration with Dennis Dougherty, Hoag Professor of Chemistry

  20. 17.5 15.0 . . . After 24 hours in nicotine, exuberant receptors are assembled more tightly. 12.5 10.0 FRET Efficiency (%) 514 nm 7.5 439 nm 5.0 439 nm 2.5 0.0 a4-b2XFP + nicotine a4-b2XFP 485 nm 535 nm 485 nm 535 nm “Stolen” photons tell us which subunits are near each other Experiments like these may show us how to develop better therapies for Parkinson’s Disease.

  21. The ultimate reductionist approach, studying nAChR traffic/regulation at the single molecule level. TIRF microscopy of nAChR geGFP in oocytes 1 3 2 2 3 1 4 4 12 μm

  22. Caltech “Alpha Club” Bruce Cohen, Ryan Drenan, Purnima Deshpande, Carlos Fonck, Sheri McKinney, Raad Nashmi, Qi Huang, Rigo Pantoja, Johannes Schwarz, Cagdas Son, Andrew Tapper, Larry Wade, Cheng Xiao Joanne Xiu, Nyssa Puskar, Jai Shanata, Shawna Frazier, Dennis A. Dougherty Sarah Lummis Stephan Pless, Joseph Lynch Sharon Grady, Al Collins, Mike Marks, Jeremy Owens, Tristan McClure-Begley, Paul Whiteaker Jim Boulter, Istvan Mody, Oliver Dorigo, Arnie Berk, Max Shao, Jack Feldman Jon Lindstrom Julie Miwa, Nathaniel Heintz Uwe Maskos, Jean-Pierre Changeux “Unnatural Amino Acid Club” Univ of Cambridge Univ Queensland Univ of Colorado, Boulder UCLA Univ. Pennsylvania Rockefeller Univ Institut Pasteur

  23. More pontifications about upregulation (“exuberance”) Increased nAChR due to chronic nicotine exposure probably confers no selective advantage . . . could be a thermodynamic necessity. A substantial, regulated pool of unassembled or cytoplasmic high-sensitivity nAChRs receptors may confer a selective advantage. If so, the selective advantage may involve responding to circadian rhythms in ACh levels. If so, is there a disease caused by faulty nAChR regulation? Autosomal dominant nocturnal frontal lobe epilepsy?

  24. “Exuberant receptors” are a thermodynamic consequence of durg-receptor Interactions Nicotine accumulates in cells Binding eventually favors high-affinity states Bound states with increasing affinity unbound + + 1 mM Nicotine+ (pKa = 7.9) + C Highest affinity bound state AC -70 mV 0 mV Free Energy A2C A2O A2D Reaction Coordinate 20 mM Nicotine+ Nicotine may stabilize subunit interfaces Covalently stabilized AR*HS ? + nicotine RHS RLS Increasingly stable assembled states Degradation Free subunits Nicotine Increased High-Sensitivity Receptors Free Energy Reaction Coordinate hr 0 20 40 60

  25. “Upregulation” Chronic exposure to nicotine causes upregulation of nicotinic receptor binding (1983: Marks & Collins; Schwartz and Kellar); Upregulation 1) Involves no change in receptor mRNA level; 2) Depends on subunit composition (Lindstrom, Kellar, Perry). Shown in experiments on clonal cell lines transfected with nAChR subunits: Nicotine seems to act as a “pharmacological chaperone” (Lukas, Lindstrom) or “maturational enhancer” (Sallette & Corringer, Heinemann) or “Novel slow stabilizer” (Green). Upregulation is “cell autonomous” and “receptor autonomous” (Henry).

  26. Midbrain slice recordings: functional upregulated receptors in a circuit produce tolerance Cheng Xiao

  27. Substantia nigra data also support the “exuberant receptor” idea Substantia Nigra Pars Compacta Chronic nicotine does not change a4 levels in dopaminergic neurons . . . Substantia Nigra Pars Reticulata . . . but does upregulate a4 levels in GABAergic inhibitory neurons

  28. Acute Nicotine Acute Saline 80 min 10 min 80 min 10 min Chronic Chronic 1 mV Saline Saline 10 ms 0.5 mV 0.5 mV 10 ms 5 ms Nicotine 1mV Nicotine 1 mV 10 ms 10 ms Simple model for cognitive sensitization: chronic nicotine + acute nicotine lowers the threshold for perforant pathway LTP Acute Nicotine Acute Saline Chronic Nicotine Acute Chronic Chronic

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