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Changes in the brain during chronic exposure to nicotine

Changes in the brain during chronic exposure to nicotine. Brookhart Lecture Henry Lester. Behavior. Circuits. Synapses. Neurons. Nicotine Addiction. May, 2009. Intracell. Binding. Parkinson’s Disease. Inadvertent therapeutic effects. Nic vs ACh. ADNFLE. Proteins. RNA. Genes.

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Changes in the brain during chronic exposure to nicotine

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  1. Changes in the brain during chronic exposure to nicotine Brookhart Lecture Henry Lester Behavior Circuits Synapses Neurons Nicotine Addiction May, 2009 Intracell. Binding Parkinson’s Disease Inadvertent therapeutic effects Nic vs ACh ADNFLE Proteins RNA Genes

  2. The nicotine video Produced for Pfizer to explain varenicline (Chantix) to physicians This summarizes knowledge in ~ 2004. “physical” addiction vs “psychological” addiction. Desensitization and “Upregulation” Behavior Circuits Synapses Neurons Nicotine Addiction 1 million channels Intracell. Binding Parkinson’s Disease Closed states(s) more stable than open states Nic vs ACh ADNFLE Proteins RNA Genes nicotine 20 seconds

  3. Focus on a42* Conclusions from hypersensitive and knockout mice (2005): Activation of a4-containing (a4b2*) receptors by nicotine Is sufficient and necessary for tolerance, sensitization, reward, (but not withdrawal) Picciotto, Marubio, Maskos, Tapper, De Biasi . . . . Behavior Circuits Synapses Neurons Nicotine Addiction Intracell. Binding Parkinson’s Disease Nic vs ACh ADNFLE Proteins (But remember that some a42* receptors contain 5, 6, or β3 subunits) RNA Genes

  4. Nicotine and ACh act on many of the same receptors, but . . . • 1. Nicotine is highly membrane-permeant. ACh is not. • Ratio unknown, probably > 1000. • 2. ACh is usually hydrolyzed by acetylcholinesterase (turnover rate ~104 /s.) In mouse, nicotine is eliminated with a half time of ~ 10 min. • Ratio: ~105 • EC50 at muscle receptors: nicotine, ~400 μM; ACh, ~ 45 μM. • Ratio, ~10. Justified to square this because nH = 2. Functional ratio, ~100. • For nicotine, EC50(muscle) / EC50(α4β2) = 400 • What causes this difference?

  5. The AChBP interfacial “aromatic box” occupied by nicotine (Sixma, 2004) aY198 C2 aW149 B aY93 A aY190 C1 non-aW55 D (Muscle Nicotinic numbering)

  6. WT WT, without cation-π interaction Nicotine makes a stronger cation-π interaction with Trp B at α4β2 receptors than at muscle receptors; this partially explains α4β2 receptors’ high binding affinity for nicotine.

  7. Nicotine makes a stronger H-bond to a backbone carbonyl at α4β2 than at muscle receptors:With amide to ester substitution: Weaker hydrogen bond C2 B A C1 Deleted hydrogen bond D

  8. Nicotine EC50 values: Muscle nAChR single component ~ 400 μM α4β2 two components ~ 1 μM, ~200 μM Underlying the 400-fold higher nicotine sensitivity of neuronal vs muscle receptors: Factor of ~16 for the cation-π interaction; Factor of ~ 12 for H-bond; 16 x 12 = 192. We still can’t explain a factor of 400/192 ~ 2. Joanne Xiu, Nyssa Puskar, Jai Shanata, Dennis Dougherty

  9. a P P P g b Activated GPCRs are sometimes phosphorylated and endocytosed. This “downregulation” terminates signalling & causes tolerance. During activation, the G protein leaves . . . P . . . revealing phosphorylation sites . . . . . .other proteins bind to the phosphates . . . kinase But continual signalling can activate genes (not a synaptic vesicle) . . . triggering endocytosis.

  10. receptor G protein i q s t kinase effector channel enzyme intracellular messenger cAMP Ca2+ phosphorylated protein Brunzell, Russell, & Piccotto, 2003 Possible molecular mechanism #1 for changes with chronic nicotine: Signal transduction triggered by a ligand-gated channel NMDA receptors and nAChRs are highly permeable to Ca2+ as well as to Na+.

  11. Possible Mechanism #2 for changes with chronic nicotine: “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, Changeux, & Corringer; Heinemann) or “Novel slow stabilizer” (Green). Upregulation is “cell autonomous” and “receptor autonomous” (Henry).

  12. Upregulation is a part of SePhaChARNS Nicotine is a “Selective Pharmacological Chaperone of Acetylcholine Receptor Number and Stoichiometry” Behavior Circuits Synapses Neurons Nicotine Addiction Intracell. Related phenomena: 1. Chronic nicotine 2. ADNFLE mutations 4. β2 vs β4 subunit Binding Parkinson’s Disease Nic vs ACh ADNFLE Proteins RNA Genes

  13. Bound states with increasing affinity unbound + + + C Highest affinity bound state AC Free Energy A2C A2O A2D Reaction Coordinate Thermodynamics of SePhaChARNS Increasingly stable assembled states Free subunits #1. Nicotine binds to subunit interfaces, favoring assembled receptors Free Energy Reaction Coordinate #2. Binding eventually favors high-affinity states HAL research 1970-2009 HAL research 2006-2009

  14. Thermodynamics of SePhaChARNS #3. Acid trapping may keep intracellular nAChRs desensitized Nicotine accumulates in cells 1 mM Nicotine+ (pKa = 7.8) pH 7.4 pH 7.0 2.5 mM Nicotine+ . . . and then in intracellular organelles. P. Paroutis, N. Touret, S Grinstein (2004) Physiology 19: 207-215 nicotine+/nicotine: 10 30 100 300

  15. Thermodynamics of SePhaChARNS, #4. Reversible stabilization amplified by covalent bonds? Covalently stabilized AR*HS ? + nicotine RHS RLS Degradation Nicotine Increased High-Sensitivity Receptors hr 0 20 40 60

  16. High-resolution fluorescence microscopy to study SePhaChARNS LTP / Opioids: regulation starts here TIRFM PM ER Pharmacological chaperoning: upregulation starts here FRET Golgi Nucleus

  17. M3 - M4 α4 N C N C loop M3 - M4 loop Ligand binding M1 M2 M3 M4 M4 HA tag XFP c - myc tag XFP b a 2 - XFP 4 - XFP FRET pairs (m = monomeric) EYFP XFP = mCerulean ECFP mEGFP mVenus mCherry mEYFP Förster resonance energy transfer (FRET): a test for subunit proximity β2 λ Neuro2a

  18. 50% α-CFP, 50% α-YFP 1/4 1/4 1/2 E No FRET b/a =1.62; 1.62-6 = 0.055 1/8 1/8 1/8 100% α3β2 E1 E2 E3 E4 100% α2β3 No FRET 1/8 1/4 1/4 % receptors with α3 Theory of FRET in pentameric receptors with αnβ(5-n)subunits

  19. Autosomal Dominant Nocturnal Frontal Lobe Epilepsy: Five M2 Domain Mutations Cause Excess Intracellular (α4)3(β2)2 Stoichiometry Behavior Circuits Synapses Neurons Nicotine Addiction Intracell. Binding Parkinson’s Disease Nic vs ACh ADNFLE Proteins RNA Genes Neuro2a

  20. A key SePhaChARNS experiment: changes in subunit stoichiometry caused by chronic nicotine Behavior Circuits Synapses Neurons Nicotine Addiction Intracell. Binding Parkinson’s Disease Nic vs ACh ADNFLE Proteins RNA Genes Neuro2a

  21. 4 hour nicotine exposure: increased (a4)2(b2)3 assembly in Golgi WHOLE CELL + 1 mM NICOTINE 4 h R2 = 0.998 y0 = 0 xc1 = 8.5 ± 0.18 w1 = 2.4 ± 0.1 A1 = 130438 ± 36122 xc2 = 10.1 ± 0.26 w2 = 2.24 ± 0.14 A2 = 64907 ± 26106 WHOLE CELL No treatment R2 = 0.999 y0 = 0 xc1 = 8.7 ± 0.06 w1 = 2.22 ± 0.12 A1 = 88465 ± 34150 xc2 = 10 ± 0.36 w2 = 2.92 ± 0.19 A2 = 109476 ± 34316 GOLGI No treatment R2 = 0.999 y0 = 0 xc1 = 8.28 ± 0.07 w1 = 1.9 ± 0.05 A1 = 6756 ± 620 xc2 = 9.72 ± 0.07 w2 = 1.8 ± 0.04 A2 = 5298 ± 621 GOLGI + 1 mM NICOTINE 4 h R2 = 0.998 y0 = 0 xc1 = 8.37 ± 0.02 w1 = 2.33 ± 0.03 A1 = 11498 ± 239 xc2 = 10.21 ± 0.04 w2 = 1.51 ± 0.06 A2 = 1986 ± 233

  22. Total Internal Reflection Fluorescence Microscopy (TIRFM) Neuro2a

  23. Differential subcellular localization and dynamics of α4GFP* receptors plasma memb. mCherry overlay α4GFPβ2 overlay α4GFPβ4 (1:1) α4GFPβ2 (1:1) 3 RXR/β subunit α4GFPβ4 (1:1) zero RXR/β subunit

  24. Strategy to evaluate the cell specificity of a4* upregulation in chronic nicotine 1. Generate knock-in mice with fully functional, fluorescent a4* receptors 2. Expose the mice to chronic nicotine 3. Find the brain regions and cell types with changed receptor levels Behavior Circuits Synapses Neurons 4. Perform physiological experiments on these regions and cells to verify function Nicotine Addiction Intracell. Binding Parkinson’s Disease Nic vs ACh ADNFLE Proteins 5. Model the cellular and circuit changes RNA Genes Leu9’Ala-YFP, YFP, CFP

  25. The Caltech a4 fluorescent mice . . . normal in all respects

  26. Cellular and subcellular specificity of SePhaChARNS CA EC MH DG IPN Medial Perforant Path Striatum SNc Thalamus, superior colliculus SNr * = upregulation shown with electrophysiology Nashmi et al J Neurosci 2007; Xiao et al, submitted

  27. Chronic nicotine 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 certain cognitive enhancements (Rusted and Warburton, 1992; Rusted et al., 1995). In the rodent context, mice show more contextual fear conditioning if, one day after withdrawal from chronic nicotine, they receive an acute nicotine dose (Davis et al., 2005); this is β2* dependent. Also chronic nicotine produces better spatial working memory performance in the radial arm maze (Levin et al., 1990; Levin et al., 1996).

  28. Chronic nicotine increases perforant path a4 fluorescence ~ 2-fold Alveus Py Or Rad • TV Bliss, T Lömo (1973) • Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path. • J Physiol. 232:331-56. LMol 200 mm Temperoammonic Path Medial Perforant Path

  29. 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

  30. DA neuron, ~ 1700 spikes 4*, 6*, and/or 7 6 Nicotine injection Frequency, Hz 4 2 A B C D VTA 0.05 mV 2 ms DAergic 0 25 4* only GABAergic 20 0.1 mV Frequency, Hz 0.5 ms 15 10 V GABAergic neuron (5 s smoothing), ~ 8300 spikes 5 0 100 200 300 400 500 600 700 0 s VTA GABAergic and DA neurons have contrasting responses to nicotine in vivo WT mouse

  31. a4-YFP knock-in: substantia nigra pars compacta neurons Spectrally unmixed background autofluorescence Spectrally unmixed a4YFP 10 mm 10 mm Raad Nashmi

  32. Midbrain data show cell specificity of SePhaChARNS Chronic nicotine does not change a4 levels in dopaminergic neurons . . . Substantia Nigra Pars Compacta (& VTA, not shown) α4 intensity per TH+ neuron . . . but does upregulate a4 levels in GABAergic inhibitory neurons. Substantia Nigra Pars Reticulata (& VTA, not shown) α4 intensity per GAD+ neuron

  33. Chronic Nicotine Tolerance Endogenous ACh Upregulated a4* nAChRs 2A Craving 1A Reward Endogenous ACh 4.0 Yoked saline 3.5 Yoked nicotine Decreased Reward 3.0 Plus Acute Nicotine (1st expsoure) 2.5 2B Dialysate DA (nM) 2.0 1B 1.5 Plus Acute Nicotine (repeated exposure) 1.0 0.5 Saline Nicotine 0.0 0 20 40 60 80 120 140 160 180 -40 -20 100 + acute nicotine Time (min) 2A 1A 1B 2B Chronic nicotine cell-specifically up-regulates functional a4* receptors: Basis for circuit-based tolerance in midbrain (Nashmi et al, 2007) Chronic Saline Endogenous ACh VTA NAc LDT DAergic Cholinergic GABAergic Rahman et al, 2004

  34. Midbrain slice recordings: functional upregulated receptors in a simple circuit Cheng Xiao

  35. In SNr of α4 knockout, chronic nicotine does not affect firing rates Cheng Xiao

  36. V Chronic nicotine increases firing rate of SNr GABAergic neurons in vivo . . . . . . we’re still gathering data for DA neurons Cheng Xiao

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

  38. a6* is Expressed in Midbrain Dopamine Neurons • Highest affinity for nicotine (function) • Involved in nicotine-stimulated DA release • Selectively lost in PD Bregma -3.08 mm Mike Marks

  39. Plasmid-based Transgenic gene of interest transgene BAC Transgenic gene of interest transgene Selective activation of DA neurons via α6 subunits & bacterial artificial chromosome (BAC) Transgenics • BACs: • 50-300kb • Easily manipulated • Includes most gene expression regulatory elements • Faithfully replicates expression pattern of endogenous gene a6 mRNA a6 BAC

  40. A carbon fiber electrode allows us to detect dopamine electrochemically in striatal slices carbon fiber A

  41. Selective Activation of DA Neurons Stimulates Locomotor Activity . . . . . . but, unlike selective a4 activation, shows no sensitization, Possibly because α6* receptors do not participate in SePhaChARNS

  42. Some changes in the brain during chronic exposure to nicotine 1. Nicotine potently activates some neuronal nAChRs (because it participates in both cation-π and H-bond interactions within the conserved aromatic box). 2. Nicotine is a selective pharmacological chaperone of acetylcholine receptor number and stoichiometry (SePhaChARNS). 3. These processes lead to α4β2* upregulation, with cellular and subcellular specificity. Behavior • 4. a. Upregulation explains tolerance to chronic nicotine, via a GABAergic-DA circuit in the midbrain. • b. A similar circuit mechanism may protect DA neurons against harmful burst firing in PD. • c. Upregulation also explains enhanced LTP in the perforant path, via a direct presynaptic mechanism. This is a simple model for cognitive sensitization Circuits Nicotine Addiction Synapses Neurons Parkinson’s Disease Intracell. ADNFLE Binding Nic vs ACh 5. Repeated selective activation of DA neurons, via hypersensitive 6* receptors, produces neither locomotor tolerance nor sensitization. Proteins RNA 6. We do not yet understand several processes, e. g. somatic signs of withdrawal, stress-induced nicotine use, and ANFLE circuitry. Genes

  43. Dennis Dougherty Kiowa Bower, Shawna Frazier, Ariel Hanek, Fraser Moss, Nyssa Puskar, Rigo Pantoja, Kristin Rule, Erik Rodriguez, Jai Shanata, Mike Torrice, Joanne Xiu Neil Harrison, Sarah Lummis, Claire Padgett, Kerry Price, Andy Thompson Romiro Salas, Mariella De Biasi Caltech “Unnatural Club” Univ. of Cambridge Baylor Coll of Medicine Caltech “Alpha Club” Bruce Cohen, Purnima Deshpande, Ryan Drenan, Carlos Fonck, Sheri McKinney, Raad Nashmi, Johannes Schwarz, Rahul Srinivasan, Cagdas Son, Andrew Tapper, Larry Wade, Cheng Xiao Al Collins, Sharon Grady, Mike Marks, Erin Meyers, Tristan McClure-Begley, Charles Wageman, Paul Whiteaker Merouane Bencherif, Greg Gatto, Daniel Yohannes Jon Lindstrom Mike McIntosh Julie Miwa, Nathaniel Heintz Robin Lester Univ of Colorado, Boulder Targacept Univ Pennsylvania Univ. Utah Rockefeller Univ Univ of Alabama

  44. FRET with fluorescent subunits qualitatively measures nAChR assembly * a4/b2Y/b2C a4Y/a4C/b2 a6Y/a6C/b2 a4/b2/b3Y/b3C Neuro2a

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