2. Changes in the brain during chronic exposure to nicotine November, 2009 Or: How Core prepares Techers for Neuroscience Henry Lester Nicotine Addiction Nicotine Addiction Parkinson’s Disease Parkinson’s Disease ADNFLE Behavior Circuits Synapses Neurons Intracell. Binding Nic vs ACh Proteins RNA Genes Inadvertent therapeutic effects of chronic nicotine --Requires chronic exposure to nicotine Total: 41 slides 1

3. Who is Henry Lester? Have always wanted to study fundamental aspects of neuroscience BA in chemistry in physics, Harvard 1966 Ph D in biophysics, Rockefeller Univ 1971 Postdoc in molecular biology, Pasteur Institute, Paris till 1973 Caltech since 1973, sabbaticals in Jerusalem & Melbourne Teach Bi/CNS 150, Intro to Neuroscience Taught Bi1, 2000-2007. “Drugs and the Brain” This increased my interest in drug addiction. My lab now concentrates on the events that accompany chronic exposure to nicotine. We also develop methods for engineering neurons, using ion channels. Chair of the Caltech faculty 2005-2007 Now Chair Caltech Task Force on Mental Health & Suicide Prevention Now President of the Biophysical Society 2

4. The nicotine video This summarizes knowledge in ~ 2004. “physical” addiction vs “psychological” addiction. Desensitization and “Upregulation” Produced for Pfizer to explain varenicline (Chantix) to physicians Behavior Circuits Synapses Neurons Intracell. Binding Nic vs ACh Proteins RNA Genes Nicotine Addiction Nicotine Addiction Parkinson’s Disease Parkinson’s Disease ADNFLE nicotine 20 seconds 1 million channels Closed states(s) more stable than open states 3

5. Nicotine Addiction Nicotine Addiction Parkinson’s Disease Parkinson’s Disease Behavior Circuits Synapses Neurons Intracell. Binding Nic vs ACh Proteins RNA ADNFLE Genes Conclusions from hypersensitive and knockout mice (2005): Activation of  4-containing (  *  receptors by nicotine Is sufficient and necessary for tolerance, sensitization, reward, (but not withdrawal) Focus on  4  2* ( But note that some a4  2* receptors contain  5,  6, or β3 subunits) 4

6. 1. Nicotine is highly membrane-permeant. ACh is not. Ratio unknown, probably > 1000. 2. ACh is usually hydrolyzed by acetylcholinesterase (turnover rate ~10 4 /s.) In mouse, nicotine is eliminated with a half time of ~ 10 min. Ratio: ~10 5 3.EC 50 at muscle receptors: nicotine, ~400 μM; ACh, ~ 45 μM. Ratio, ~10. Justified to square this because nH = 2. Functional ratio, ~100. For nicotine, EC 50 (muscle) / EC 50 (α4β2) = 400 What causes this difference? Nicotine and ACh act on many of the same receptors, but... 5

7. The AChBP interfacial “aromatic box” occupied by nicotine (Sixma, 2004)  W149 B  Y93 A non-  W55 D  Y198 C2  Y190 C1 (Muscle Nicotinic numbering) Hydrogen bond 12-fold tighter binding vs muscle Cation - π interaction 16-fold tighter binding vs muscle Joanne Xiu. Nyssa Puskar, Jai Shanata Xiu et al, Nature 2009 6

8. kinase phosphorylated protein cAMP Ca 2+ intracellular messenger receptor tsqi G protein enzymechannel effector NMDA receptors and nAChRs are highly permeable to Ca 2+ as well as to Na +. Possible molecular mechanism #1 for changes with chronic nicotine: Signal transduction triggered by a ligand-gated channel Brunzell, Russell, & Piccotto, 2003 7

9. Chronic exposure to nicotine causes upregulation of nicotinic receptor binding; Upregulation 1) Involves no change in receptor mRNA level; 2) Depends on subunit composition. Possible Mechanism #2 for changes with chronic nicotine: “Upregulation” Shown in experiments on clonal cell lines transfected with nAChR subunits: Nicotine seems to act as a “pharmacological chaperone” or “maturational enhancer” or “Novel slow stabilizer”. Upregulation is “cell autonomous” and “receptor autonomous”. 8

10. Behavior Circuits Synapses Neurons Intracell. Binding Nic vs ACh Proteins RNA Genes Upregulation is a part of SePhaChARNS Nicotine is a “Selective Pharmacological Chaperone of Acetylcholine Receptor Number and Stoichiometry” Nicotine Addiction Nicotine Addiction Parkinson’s Disease Parkinson’s Disease ADNFLE Related phenomena: 1. Chronic nicotine 2. ADNFLE mutations 4. β2 vs β4 subunit 9

11. + + Free Energy Reaction Coordinate Free subunits Increasingly stable assembled states #1. Nicotine binds to subunit interfaces, favoring assembled receptors + Bound states with increasing affinity Free Energy Reaction Coordinate C AC A2CA2C A2OA2O A2DA2D Highest affinity bound state unbound #2. Binding eventually favors high-affinity states Thermodynamics of SePhaChARNS 10

12. 2.5  M Nicotine + 1  M Nicotine + (pKa = 7.8) pH 7.4 pH 7.0 Nicotine accumulates in cells P. Paroutis, N. Touret, S Grinstein (2004) Physiology 19: 207-215 nicotine + /nicotine: 1010030300... and then in intracellular organelles. Thermodynamics of SePhaChARNS #3. Acid trapping may keep intracellular nAChRs desensitized 11

13. Thermodynamics of SePhaChARNS, #4. Reversible stabilization amplified by covalent bonds? Nicotine hr 0204060 Increased High- Sensitivity Receptors R LS R HS Covalently stabilized AR* HS Degradation + nicotine ? 12

14. Secretory pathway Overview of membrane protein traffic Pharmacological chaperoning: upregulation starts here (Oversimplification) Early LTP / Opioids: regulation starts here (Oversimplification) out 13

15. Förster resonance energy transfer (FRET): a test for subunit proximity ECFPXFP = EYFP mEYFP mVenusmCeruleanmEGFPmCherry Ligand binding M1M2M3 M4 M3-M4 loop M4 M3-M4 loop α4α4 c-myc tagXFP  4-  2- HA tagXFP FRET pairs (m = monomeric) NCNC β2β2 Neuro2a λ 14

16. Theory of FRET in pentameric receptors with α n β (5-n) subunits No FRET E 1/2 1/4 E1E2E3E4 1/8 1/4 1/8 50% α-CFP, 50% α-YFP b/a =1.62; 1.62 -6 = 0.055 100% α 3 β 2 100% α 2 β 3 % receptors with α 3 15

17. Whole-cell donor photobleach experiments suggest: 24 hr nicotine (1 μM) Partially shifts nAChR stoichiometry from ( α 4) 3 ( β 2) 2 to ( α 4) 2 ( β 2) 3 Neuro2a Cagdas Son α 4CFP + α 4YFP 1:1 plus β 2 α 4 plus β 2CFP + β 2YFP 1:1 16

18. 4 hour nicotine exposure: increased (  4) 2 (  2) 3 assembly in Golgi WHOLE CELL No treatment R 2 = 0.999 y 0 = 0 x c1 = 8.7 ± 0.06 w 1 = 2.22 ± 0.12 A1 = 88465 ± 34150 x c2 = 10 ± 0.36 w 2 = 2.92 ± 0.19 A2 = 109476 ± 34316 GOLGI No treatment R 2 = 0.999 y 0 = 0 x c1 = 8.28 ± 0.07 w 1 = 1.9 ± 0.05 A1 = 6756 ± 620 x c2 = 9.72 ± 0.07 w 2 = 1.8 ± 0.04 A2 = 5298 ± 621 WHOLE CELL + 1  M NICOTINE 4 h R 2 = 0.998 y 0 = 0 x c1 = 8.5 ± 0.18 w 1 = 2.4 ± 0.1 A1 = 130438 ± 36122 x c2 = 10.1 ± 0.26 w 2 = 2.24 ± 0.14 A2 = 64907 ± 26106 GOLGI + 1  M NICOTINE 4 h R 2 = 0.998 y 0 = 0 x c1 = 8.37 ± 0.02 w 1 = 2.33 ± 0.03 A1 = 11498 ± 239 x c2 = 10.21 ± 0.04 w 2 = 1.51 ± 0.06 A2 = 1986 ± 233 17

19. Most membrane proteins exit the ER in a COPII-dependent manner COP II: Sec23/24 heterodimer GTPase: Sar1 scission ER lumen ER membrane Cytosolic compartment Mancias & Goldberg, Traffic 2005 18

20. Total Internal Reflection Fluorescence Microscopy (TIRFM) Neuro2a 19

21. Differential subcellular localization and dynamics of α4GFP* receptors α4GFPβ2 α4GFPβ4 (1:1) α4GFPβ2 (1:1)overlayplasma memb. mCherry α4GFPβ4 (1:1) overlay 3 RXR/β subunit zero RXR/β subunit 20

22. Strategy to evaluate the cell specificity of  4* upregulation in chronic nicotine 1. Generate knock-in mice with fully functional, fluorescent  4* receptors 2. Expose the mice to chronic nicotine 3. Find the brain regions and cell types with changed receptor levels 4. Perform physiological experiments on these regions and cells to verify function 5. Model the cellular and circuit changes YFP,Leu9’Ala-YFP, CFP Behavior Circuits Synapses Neurons Intracell. Binding Nic vs ACh Proteins RNA Genes Nicotine Addiction Nicotine Addiction Parkinson’s Disease Parkinson’s Disease ADNFLE 21

23. The Caltech  4 fluorescent mice... normal in all respects 22

24. Cellular and subcellular specificity of SePhaChARNS Thalamus, superior colliculus SNc SNr Striatum Upregulation? TransmitterSomaTerm.Region / projection Glu??Yes* Entorhinal cortex → dentate gyrus AChNo Medial habenula → Interpeduncular nucleus DANo*Yes* Ventral tegmental area, substantia nigra pars compacta → Striatum GABAAYes* SN pars reticulata, VTA → SNC, VTA CA DG EC MH IPN Medial Perforant Path * = upregulation shown with electrophysiology Nashmi et al J Neurosci 2007; Xiao et al, submitted 23

25. 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). 24

26. 200  m Medial Perforant Path Py Or Rad LMol Alveus Temperoammonic Path Chronic nicotine increases perforant path  4 fluorescence ~ 2-fold TV Bliss, T L ö mo (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. 25

27. 1 mV 10 ms 1 mV 10 ms Chronic Chronic Nicotine Chronic Acute Nicotine 10 min80 min 1mV 10 ms Saline Nicotine 0.5 mV Acute Nicotine Chronic 10 min 80 min Saline Nicotine 0.5 mV 5 ms Acute Saline Chronic Acute Saline Acute Simple model for cognitive sensitization: chronic nicotine + acute nicotine lowers the threshold for perforant pathway LTP 26

28. VTA GABAergic and DA neurons have contrasting responses to nicotine in vivo V GABAergic DAergic VTA WT mouse 27

29. VTA Recordings from a 16-channel microprobe Sotiris Masmanidis T1 - T4, s C. Cross-correlation (cluster 1 vs cluster 4) 50 μ m 28

30.  4-YFP knock-in: substantia nigra pars compacta neurons Raad Nashmi Spectrally unmixed  4YFP Spectrally unmixed background autofluorescence 10  m 29

31. Substantia Nigra Pars Reticulata (& VTA, not shown)... but does upregulate  4 levels in GABAergic inhibitory neurons. Chronic nicotine does not change  4 levels in dopaminergic neurons... Substantia Nigra Pars Compacta (& VTA, not shown) Midbrain data show cell specificity of SePhaChARNS α4 intensity per TH+ neuron α4 intensity per GAD+ neuron 30

32. Chronic Saline 1A Endogenous ACh 1A 2A 1B 2B 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Yoked saline Yoked nicotine SalineNicotine -40-20 020406080 100 120140160180 Time (min) Dialysate DA (nM) Rahman et al, 2004 2B Decreased Reward Plus Acute Nicotine (repeated exposure) Chronic nicotine cell-specifically up-regulates functional  4* receptors: Basis for circuit-based tolerance in midbrain (Nashmi et al, 2007) Endogenous ACh VTA LDT Cholinergic NAc DAergic GABAergic Chronic Nicotine  Tolerance 2A Upregulated  4* nAChRs Craving Endogenous ACh 1B Reward Plus Acute Nicotine (1 st expsoure) + acute nicotine 31

33. Midbrain slice recordings: functional upregulated receptors in a simple circuit Cheng Xiao 32

34. In SNr of α4 knockout, chronic nicotine does not affect firing rates Cheng Xiao 33

35. Chronic nicotine increases firing rate of SNr GABAergic neurons in vivo... V Cheng Xiao... we’re still gathering data for DA neurons 34

36. GABAergic neurons have increased (or more regular?) firing in chronic nicotine... Thalamus, superior colliculus GABAergic DAergic SNc SNr Endogenous ACh PPTg Cholinergic Striatum Upregulated a4* nAChRs... Analogous to “deep brain stimulation” in subthalamic nucleus? STN Hypothesis: Circuit-based neuroprotection by chronic nicotine in substantia nigra via Cholinergic, Dopaminergic, and GABAergic neurons in Hindbrain & Midbrain 35

37.  6* is Expressed in Midbrain Dopamine Neurons Bregma -3.08 mm Highest affinity for nicotine (function) Involved in nicotine-stimulated DA release Selectively lost in PD Mike Marks 36

38. Selective activation of DA neurons via α6 subunits & bacterial artificial chromosome (BAC) Transgenics transgene Plasmid-based Transgenic gene of interest BAC Transgenic gene of interest transgene BACs: 1.50-300kb 2.Easily manipulated 3.Includes most gene expression regulatory elements 4.Faithfully replicates expression pattern of endogenous gene  6 mRNA  6 BAC 37

39. A carbon fiber electrode allows us to detect dopamine electrochemically in striatal slices carbon fiber A 38

40. Selective Activation of DA Neurons Stimulates Locomotor Activity...... but, unlike selective  activation, shows no sensitization, Possibly because α6* receptors do not participate in SePhaChARNS 39

41. Behavior Circuits Synapses Neurons Intracell. Binding Nic vs ACh Proteins RNA Genes Nicotine Addiction Nicotine Addiction Parkinson’s Disease Parkinson’s Disease ADNFLE 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. 4.a. Upregulation explains enhanced LTP in the perforant path, via a direct presynaptic mechanism. This is a simple model for cognitive sensitizationa. b.Upregulation explains tolerance to chronic nicotine, via a GABAergic-DA circuit in the midbrain. c.A similar circuit mechanism may protect DA neurons against harmful burst firing in PD. 6. We do not yet understand several processes, e. g. somatic signs of withdrawal, stress-induced nicotine use, and ANFLE circuitry. 5. Repeated selective activation of DA neurons, via hypersensitive  6* receptors, produces neither locomotor tolerance nor sensitization. 40

42. Bruce Cohen, Purnima Deshpande, Ryan Drenan, Carlos Fonck, Sotiris Masmanidis, Sheri McKinney, Raad Nashmi, Johannes Schwarz, Kim Scott, Rahul Srinivasan, Cagdas Son, arry 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 Caltech “Alpha Club” Targacept Univ Pennsylvania Univ. Utah Rockefeller Univ 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” Baylor Coll of Medicine Univ. of Cambridge Univ of Alabama 41