Bi 202 October 2010 Drug Addiction Henry Lester This lecture is at

1. Bi 202 October 2010 Drug Addiction Henry Lester This lecture is at www.its.caltech.edu/~lester/Recent-lectures

2. Recreational drugs Addictive drugs

3. “poppy that brings sleep” (opium) marijuana, hemp tobacco coca yeast wheat fungus; Salem witch trials? coffee tea Based on plant ergot

4. H+ cocaine in the body exemplifies permeation by weak acids & bases blood Lipid barrier, e. g. membrane(s) lungs, nose, stomach cocaine base (crack) cocaine hydrochloride H+ Cl- South American Indians use Ca(OH)2 from limestone to shift this equilibrium

5. Neurons that make dopamine: “pleasure-reward” system highlighted in a sagittal view (human brain). Most addictive drugs affect this system

6. Goal-seeking behavior controlled by the dopamine pleasure / reward system Nature 2002 417:37 Blue arrows indicate positions where the experimenter gave right (R) and left (L) directional cues, by stimulating the part of the brain that receives left or right whisker signals. Red dots indicate rat head positions at 1-s intervals. Green dots indicate positions at which reward stimulations were administered to dopaminergic cells Black arrows indicate positions 0.5 s after directional commands. Examples of guided rat navigation using brain microstimulation. a, Route followed by a rat guided through a slalom course. Inset, detail of the events that took place inside the dashed enclosure. b, Route taken by a rat guided over a three-dimensional obstacle course. The animal was instructed to climb a vertical ladder, cross a narrow ledge, descend a flight of steps, pass through a hoop and descend a steep (70°) ramp. Two rounds of high-density dopamine cell stimulation were required to guide the rat successfully down the ramp, demonstrating the motivational qualities of stimulation.

7. “Network Model of Pathways by which Acute Exposure to Addictive Drugs Uncouples Behaviorally Relevant Control of DA Neurotransmission” (Sulzer, 2011)

8. 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

9. Three general components of addiction • 1. Tolerance • Dependence • Goal-seeking behavior Tolerance a. Metabolic tolerance: Metabolism of the drug proceeds more efficiently. This occurs primarily in the liver. It occurs for many types of drugs, including aspirin and penicillin. b. Cellular tolerance: Individual neurons or neuronal circuits become less responsive to the drug.

11. Morphine dependence at the single-cell level Rats were exposed to morphine for 5 days and then observed for “withdrawal behavior”: irritability, jumping, wet-dog shakes, head-bobbing, sweeping tail movements, yawning. Action potential frequencies were recorded in the noradrenergic neurons of the locus coeruleus. When morphine was withdrawn from the receptors, with the help of a morphine antagonist, the firing frequency in the neurons increased in parallel with the withdrawal behavior. Rasmussen et al J Neurosci 10, 238 (1990) withdrawal behavior rats morphine-treated control firing frequency in locus coeruleus morphine antagonist (naltrexone)

12. a P P P g b Activated GPCRs are sometimes phosphorylated and endocytosed. This “downregulation” terminates signalling. 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.

13. 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+.

14. 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” 1 million channels Closed states(s) more stable than open states nicotine 20 seconds

15. 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. • HAL & Dennis Dougherty (Chem) study this difference

16. The AChBP interfacial “aromatic box” occupied by nicotine (Sixma, 2004), Probed functionally by unnatural amino acid mutagenesis H-bond 12-fold tighter binding vs muscle Additional H-bond to non-α subunit aY198 C2 aW149 B aY93 A Cation-πinteraction 16-fold tighter binding vs muscle aY190 C1 non-aW55 D (Muscle Nicotinic numbering)

17. Components of nicotine dependence Reward Cognitive Sensitization Stress Relief Weight control Self-medication in schizophrenia (HAL, PHP project) Behavior Circuits Synapses Neurons Changes in the Brain during Chronic Exposure to Nicotine Intracell. Binding Nic vs ACh Proteins RNA Genes

18. lynx α agonist agonist α aux DA

19. Complexity of nicotine dependence May arise from the widespread distribution of Highly nicotine-sensitive nAChRs

20. V The tactic of fluorescent knock-in mice for evaluating cellular and subcellular specificity of nAChR upregulation 1. Generate knock-in mice with fully functional, fluorescent nAChRs 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 ACh, nicotine puffs YFP

21. Cellular and subcellular specificity of Selective α4* nAChR Upregulation CA EC MH DG IPN Medial Perforant Path STN Striatum SNc, VTA Thalamus, superior colliculus SNr, VTA Nashmi et al J Neurosci 2007; Xiao et al, J. Neurosci 2009; Xiao et al, in review

22. Either activation and/or desensitization can be amplified by upregulation nAChR activation Nicotine activates quiescent nAChRs upregulated nAChRs ~ 2 min naïve nAChRs Nicotine desensitizes ongoing activation nAChR activation

23. Like most drugs, nicotine is a weak base. Its neutral form passes through 6 plasma membranes in ~ 20 s Alvelolar epithelium Brain capillary Lungs Blood CSF H+ logP = 1.1 = log (solubility in octanol / water)

24. Na+ “Inside-out” Drug Action by Nicotine at α4β2 nAChRs nAChR membrane Classical Pathway: Channel activation & desensitization Ca2+ Plasma Golgi Clathrin Early endosome Secretory vesicle Nicotine in CSF COPI ATF6 Lysosome Golgi complex ATF6 COPI Pharmacological Chaperoning→ upregulation COPII Endoplasmic reticulum COPII vesicle Sec 13/31 nAChR PERK IRE1 M3-M4 loop ATF4 Sec24 Sec23 eIF2α Unfolded protein response Sar1 → Do neurons survive Despite stressors? H+ XBP1 UPRE + BiP ER PERK Nucleus IRE1

25. Three possible results of nicotine-nAChR binding in the endoplasmic reticulum 2. Nicotine binding at subunit interface favors assembled nAChRs (a “matchmaker”) • Agonist binding eventually favors stable, • high-affinity states (a “chaperone”) agonist Bound states with increasing affinity unbound 106 channels “closed” AC ? “activated” Highest affinity nicotine 20 sec Free Energy “desensitized” Reaction Coordinate 3. Nicotine may displace lynx, directing nAChRs toward cholesterol-poor domains (an “escort”) nicotine lynx

26. Chronic nicotine causes tolerance of dopamine release 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

27. 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

28. Chronic nicotine increases medial perforant path a4 fluorescence ~ 2-fold. Relevant to cognitive sensitization? Humans: Some smokers report that they think better when they smoke; smokers who smoke nicotine cigarettes (but not nicotine-free cigarettes) display certain cognitive enhancements (Rusted and Warburton, 1992; Rusted et al., 1995). Rodents: 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 α4β2* dependent. Also chronic nicotine produces better spatial working memory performance in the radial arm maze (Levin et al., 1990; Levin et al., 1996). Alveus Py Or Rad LMol 200 mm Temperoammonic Path Medial Perforant Path

29. Changes in the brain during chronic exposure to nicotine 1. The modern hypothesis: selective upregulation of nAChRs (via SePhaChARNS) is necessary and sufficient for the early stages of nicotine dependence (hours, days, and weeks) • Selective upregulation thus instantiates some phenomena typically invoked to explain the neuroscience of drug abuse: • adaptation, neuroadaptation, plasticity, compensation, and homeostasis • We do not yet understand several processes, including • somatic signs of withdrawal and • stress-induced nicotine use.