Three responses to chronic nicotine exposure:

Studies on

genes, proteins, drugs, cells, circuits, and behaviorNovember, 2007Henry Lester

1. How does one explain nicotine addiction?Does it matter? Wont everyone stop smoking soon?

Smokeless tobacco?2. Nicotine as an imperfect therapeutic drug

Best example: Parkinsons disease

3. Cellular / molecular approaches to better therapies

Prevalence of current smoking among adults aged 18 years and overProgress on smoking cessation is very slowSelf-medicationmay be the reasonIn 2002, individuals with a current psychiatric disorder comprised 7% of the US population,

but they smoked 34-46% of all cigarettes in the US.

The nicotine videoProduced for Pfizer to explain varenicline (Chantix) to physiciansThis summarizes knowledge in ~ 2004.ligand is a molecule that binds to another.physical addiction vs psychological addiction.Desensitization and UpregulationSome abbreviations on future slides:ACh, acetylcholinenAChR, nicotinic acetylcholine receptorDA, dopamine

Conclusions from knockout and hypersensitive mice (2005): Activation of a4b2-containing (a4b2*) receptors by nicotineIs necessary and sufficient for sensitization, tolerance, reward, (but withdrawal?)

Focus on a42 receptors

Chronic nicotine exposure causes tolerance of dopamine releaseThe yoked self-administration experimentMaster animalYoked animalRahman, Zhang, Engleman, & Corrigall, 20040.00.51.01.52.02.53.03.54.0Yoked salineYoked nicotineSalineNicotine-4004080120160Time (min)Dialysate DA (nM)

2. Chronic nicotine exposure causes cognitive sensitizationIn 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.

3. Inverse correlation between long-term tobacco smoking and Parkinsons diseaseIn identical twins discordant for both Parkinsons 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 effectsBeneficial results of short-term nicotine exposure:Pain reduction.Increased concentration: ADHD, Schizophrenia. Alzheimer (Aricept = donepezil, a cholinesterase inhibitor; Reminyl = galantamine)Decreased inflammation. Antidepressant actions.

nAChRsare highly permeable to Ca2+as well as to Na+. Possible mechanism 1: The Molecular Relay Race: Signal transduction triggered by a ligand-gated channel

Chronic exposure to nicotine induces more nicotinic receptors Possible Mechanisms 2a, 2b: 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.nicotine addiction? The Receptor Dilemma: How (if at all) do changed receptors contribute to . . .The Bored Receptor (desensitized)versus b. The Exuberant Receptor (upregulated)

Strategy to choose between the bored or exuberant receptors in the response to chronic nicotine exposure1. 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

200 mmMedial Perforant PathPyOrRadLMolAlveusTemperoammonic PathChronic nicotine increases a4 fluorescence ~ 2-fold in hippocampus--a brain area that provides a good model for cognition.

Modified from Laviolette and van der Kooy, Nature Reviews Neuroscience, 2004 Nicotinic receptors in the reward pathwayforebrain midbrain hindbrainLDT,PPTg

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)

a4-YFP knock-in: substantia nigra pars compacta neuronsSpectrally unmixed a4YFPSpectrally unmixed background autofluorescence10 mm10 mm500520540560580600050010001500YFP IntensityWavelength (nm) a4YFP Background

Substantia Nigra Pars Reticulata. . . but does upregulate a4 levels in GABAergic inhibitory neurons. Chronic nicotine does not change a4 levels in dopaminergic neurons . . . Substantia Nigra Pars CompactaSubstantia nigra data also support the exuberant receptor idea4 intensity per TH+ neuron 4 intensity per GAD+ neuron

Chronic Saline1A1B0.00.51.01.52.02.53.03.54.0Yoked salineYoked nicotineSalineNicotine-40-20020406080100120140160180Time (min)Dialysate DA (nM) Rahman et al, 2004Chronic nicotine cell-specifically upregulates a4* receptors: Basis for circuit-based tolerance in midbrainvia exuberant inhibitionEndogenous AChVTA

LDTCholinergicNAcDAergicGABAergic1A2A2B

GABAergic neurons may have increased or more regular firing in chronic nicotine. . .Hypothesis: Circuit-based neuroprotection by chronic nicotine in substantia nigraviaCholinergic, Dopaminergic, and GABAergic neurons in Hindbrain & MidbrainUpregulated a4* nAChRs

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.

Conclusions from hypersensitive & fluorescent miceHow do we develop better therapeutics based on these ideas?

The nicotinic receptors interfacial aromatic box occupied by nicotine Showing the cation-p interaction with unnatural amino acidsaY198C2aY190C1aY93AaW149Bnon-aW55DCollaboration with Dennis Dougherty, Hoag Professor of Chemistry

Stolen photons tell us which subunits are near each otherExperiments like these may show us how to develop better therapies for Parkinsons Disease.

12 m14321234The ultimate reductionist approach,studying nAChR traffic/regulation at the single molecule level.TIRF microscopy of nAChR geGFP in oocytes

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 XiaoJoanne Xiu, Nyssa Puskar, Jai Shanata, Shawna Frazier, Dennis A. DoughertySarah LummisStephan Pless, Joseph Lynch Sharon Grady, Al Collins, Mike Marks, Jeremy Owens, Tristan McClure-Begley, Paul WhiteakerJim Boulter, Istvan Mody, Oliver Dorigo, Arnie Berk, Max Shao, Jack FeldmanJon LindstromJulie Miwa, Nathaniel HeintzUwe Maskos, Jean-Pierre ChangeuxUniv QueenslandUniv of Colorado, BoulderCaltech Alpha ClubUCLA

Univ. PennsylvaniaRockefeller UnivInstitut Pasteur Unnatural Amino Acid ClubUniv of Cambridge

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?

Exuberant receptors are a thermodynamic consequence of durg-receptor Interactions

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).Upregulation Shown in experiments on clonal cell lines transfected with nAChR subunits:Nicotine seems to act as a pharmacological chaperone (Lukas, Lindstrom)ormaturational enhancer (Sallette & Corringer, Heinemann)orNovel slow stabilizer (Green). Upregulation is cell autonomous and receptor autonomous (Henry).

Midbrain slice recordings: functional upregulated receptors in a circuit produce toleranceCheng Xiao

Substantia Nigra Pars Reticulata. . . but does upregulate a4 levels in GABAergic inhibitoryneurons Chronic nicotine does not change a4 levels in dopaminergic neurons . . . Substantia Nigra Pars CompactaSubstantia nigra data also support the exuberant receptor idea

ChronicChronic NicotineChronicAcute NicotineAcute SalineAcuteSimple model forcognitivesensitization:

chronic nicotine + acute nicotine lowers the threshold for perforant pathway LTP