Prof. Uri Littauer Lecture:

Changes in the brain during chronic exposure to nicotine

Studies on

genes, receptor binding, proteins, drugs, cells, circuits, and behavior

February, 2009Henry Lester

1/33

Conclusions from hypersensitive and knockout mice (2005):

Activation of 4-containing (*receptors by nicotine

Is sufficient and necessary for

tolerance, sensitization, reward, (but withdrawal?)

Picciotto, Marubio, Maskos, Tapper . . . . Caltech, Pasteur

There are good reasons to focus on 42* nAChRs

What are the mechanisms?

(But remember that some 42* receptors contain:≥ one 5, 6, or β3 subunit)

There are excellent reasons to focus on

the nicotinic acetylcholine receptors (nAChR) themselves:

subtypes: 1-10, 1 - 4, γ, δ, ε

2/33

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

3. EC50 at muscle receptors: nicotine, ~400 μM; ACh, ~ 45 μM.

Ratio, ~10. Justified to square this because nH = 2.

Functional ratio, ~100. What causes this difference?

Nicotine and ACh act on many of the same receptors, but . . .

3/33

Binding region

Membrane region

Cytosolicregion

(incomplete)

Colored by secondary

structure

Colored by subunit(chain)

Nearly Complete Nicotinic Acetylcholine Receptor, a Well-Studied Cys-loop Receptor

~ 2200 amino acids in 5 chains

(“subunits”),

MW ~ 2.5 x 106

4/33

5/30

The AChBP interfacial “aromatic box” occupied by nicotine (Sixma, 2004)

W149BY93

A

non-W55D

Y198C2

Y190C1

(Muscle Nicotinic numbering)

Nicotine makes a stronger cation-π interaction with Trp Bat α4β2 receptors than at muscle receptors;

this partially explains α4β2 receptors’ high binding affinity for nicotine.

4 3 2 1 01

10

100

1,000

nic

otin

e E

C50

,M

Number of F-Trp atoms

Receptor muscle (~3-fold) (47-fold)

WT

WT, without cation-π

interaction

6/33

NH

HN

NH

W149

O T150

O

replace i+1 byanalogous

-hydroxy acid

NH

ONH

W149

O Tah150

O

HN+ H

N+ weakenedhydrogen bond

A BC1

C2

D

7/30

Nicotine makes a stronger H-bond to a backbone carbonyl

at α4β2 than at muscle receptors:With amide to ester substitution,

EC50 increases 20-fold vs 1.5-fold

Weaker hydrogen bond

Deleted hydrogen bond

8/30Xiu, Puskar, Shanata, Lester, Dougherty. 2009. Nature in press

Nicotine EC50 values:

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.

Muscle nAChR single component ~ 400 μM

α4β2 two components ~ 1 μM, ~200 μM

kinase

phosphorylatedprotein

cAMPCa2+

intracellularmessenger

receptor

tsqiG protein

enzymechannel effector

NMDA receptors

and

nAChRs

are highly permeable to Ca2+

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

9/33

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).

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” (Lukas, Lindstrom)

or

“maturational enhancer”

(Sallette, Changeux, & Corringer; Heinemann)

or

“Novel slow stabilizer” (Green).

Upregulation is “cell autonomous” and “receptor

autonomous” (Henry). 10/62

+ +

Fre

e E

nerg

y

Reaction Coordinate

Free subunits

Increasingly stable

assembled states

Nicotine stabilizes subunit interfaces

2.5 M Nicotine+

1 M Nicotine+

(pKa = 7.8)

pH 7.4

pH 7.0

Nicotine accumulates in acidic cells & organelles

+Boundstates with

increasing affinity

Fre

e E

ne

rgy

Reaction Coordinate

C

AC

A2C A2OA2D

Highest affinity bound state

unbound

Binding eventually favors high-affinity states

Nicotine

hr0 20 40 60

Incr

ease

d H

igh-

Sensi

tivit

y R

ece

pto

rs

RLS RHS

Covalently stabilized

AR*HSDegradation

+ nicotine ?

Upregulation is a thermodynamic consequence of nicotine-nAChR interactions

SePhaCHARNS“Selective Pharmacological Chaperoning of Acetylcholine Receptor Number and Stoichiometry”

11/33

12

Nucleus

TIRFM

FRET

High-resolution fluorescence microscopy to study SePhaChARNS

Golgi

ER

PMLTP / Opioids: regulation starts herePharmacological chaperoning: upregulation starts here

Förster resonance energy transfer (FRET): a test for subunit proximity

ECFPXFP =EYFP

mEYFPmVenusmCerulean mEGFP mCherry

Ligand binding M1 M2 M3 M4M3-M4 loop

M4

M3 - M4loopα4

c-myc tag XFP

4-XFP 2-XFP

HA tag XFP

FRET pairs(m = monomeric)

N C N Cβ2

-20 0 20 40 60 800

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

Data: Data1_C54Model: GaussEquation: y=y0 + (A/(w*sqrt(PI/2)))*exp(-2*((x-xc)/w)^2)Weighting: y No weighting Chi^2/DoF = 288.49226R^2 = 0.9912 y0 4.19078 ±1.65095xc 12.03086 ±0.06603w 20.05847 ±0.15945A 12986.99416 ±114.84783

Nu

mb

er o

f P

ixel

s

% FRET Efficiency

2ECFP 4EYFP

FRET NFRET

13/33

Theory of FRET in pentameric receptors with αnβ(5-n) subunits

No FRET

No FRETE

1/2 1/4 1/4

E1 E2 E3 E4

1/8

1/4

1/4

1/8 1/8 1/8

50% α-CFP, 50% α-YFP

b/a =1.62; 1.62-6 = 0.055

0

20

40

60

80

0 20 40 60 80 100Distance between adjacent subunits, A

FR

ET

Eff

icie

ncy 100%(3(

100%((3

100% α3β2100% α2β3

% receptors with α3 14/33

A key experiment: changes in subunit stoichiometry caused by chronic nicotine!

Cagdas Son

0

2

4

6

8

10

12

14

16

(4CFP + YFP) : 21:1

+ Nicotine

% F

RE

T E

ffic

ienc

y

control

(CFP + YFP)1:1

+ Nicotinecontrol

Neuro2a 15/33

16

4 hour nicotine exposure causes an increase in (4)2(2)3 assembly

0 2 4 6 8 10 12 14 16 18 200

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

55000

Pix

el c

ount

NFRET (%)

0 2 4 6 8 10 12 14 16 18 200

500

1000

1500

2000

2500

3000

3500

4000

Pix

el c

ount

NFRET (%)

0 2 4 6 8 10 12 14 16 18 200

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

55000

Pix

el c

ount

NFRET (%)

WHOLE CELLNo treatment

R2 = 0.999y0 = 0 xc1 = 8.7 ± 0.06w1 = 2.22 ± 0.12A1 = 88465 ± 34150xc2 = 10 ± 0.36w2 = 2.92 ± 0.19A2 = 109476 ± 34316

GOLGINo treatment

R2 = 0.999y0 = 0 xc1 = 8.28 ± 0.07w1 = 1.9 ± 0.05A1 = 6756 ± 620xc2 = 9.72 ± 0.07w2 = 1.8 ± 0.04A2 = 5298 ± 621

WHOLE CELL+ 1 M NICOTINE 4 h

R2 = 0.998y0 = 0 xc1 = 8.5 ± 0.18w1 = 2.4 ± 0.1A1 = 130438 ± 36122xc2 = 10.1 ± 0.26w2 = 2.24 ± 0.14A2 = 64907 ± 26106

0 2 4 6 8 10 12 14 16 18 200

500

1000

1500

2000

2500

3000

3500

4000

Pix

el c

ount

NFRET (%)

GOLGI+ 1 M NICOTINE 4 h

R2 = 0.998y0 = 0 xc1 = 8.37 ± 0.02w1 = 2.33 ± 0.03A1 = 11498 ± 239xc2 = 10.21 ± 0.04w2 = 1.51 ± 0.06A2 = 1986 ± 233

PM-mCherry α4-GFPβ2 Overlay

A. Control

B. 0.1 µM nic 48 h

TotalInternal

ReflectionFluorescenceMicroscopy

(TIRFM)

17/33

18

Differential subcellular localization and dynamics of α4GFP* receptors

α4GFPβ2

α4GFPβ4 (1:1)

α4GFPβ2 overlayplasma memb. mCherry

α4GFPβ4 overlay

3 RXR per β subunit

0 RXR per β subunit

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,CFP19/33

Cellular and subcellular specificity of SePhaChARNS

Thalamus,

superior colliculus

SNc

SNr

Striatum

Upregulation?

Transmitter Soma Term. Region / projection

Glu ?? Yes* Entorhinal cortex → dentate gyrus

ACh No No Medial habenula → Interpeduncular nucleus

DA No* Yes* Ventral tegmental area, substantia nigra pars compacta →

Striatum

GABAA Yes* Yes* SN pars reticulata, VTA → SNC, VTA

CA

DG

ECMH

IPNMedial Perforant Path

* = upregulation shown with electrophysiology

Nashmi et al J Neurosci 2007; Xiao et al, in prep.20/33

The Caltech 4 fluorescent mice . . . normal in all respects

21/33

VTA GABAergic and DA neurons have contrasting responses to nicotine in vivo

DA neuron, ~ 1700 spikes

Nicotineinjection

GABAergic neuron (5 s smoothing), ~ 8300 spikes

0.05 m V2 m s

0.05 m V2 m sF

requ

ency

, H

z

0 100 200 300 400 500 600 700

0

2

4

6

0

5

10

15

20

25

s

Fre

quen

cy,

Hz

0.1 m V

0.5 m s

0.1 mV0.5 ms

A B C D0.05 m V2 m s

0.05 mV2 ms

4*, 6*, and/or 7

4* only

V

GABAergic

DAergic

VTA

WT mouse

22/30

4-YFP knock-in: substantia nigra pars compacta neurons

Raad Nashmi

Spectrally unmixed 4YFP Spectrally unmixed background autofluorescence

10 m 10 m

Shortcut to Projections of 32-32-LS5unmix.avi.lnk

23/33

24/24

0 500 1000 1500 2000 2500 30000

20

40

60

80

100

Cu

mu

lativ

e P

erc

en

tag

e

0 500 1000 15000

20

40

60

80

100C

um

ula

tive

Pe

rce

nta

ge

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

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 salineYoked nicotine

Saline Nicotine

-40 -20 0 20 40 60 80 100120140160180

Time (min)

Dia

lysa

te D

A (

nM

)

Rahman et al, 2004

2BDecreased 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(1st expsoure)

+ acute nicotine25/33

Midbrain slice recordings: functional upregulated receptors in a simple circuit

Cheng Xiao

In SNr of α4 knockout, these effects of chronic nicotine vanish

26/33

Chronic nicotine increases firing rate of SNr GABAergic neurons in vivo . . .

V

Cheng Xiao

. . . we’re still gathering data for DA neurons

27/33

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); β* dependent

also chronic nicotine produces better spatial working memory performance in

the radial arm maze (Levin et al., 1990; Levin et al., 1996).

28/33

200 m

Medial Perforant Path

Py Or Rad

LMol

Alveus

Temperoammonic Path

Chronic nicotine increases perforant path 4 fluorescence ~ 2-fold

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.

29/33

0 10 20 30 400.0

0.5

1.0

1.5

2.0

2.5

Slo

pe (-m

V/m

s)

Stimulus Strength (A)

1 mV

10 ms

1 mV

10 ms

Saline Mecamylamine

-20

-10

0

10

20

30

40

LTP

Indu

ctio

n (%

incr

ease

)

p < 0.001

0 20 40 60 80 10080

90

100

110

120

130

140

150

160

fEP

SP

Slo

pe

(%

)

Time (min)

Chronic Acute Nicotine Nicotine Saline Nicotine

1 M Nicotine

0 20 40 60 80 10080

90

100

110

120

130

140

fEP

SP

Slo

pe (

%)

Time (min)

Chronic Acute Nicotine Saline Saline Saline

Saline Nicotine0

10

20

30

40

LTP

Indu

ctio

n (%

incr

ease

)

Chronic

Chronic Nicotine

Saline Nicotine0

10

20

30

40

LTP

Indu

ctio

n (%

incr

ease

)

P < 0.001

Chronic

Acute Nicotine

10 min 80 min

1mV

10 ms

10 ms

Saline

Nicotine

0.5 mV

Acute Nicotine

Chronic10 min 80 min

Saline

Nicotine

0.5 mV

5 ms

Acute SalineChronic

Acute Saline

Acute

Simple model forcognitive

sensitization:

chronic nicotine +

acute nicotine lowers the threshold

for perforant pathway LTP

30/33

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. Chronic exposure to nicotine chaperones α4β2* number and stoichiometry.

3. These processes lead to cell-specific α4β2* upregulation.

4. a. Upregulation explains tolerance to chronic nicotine, via a GABAergic-DA circuit in the midbrain.b. Upregulation also explains enhanced LTP in the perforant path, via a direct presynaptic mechanism. This is a simple model for cognitive sensitization

Genes

Binding

Proteins

Cells

Circuits

Behavior

5. We do not yet understand several processes, including somatic signs of withdrawal and stress-induced nicotine use.

31/62

Understanding the changes in the brain during chronic exposure to nicotine:Translational relevance of SePhaChARNS

1. Nicotine addiction (~1 billion people, since 1540)

2. Strong inverse correlation between smoking & Parkinson’s disease (~ 50 million people, since 1819)

3. Tobacco use suppresses seizures in patients with autosomal dominant nocturnal frontal-lobe epilepsy, linked to the α4 and β2 subunits (~1000 patients, since 1994)

32/62

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

Univ of Colorado, Boulder

Caltech“Alpha Club”

Targacept

Univ. Pennsylvania

Univ. Utah

Rockefeller Univ

Dennis DoughertyKiowa 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

Stephan Pless, Joe Lynch

Caltech“Unnatural Club”

Uni Queensland

Univ. of Cambridge

33/33

kinase

P P. . .other proteins

bind to the phosphates . . .

Activated GPCRs are sometimes phosphorylated and endocytosed. This “downregulation” terminates signalling.

P P

But continual signalling can activate genes

During activation, the G protein leaves . . .

. . . revealingphosphorylationsites . . .

(not a synaptic vesicle)

. . . triggeringendocytosis.

34/31

FRET with fluorescent subunits tells us about nicotinic receptor assembly

4Y/

4C/2

4/2

Y/2C

6Y/

6C/2

4/2

/3Y/3

C

*

Neuro2a35/62

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”

Some abbreviations on future slides:

ACh, acetylcholine

nAChR, nicotinic acetylcholine receptor

DA, dopamine

nicotine20 seconds

1 millionchannels

36/31

Chronic nicotine causes tolerance of dopamine release

Master animal

Yoked animal

Rahman, Zhang, Engleman, & Corrigall, 2004

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0 Yoked salineYoked nicotine

Saline Nicotine

-40 0 40 80 120 160

Time (min)

Dia

lysa

te D

A (

nM)

37/31