Changes in the brain during chronic exposure to nicotine

November, 2009

Or:

How Core prepares Techers for Neuroscience

Henry LesterNicotine

AddictionNicotine

AddictionParkinson’s

DiseaseParkinson’s

DiseaseADNFLEADNFLE

BehaviorBehavior

CircuitsCircuits

SynapsesSynapses

NeuronsNeurons

Intracell.Intracell.

BindingBinding

Nic vs AChNic vs ACh

ProteinsProteins

RNARNA

GenesGenes

Inadvertent therapeutic effects of chronic nicotine

--Requires chronic exposure to nicotine

Total: 41 slides1

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 Society2

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

BehaviorBehavior

CircuitsCircuits

SynapsesSynapses

NeuronsNeurons

Intracell.Intracell.

BindingBinding

Nic vs AChNic vs ACh

ProteinsProteins

RNARNA

GenesGenes

NicotineAddiction

NicotineAddiction

Parkinson’s Disease

Parkinson’s Disease

ADNFLEADNFLE

nicotine20 seconds

1 millionchannels

Closed states(s) more stable than open states

3

NicotineAddictionNicotine

Addiction

Parkinson’s Disease

Parkinson’s Disease

BehaviorBehavior

CircuitsCircuits

SynapsesSynapses

NeuronsNeurons

Intracell.Intracell.

BindingBinding

Nic vs AChNic vs ACh

ProteinsProteins

RNARNA

ADNFLEADNFLE

GenesGenes

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 42*

(But note that some a42* receptors contain 5, 6, or β3 subunits)

4

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.

For nicotine, EC50

(muscle) / EC50

(α4β2) = 400

What causes this difference?

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

5

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

W149BY93

A

non-W55D

Y198C2

Y190C1

(Muscle Nicotinic numbering)

Hydrogen bond12-fold tighter binding vs

muscle

Cation-π interaction16-fold tighter binding vs muscle

Joanne Xiu. Nyssa Puskar, Jai Shanata Xiu et al, Nature 2009 6

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

7

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

BehaviorBehavior

CircuitsCircuits

SynapsesSynapses

NeuronsNeurons

Intracell.Intracell.

BindingBinding

Nic vs AChNic vs ACh

ProteinsProteins

RNARNA

GenesGenes

Upregulation is a part of SePhaChARNS

Nicotine is a

“Selective Pharmacological Chaperone

of

Acetylcholine Receptor Number

and

Stoichiometry”NicotineAddiction

NicotineAddiction

Parkinson’s Disease

Parkinson’s Disease

ADNFLEADNFLE

Related phenomena:

1. Chronic nicotine2. ADNFLE mutations

4. β2 vs β4 subunit

9

+ +

Fre

e E

nerg

y

Reaction Coordinate

Free subunits

Increasingly stable

assembled states

#1. Nicotine binds to subunit interfaces, favoring assembled receptors

+Boundstates with

increasing affinity

Fre

e E

nerg

y

Reaction Coordinate

C

AC

A2C A2O

A2D

Highest affinity bound state

unbound

#2. Binding eventually favors high-affinity states

Thermodynamics of SePhaChARNS

10

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: 10 10030 300

. . . and then in intracellular organelles.

Thermodynamics of SePhaChARNS#3. Acid trapping may keep intracellular nAChRs desensitized

11

Thermodynamics of SePhaChARNS, #4.

Reversible stabilization amplified by covalent bonds?

Nicotine

hr0 20 40 60

Increased High-Sensitivity

Receptors

RLS RHS

Covalently stabilized

AR*HSDegradation

+ nicotine

?

12

Secretory pathway

Overview of membrane protein traffic

Pharmacological chaperoning: upregulation starts here(Oversimplification)

Early LTP / Opioids: regulation starts here(Oversimplification)

out

13

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

Neuro2a

λ

14

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 15

Whole-cell donor photobleach experiments suggest:24 hr nicotine (1 μM)

Partially shifts nAChR stoichiometryfrom (α4)3(β2)2 to (α4)2(β2)3

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

Neuro2aCagdas Son

α4CFP + α4YFP 1:1plus β2

α4 plus β2CFP + β2YFP

1:1 16

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 (%)

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 (%)

4 hour nicotine exposure: increased (4)2(2)3 assembly in Golgi

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

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

17

Most membrane proteins exit the ER in a COPII-dependent manner

COP II: Sec23/24 heterodimer

GTPase: Sar1

scission

ER lumenER membrane

Cytosolic compartmentMancias & Goldberg, Traffic 2005

18

PM-mCherry α4-GFPβ2 Overlay

A. Control

B. 0.1 µM nic 48 h

TotalInternal

ReflectionFluorescenceMicroscopy

(TIRFM)

Neuro2a19

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

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

BehaviorBehavior

CircuitsCircuits

SynapsesSynapses

NeuronsNeurons

Intracell.Intracell.

BindingBinding

Nic vs AChNic vs ACh

ProteinsProteins

RNARNA

GenesGenes

NicotineAddiction

NicotineAddiction

Parkinson’s Disease

Parkinson’s Disease

ADNFLEADNFLE

21

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

22

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, submitted23

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

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.

25

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

26

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

27

VTA Recordings from a 16-channel microprobe

A. Probable DA neuron (“cluster 1”) B. Probable GABA neuron “cluster 4”)

T1 – T4, s

nicotine3 mg/kg

quinpirole0.3 mg/kg

C. Cross-correlation(cluster 1 vs cluster 4)

Sotiris MasmanidisT1 - T4, s

C. Cross-correlation (cluster 1 vs cluster 4) 50 μm

28

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

29

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 30

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 nicotine31

Midbrain slice recordings: functional upregulated receptors in a simple circuit

Cheng Xiao 32

1

GABA neurons

Firi

ng r

ate

(Hz)

0

10

20

30

40

Chronic nicotineChronic saline(5 animals each)

DA neurons

0

1

2

3

4

5

Firi

ng r

ate

(Hz)

0

3

6

9

12

15

18

0.0

0.5

1.0

1.5

2.02

4 ce

lls

22

cells

29

cells

26

cells

ChronicNicotine

ChronicSaline

ChronicNicotine

ChronicSaline

In SNr of α4 knockout, chronic nicotine does not affect firing rates

Cheng Xiao 33

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

V

Cheng Xiao

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

34

GABAergic neuronshave increased

(or more regular?) firing

in chronic nicotine. . .

Thalamus,

superior colliculus

GABAergic

DAergic

SNc

SNrEndogenous 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

viaCholinergic, Dopaminergic, and GABAergic neurons in Hindbrain & Midbrain

35

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

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. 50-300kb2. Easily manipulated3. Includes most gene expression

regulatory elements4. Faithfully replicates expression

pattern of endogenous gene

6 mRNA

6 BAC

37

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

carbon fiber

A

HO

HO

H2C

CH2

NH3+

38

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

BehaviorBehavior

CircuitsCircuits

SynapsesSynapses

NeuronsNeurons

Intracell.Intracell.

BindingBinding

Nic vs AChNic vs ACh

ProteinsProteins

RNARNA

GenesGenes

NicotineAddictionNicotine

Addiction

Parkinson’s Disease

Parkinson’s Disease

ADNFLEADNFLE

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

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 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

Romiro Salas, Mariella De Biasi

Caltech“Unnatural Club”

Baylor Coll of Medicine

Univ. of Cambridge

Univ of Alabama

41