1/24

Three responses to chronic nicotine exposure:

Studies on

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

November, 2007

Henry Lester

2/24

1. How does one explain nicotine addiction?

Does it matter? Won’t everyone stop smoking soon?

Smokeless tobacco?

2. Nicotine as an imperfect therapeutic drug

Best example: Parkinson’s disease

3. Cellular / molecular approaches to better therapies

3/24

Prevalence of current smoking among adults aged 18 years and over

(National Health Interview Survey)

United States, 1997–2006

1996 1998 2000 2002 2004 20060

5

10

15

20

25

30

perc

ent

year

Progress on smoking cessation is very slow

“Self-medication”may be the reason

In 2002, individuals with a current psychiatric disorder comprised 7%

of the US population,

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

4/24

The nicotine video

Produced for Pfizer to explain varenicline (Chantix) to

physicians

This summarizes knowledge in ~ 2004.

“ligand” is a molecule that binds to another.

“physical” addiction vs “psychological” addiction.

“Desensitization“ and “Upregulation”

Some abbreviations on future slides:

ACh, acetylcholine

nAChR, nicotinic acetylcholine

receptor

DA, dopamine

receptors become “bored”

nicotine20 seconds

1 millionchannels

5/24

Conclusions from knockout and hypersensitive mice (2005):

Activation of 42-containing (*receptors by nicotine

Is necessary and sufficient for

sensitization, tolerance, reward, (but withdrawal?)

Focus on 4β2 receptors

What are the mechanisms?

6/24

1. Chronic nicotine exposure causes tolerance of dopamine releaseThe “yoked self-administration” experiment

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)

7/24

2. Chronic nicotine exposure 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 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.

8/24

3. Inverse correlation between long-term tobacco smoking and Parkinson’s disease

In identical twins discordant for both Parkinson’s 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 effects

Beneficial results of short-term nicotine exposure:

Pain reduction.

Increased concentration: ADHD, Schizophrenia.

Alzheimer (Aricept = donepezil, a cholinesterase inhibitor; Reminyl = galantamine)

Decreased inflammation.

Antidepressant actions.

9/24

kinase

phosphorylatedprotein

cAMPCa2+

intracellularmessenger

receptor

tsqiG protein

enzymechannel effector

nAChRs

are highly permeable to Ca2+

as well as to Na+.

Possible mechanism 1: The “Molecular Relay Race”:

Signal transduction triggered by a ligand-gated channel

10/24

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.

If the upregulated receptors are Desensitized (“bored”),

this might cause decreased synaptic transmission and excitation, leading to tolerance.

But this does not explain cognitive sensitization.

nicotine addiction?

The “Receptor Dilemma”: How (if at all) do changed receptors contribute to . . .

If the upregulated receptors are active,

excitotoxicity might exacerbate Parkinson’s disease.

If the upregulated receptors are desensitized, this might be neuroprotective.

neuroprotection?

a. The “Bored Receptor” (desensitized)

versus b. The “Exuberant Receptor”

(upregulated)

11/24

Strategy to choose between the “bored” or “exuberant” receptors in the response to chronic nicotine exposure

1. Generate mice with fully functional, fluorescent 4* 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

12/24

200 m

Medial Perforant Path

Py Or Rad

LMol

Alveus

Temperoammonic Path

Chronic nicotine increases 4 fluorescence ~ 2-fold in hippocampus--a brain area that provides a good model for cognition.

Functional studies show: the new receptors are “exuberant”, not “bored”

V

14/24

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)

15/24

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

Spectrally unmixed 4YFP Spectrally unmixed background autofluorescence

10 m 10 m

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

500 520 540 560 580 6000

500

1000

1500

YF

P In

tens

ity

Wavelength (nm)

4YFP Background

16/24

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

. . . but does upregulate 4 levels in GABAergic inhibitory neurons.

Chronic nicotine does not change 4 levels in dopaminergic neurons . . . Substantia Nigra

Pars Compacta

Substantia nigra data also support the “exuberant receptor” idea

α4 intensity per TH+ neuron

α4 intensity per GAD+ neuron

17/24

Chronic Saline

1A

Endogenous ACh

1B

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 upregulates 4* receptors: Basis for circuit-based tolerance in midbrain

via “exuberant inhibition”

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 nicotine

1A

2A2B

18/24

. . . As produced by “deep brain stimulation” in subthalamic nucleus

GABAergic neurons may have increased or more regular firing

in chronic nicotine. . .

Hypothesis: Circuit-based neuroprotection by chronic nicotine in substantia nigra

viaCholinergic, Dopaminergic, and GABAergic neurons in Hindbrain & Midbrain

Thalamus,

superior colliculus

GABAergic

DAergicSNc

SNrEndogenous ACh

PPTg

Cholinergic

Striatum

Upregulated 4* nAChRs

19/24

When 4* 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 mice

How do we develop better therapeutics based on these ideas?

20/24

The nicotinic receptor’s interfacial “aromatic box” occupied by nicotine Showing the cation- interaction with unnatural amino acids

Y198C2

Y190C1

Y93A

W149B

non-W55D

Collaboration with Dennis Dougherty, Hoag Professor of Chemistry

21/24

485 nm535 nm 485 nm535 nm

439 nm 439 nm

514 nm

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

FR

ET

Eff

icie

nc

y (%

)

4-2XFP 4-2XFP+

nicotine

. . . After 24 hours in nicotine,exuberant receptors

are assembled more tightly.

“Stolen” photons tell us which subunits are near each other

Experiments like these may show us

how to develop better therapies for Parkinson’s Disease.

22/24

12 μm

1

4

3

2

1

2

3

4

The ultimate reductionist approach,studying nAChR traffic/regulation at the single molecule level.

TIRF microscopy of nAChR eGFP in oocytes

23/24

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 Xiao

Joanne Xiu, Nyssa Puskar, Jai Shanata, Shawna Frazier, Dennis A. Dougherty

Sarah Lummis

Stephan Pless, Joseph Lynch

Sharon Grady, Al Collins, Mike Marks, Jeremy Owens, Tristan McClure-Begley, Paul Whiteaker

Jim Boulter, Istvan Mody, Oliver Dorigo, Arnie Berk, Max Shao, Jack Feldman

Jon Lindstrom

Julie Miwa, Nathaniel Heintz

Uwe Maskos, Jean-Pierre Changeux

Univ Queensland

Univ of Colorado, Boulder

Caltech “Alpha Club”

UCLA

Univ. Pennsylvania

Rockefeller Univ

Institut Pasteur

“Unnatural Amino Acid Club”

Univ of Cambridge

24/24

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?

25/24

+ +

Fre

e E

nerg

y

Reaction Coordinate

Free subunits

Increasingly stable

assembled states

Nicotine may stabilize subunit interfaces

20 M Nicotine+

1 M Nicotine+

(pKa = 7.9)

0 mV-70 mV

Nicotine accumulates in cells

+Boundstates with

increasing affinity

Fre

e E

nerg

y

Reaction Coordinate

C

AC

A2C A2O

A2D

Highest affinity bound state

unbound

Binding eventually favors high-affinity states

“Exuberant receptors” are a thermodynamic consequence of durg-receptor Interactions

Nicotine

hr0 20 40 60

Incr

ease

d H

igh-

Sensi

tivit

y R

ece

pto

rs

RLS RHS

Covalently stabilized

AR*HSDegradation

+ nicotine

?

26/24

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)

or

“maturational enhancer” (Sallette & Corringer, Heinemann)

or

“Novel slow stabilizer” (Green).

Upregulation is “cell autonomous” and “receptor autonomous” (Henry).

27/24

Midbrain slice recordings: functional upregulated receptors in a circuit produce tolerance

Cheng Xiao

28/24

0 500 1000 1500 2000 25000

10

20

30

40

50

Nu

mb

er

of N

eu

ron

s

Mean 4YFP Intensity per TH+ Neuron

Saline (n=612) Nicotine (n=581)

0 250 500 750 1000 1250 15000

10

20

30

40

50

Num

ber

of N

euro

ns

Mean 4YFP Intensity per GAD67+ Neuron

Saline (n=256) Nicotine (n=237)

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

100

Cu

mu

lativ

e P

erc

en

tag

e

Substantia Nigra Pars Reticulata. . . but does upregulate 4 levels in GABAergic inhibitoryneurons

Chronic nicotine does not change 4 levels in dopaminergic neurons . . .

Substantia Nigra Pars Compacta

Substantia nigra data also support the “exuberant receptor” idea

29/24

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