Changes in the brain during chronic exposure to nicotine:

Cellular and subcellular level selectivity of upregulation

Mouse models

October, 2009

Henry LesterNicotineAddictionNicotine

AddictionParkinson’s

DiseaseParkinson’s

DiseaseADNFLEADNFLE

BehaviorBehavior

CircuitsCircuits

SynapsesSynapses

NeuronsNeurons

Subcell.Subcell.

BindingBinding

Nic vs AChNic vs ACh

ProteinsProteins

RNARNA

GenesGenes

Inadvertent therapeutic effects of chronic nicotine

1

Upregulation is a part of

SePhaChARNS (discussed by J.

Lindstrom) Nicotine is a

“Selective Pharmacological Chaperone

of

Acetylcholine Receptor Number and Stoichiometry”

Related phenomena:

1. Chronic nicotine (today’s topic)

2.ADNFLE mutations

3. β2 vs β4 subunit

4.Trafficking motifs

5.Lynx proteins

6.Single molecules

2

Cellular and subcellular specificity of SePhaChARNS

Thalamus,

superior colliculus

SNc, VTA

SNr,VTA

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

EC

Medial Perforant Path

Raad Nashmi et al J Neurosci 2007; Cheng Xiao et al, J. Neurosci 2009 3

MH

IPN

Strategy to evaluate the cellular and subcellular specificity of 4* upregulation

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,CFP4

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

5

200 m

Medial Perforant Path

Py Or Rad

LMol

Alveus

Temperoammonic Path

6

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).

Chronic nicotine increases medial perforant path 4 fluorescence ~ 2-fold.Relevant to cognitive sensitization?

0 10 20 30 400.0

0.5

1.0

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pe (-m

V/m

s)

Stimulus Strength (A)

1 mV

10 ms

1 mV

10 ms

Saline Mecamylamine

-20

-10

0

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LTP

Indu

ctio

n (%

incr

ease

)

p < 0.001

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fEP

SP

Slo

pe

(%

)

Time (min)

Chronic Acute Nicotine Nicotine Saline Nicotine

1 M Nicotine

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SP

Slo

pe (

%)

Time (min)

Chronic Acute Nicotine Saline Saline Saline

Saline Nicotine0

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LTP

Indu

ctio

n (%

incr

ease

)

Chronic

Chronic Nicotine

Saline Nicotine0

10

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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

7

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

8

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

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quen

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0.5 m s

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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

9

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nta

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Substantia Nigra Pars Reticulata(& VTA, not shown)

. . . but does upregulate 4 levels in GABAergic inhibitory neuron somata.

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

Pars Compacta(& VTA, not shown)

Midbrain data show cell specificity of SePhaChARNS

α4 intensity per TH+ neuron

α4 intensity per GAD+ neuron 10

Test for functional α4* upregulation:Electrophysiology in slices and intact anesthetized mice

Cheng Xiao

V

11

Including studies with α4 knockout (KO) mice (J. Drago)

(Tyrosine hydroxylase immunostain)

ACh, nicotine puffs

Chronic nicotine modifies α4* currents in substantia nigra neurons

SN pars compacta DA somataSN pars reticulata GABAergic somata

α4KO

Chronic Saline

1A

Endogenous ACh

1A

2A

1B

2B

0.0

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1.0

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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:

Hyothesis 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 nicotine13

Chronic nicotine modifies firing rates in substantia nigra neurons:

the role of α4* nAChRs on GABAergic neuronsSN pars compacta DA neurons

α4 KO

α4 KO

SN pars reticulata GABAergic neurons

vv Also Tan . . . Laviolette Neuropharm 2009

vv

α4 KO

α4 KO

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

15

We sought α4* nAChRs in striatal neurons, using fluorescence and electrophysiology.

We found none. Therefore,

we developed assays for the α4* nAChRs on dopaminergic nerve terminals in striatum . . .

α4β2* nAChRs may modulate eEPSPs onto medium spiny neurons

V

α4 KO

α4* nAChRs and dopamine D2/D3 receptors modulate sEPSCs in MSNs

α4 KO

19

Chronic nicotine augments nicotinic modulation of sEPSCs in MSNs

20

Chronic nicotine regulates the nigrostriatal pathwayvia α4β2* nAChR upregulation, with cellular and subcellular

selectivity

In the planning & construction phases

21

Knock-in mice with fluorescent nAChR subunits:

Monomeric GFP and Cherryfor studies on localization and on assembly (FRET)

α3, α4, α5, α6, α7, β2, β3, β4

α6* 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

22

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

23

TM2 Pore-Lining Leu9’ Residue Controls Receptor Sensitivity

Miyazawa, Fujiyoshi, Unwin, Nature 2003

Fonck, et al. J. Neurosci. 2005

•Leu9’ Lines the Ion Channel Pore

•Leu9’ Mutations Shift Dose-Response Curve to Left

•Leu9’ Mutations are Dominant & Gain of Function

24

α4 data, not α6!

Recapitulation of Endogenous α6 Expression in Tg Mice:α6 is Expressed in DA Neurons but not GABA Neurons;

But 4 is expressed in both

Selective Activation of DA Neurons Stimulates Locomotor Activity . . .

. . . but, unlike selective α4 activation, shows no sensitization,possibly because α6* receptors do not participate in SePhaChARNS

26

BehaviorBehavior

CircuitsCircuits

SynapsesSynapses

NeuronsNeurons

Subcell.Subcell.

BindingBinding

Nic vs AChNic vs ACh

ProteinsProteins

RNARNA

GenesGenes

Selective nAChR upregulation during chronic exposure to nicotine

1. Nicotine is a selective pharmacological chaperone of acetylcholine receptor number and stoichiometry (SePhaChARNS)(discussed by J. Lindstrom).

2. In the medial perforant path, α4* upregulation explains enhanced LTP, via a direct presynaptic mechanism. This is a simple model for cognitive sensitization.

6. We do not yet understand several processes, e. g. somatic signs of withdrawal, stress-induced nicotine use, and ANFLE circuitry.

NicotineAddictionNicotine

Addiction

Parkinson’s Disease

Parkinson’s Disease

ADNFLEADNFLE

5. Repeated selective activation of DA neurons, via hypersensitive 6* receptors, produces neither locomotor tolerance nor sensitization.

27

3. a. In midbrain, α4* upregulation in GABAergic neurons explains tolerance to chronic nicotine, via the GABAergic-DA circuit.

b. A similar circuit mechanism may protect DA neurons against harmful burst firing in PD.

4. In striatal DA terminals, α4* upregulation may increase the influence of cholinergic interneurons on DA release.

Bruce Cohen, Purnima Deshpande, Ryan Drenan, Carlos Fonck,

Sheri McKinney, Raad Nashmi, Johannes Schwarz,

Rahul Srinivasan, Cagdas Son, Andrew Tapper, Cheng Xiao

Al Collins, Sharon Grady, Mike Marks, Erin Meyers,

Tristan McClure-Begley, Charles Wageman, Paul Whiteaker

Univ of Colorado,

Boulder

Caltech

Univ. of Colorado,

Denver

28

J. Michael McIntoshUniv. Utah

Robert Freedman, Sherry Leonard

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

carbon fiber

A

HO

HO

H2C

CH2

NH3+

29