changes in the brain during chronic exposure to nicotine: cellular and subcellular level selectivity...
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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
1.5
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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
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90
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fEP
SP
Slo
pe
(%
)
Time (min)
Chronic Acute Nicotine Nicotine Saline Nicotine
1 M Nicotine
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fEP
SP
Slo
pe (
%)
Time (min)
Chronic Acute Nicotine Saline Saline Saline
Saline Nicotine0
10
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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
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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0
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4
6
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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
9
0 500 1000 1500 2000 2500 30000
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mu
lativ
e P
erc
en
tag
e
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um
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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
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:
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