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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
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