Changes%20in%20the%20brain%20during%20chronic%20exposure%20to%20nicotine

1
November, 2009
Changes in the brain during chronic exposure to
nicotine
Or How Core prepares Techers for
Neuroscience Henry Lester
--Requires chronic exposure to nicotine
Total 41 slides
2
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 Society
3
The nicotine video
Produced for Pfizer to explain varenicline
(Chantix) to physicians
This summarizes knowledge in 2004. physical
addiction vs psychological addiction. Desensiti
zation and Upregulation

Behavior
Circuits
Synapses
Neurons
Nicotine Addiction
Intracell.
Binding
Parkinsons Disease
Nic vs ACh
ADNFLE
Proteins
RNA
Genes
4
Focus on a4?2
Conclusions from hypersensitive and knockout mice
(2005) Activation of a4-containing (a4b2)
receptors by nicotine Is sufficient and
necessary for tolerance, sensitization, reward,
(but not withdrawal)
Behavior
Circuits
Synapses
Neurons
Nicotine Addiction
Intracell.
Binding
Parkinsons Disease
Nic vs ACh
ADNFLE
Proteins
(But note that some a4?2 receptors contain ?5,
?6, or ß3 subunits)
RNA
Genes
5
Nicotine and ACh act on many of the same
receptors, but . . .

• 1. Nicotine is highly membrane-permeant. ACh is
  not.
• Ratio unknown, probably gt 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
• 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(a4ß2) 400
• What causes this difference?

6
The AChBP interfacial aromatic box occupied by
nicotine (Sixma, 2004)
Hydrogen bond 12-fold tighter binding vs muscle
aY198 C2
aW149 B
aY93 A
aY190 C1
non-aW55 D
Joanne Xiu. Nyssa Puskar, Jai Shanata Xiu et al,
Nature 2009
(Muscle Nicotinic numbering)
7
Possible molecular mechanism 1 for changes with
chronic nicotine Signal transduction triggered
by a ligand-gated channel
NMDA receptors and nAChRs are highly permeable
to Ca2 as well as to Na.
8
Possible Mechanism 2 for changes with chronic
nicotine Upregulation
Chronic exposure to nicotine causes upregulation
of nicotinic receptor binding Upregulation 1)
Involves no change in receptor mRNA level 2)
Depends on subunit composition.
Shown in experiments on clonal cell lines
transfected with nAChR subunits Nicotine seems
to act as a pharmacological chaperone or matur
ational enhancer or Novel slow stabilizer.
Upregulation is cell autonomous and receptor
autonomous.
9
Upregulation is a part of SePhaChARNS
Nicotine is a Selective Pharmacological
Chaperone of Acetylcholine Receptor Number
and Stoichiometry

Behavior
Circuits
Synapses
Neurons
Nicotine Addiction
Intracell.
Related phenomena 1. Chronic nicotine 2.
ADNFLE mutations 4. ß2 vs ß4 subunit
Binding
Parkinsons Disease
Nic vs ACh
ADNFLE
Proteins
RNA
Genes
10
Thermodynamics of SePhaChARNS
Increasingly stable assembled states
Free subunits
1. Nicotine binds to subunit interfaces,
favoring assembled receptors
Free Energy
Reaction Coordinate
2. Binding eventually favors high-affinity
states
11
Thermodynamics of SePhaChARNS 3. Acid trapping
may keep intracellular nAChRs desensitized
. . . and then in intracellular organelles.
P. Paroutis, N. Touret, S Grinstein (2004)
Physiology 19 207-215
nicotine/nicotine
10
100
30
300
12
Thermodynamics of SePhaChARNS, 4. Reversible
stabilization amplified by covalent bonds?
Covalently stabilized ARHS
nicotine
?
RHS
RLS
Degradation
Nicotine
Increased High-Sensitivity Receptors
hr
0
20
40
60
13
Overview of membrane protein traffic
Secretory pathway
14
Förster resonance energy transfer (FRET) a test
for subunit proximity
?
Neuro2a
15
Theory of FRET in pentameric receptors with
anß(5-n) subunits
16
Whole-cell donor photobleach experiments
suggest 24 hr nicotine (1 µM) Partially shifts
nAChR stoichiometry from (a4)3(ß2)2 to (a4)2(ß2)3
a4 plus ß2CFP ß2YFP 11
a4CFP a4YFP 11 plus ß2
Neuro2a
Cagdas Son
17
4 hour nicotine exposure increased (a4)2(b2)3
assembly in Golgi
WHOLE CELL 1 mM NICOTINE 4 h R2 0.998 y0
0 xc1 8.5 0.18 w1 2.4 0.1 A1 130438
36122 xc2 10.1 0.26 w2 2.24 0.14 A2
64907 26106
WHOLE CELL No treatment R2 0.999 y0 0 xc1
8.7 0.06 w1 2.22 0.12 A1 88465
34150 xc2 10 0.36 w2 2.92 0.19 A2
109476 34316
GOLGI No treatment R2 0.999 y0 0 xc1
8.28 0.07 w1 1.9 0.05 A1 6756 620 xc2
9.72 0.07 w2 1.8 0.04 A2 5298 621
GOLGI 1 mM NICOTINE 4 h R2 0.998 y0 0
xc1 8.37 0.02 w1 2.33 0.03 A1 11498
239 xc2 10.21 0.04 w2 1.51 0.06 A2 1986
233
18
Most membrane proteins exit the ER in a
COPII-dependent manner
Mancias Goldberg, Traffic 2005
19
Total Internal Reflection Fluorescence Microscopy
(TIRFM)
Neuro2a
20
Differential subcellular localization and
dynamics of a4GFP receptors
a4GFPß2 (11)
3 RXR/ß subunit
a4GFPß4 (11)
zero RXR/ß subunit
21
Strategy to evaluate the cell specificity of a4
upregulation in chronic nicotine
1. Generate knock-in mice with fully functional,
fluorescent a4 receptors
2. Expose the mice to chronic nicotine

3. Find the brain regions and cell types with
changed receptor levels
Behavior
Circuits
Synapses
Neurons
4. Perform physiological experiments on these
regions and cells to verify function
Nicotine Addiction
Intracell.
Binding
Parkinsons Disease
Nic vs ACh
ADNFLE
Proteins
5. Model the cellular and circuit changes
RNA
Genes
YFP,
Leu9Ala-YFP,
CFP
22
The Caltech a4 fluorescent mice . . . normal in
all respects
23
Cellular and subcellular specificity of
SePhaChARNS
CA
EC
DG
Upregulation? 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
Striatum
SNc
Thalamus, superior colliculus
SNr
upregulation shown with electrophysiology
Nashmi et al J Neurosci 2007 Xiao et al,
submitted
24
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 ß2 dependent. Also chronic
nicotine produces better spatial working memory
performance in the radial arm maze (Levin et al.,
1990 Levin et al., 1996).
25
Chronic nicotine increases perforant path a4
fluorescence 2-fold
Alveus
Py
Or
Rad
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. 232331-56.
LMol
200 mm
Medial Perforant Path
Temperoammonic Path
26
Simple model for cognitive sensitization chronic
nicotine acute nicotine lowers the
threshold for perforant pathway LTP
Chronic Nicotine
Acute Nicotine
Acute Saline
Acute
Chronic
Chronic
27
VTA GABAergic and DA neurons have contrasting
responses to nicotine in vivo
WT mouse
28
VTA Recordings from a 16-channel microprobe
Sotiris Masmanidis
29
a4-YFP knock-in substantia nigra pars compacta
neurons
Spectrally unmixed background autofluorescence
Spectrally unmixed a4YFP
10 mm
10 mm
Raad Nashmi
30
Midbrain data show cell specificity of SePhaChARNS
Chronic nicotine does not change a4 levels in
dopaminergic neurons . . .
Substantia Nigra Pars Compacta ( VTA, not shown)
a4 intensity per TH neuron
. . . but does upregulate a4 levels in
GABAergic inhibitory neurons.
Substantia Nigra Pars Reticulata ( VTA, not
shown)
a4 intensity per GAD neuron
31
Chronic nicotine cell-specifically up-regulates
functional a4 receptors Basis for
circuit-based tolerance in midbrain (Nashmi et
al, 2007)
Chronic Saline
Endogenous ACh
VTA
NAc
LDT
DAergic
Cholinergic
GABAergic
Rahman et al, 2004
32
Midbrain slice recordings functional upregulated
receptors in a simple circuit
Cheng Xiao
33
In SNr of a4 knockout, chronic nicotine does not
affect firing rates
Cheng Xiao
34
Chronic nicotine increases firing rate of SNr
GABAergic neurons in vivo . . .
. . . were still gathering data for DA neurons
Cheng Xiao
35
Hypothesis Circuit-based neuroprotection by
chronic nicotine in substantia nigra via Cholinerg
ic, Dopaminergic, and GABAergic neurons in
Hindbrain Midbrain
Striatum
SNc
DAergic
PPTg
GABAergic neurons have increased (or more
regular?) firing in chronic nicotine. . .
Thalamus, superior colliculus
Cholinergic
GABAergic
SNr
Endogenous ACh
Upregulated a4 nAChRs
36
a6 is Expressed in Midbrain Dopamine Neurons

• Highest affinity for nicotine (function)
• Involved in nicotine-stimulated DA release
• Selectively lost in PD

Bregma -3.08 mm
Mike Marks
37
Selective activation of DA neurons via a6
subunits bacterial artificial chromosome (BAC)
Transgenics

• BACs
• 50-300kb
• Easily manipulated
• Includes most gene expression regulatory elements
• Faithfully replicates expression pattern of
  endogenous gene

a6 mRNA
a6 BAC
38
A carbon fiber electrode allows us to detect
dopamine electrochemically in striatal slices
carbon fiber
39
Selective Activation of DA Neurons Stimulates
Locomotor Activity . . .
. . . but, unlike selective a4 activation, shows
no sensitization, Possibly because a6 receptors
do not participate in SePhaChARNS
40
Some changes in the brain during chronic exposure
to nicotine
1. Nicotine potently activates some neuronal
nAChRs (because it participates in both cation-p
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 a4ß2 upregulation,
with cellular and subcellular specificity.
Behavior

• 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.

Circuits
Nicotine Addiction
Synapses
Neurons
Parkinsons Disease
Intracell.
ADNFLE
Binding
Nic vs ACh
5. Repeated selective activation of DA neurons,
via hypersensitive ?6 receptors, produces
neither locomotor tolerance nor sensitization.
Proteins
RNA
6. We do not yet understand several processes,
e. g. somatic signs of withdrawal, stress-induced
nicotine use, and ANFLE circuitry.
Genes
41
Dennis Dougherty Kiowa 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
Univ. of Cambridge
Baylor Coll of Medicine
Caltech Alpha Club
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
Targacept Univ Pennsylvania Univ.
Utah Rockefeller Univ
Univ of Alabama