Changes in the brain during chronic exposure to nicotine:

1
Changes in the brain during chronic exposure to
nicotine Cellular and subcellular level
selectivity of upregulation Mouse models
Henry Lester
October, 2009
2
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 (todays topic)
• ADNFLE mutations
• 3. ß2 vs ß4 subunit
• Trafficking motifs
• Lynx proteins
• Single molecules

3
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, VTA
Thalamus, superior colliculus
SNr, VTA
Raad Nashmi et al J Neurosci 2007 Cheng Xiao et
al, J. Neurosci 2009
4
Strategy to evaluate the cellular and subcellular
specificity of a4 upregulation
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
4. Perform physiological experiments on these
regions and cells to verify function
5. Model the cellular and circuit changes
YFP,
Leu9Ala-YFP,
CFP
5
The Caltech a4 fluorescent mice . . . normal in
all respects
6
Chronic nicotine increases medial perforant path
a4 fluorescence 2-fold. Relevant to cognitive
sensitization?
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
a4ß2 dependent. Also chronic nicotine produces
better spatial working memory performance in the
radial arm maze (Levin et al., 1990 Levin et
al., 1996).
Alveus
Py
Or
Rad
LMol
200 mm
Medial Perforant Path
Temperoammonic Path
7
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
8
a4-YFP knock-in substantia nigra pars compacta
neurons
Spectrally unmixed background autofluorescence
Spectrally unmixed a4YFP
10 mm
10 mm
Raad Nashmi
9
VTA GABAergic and DA neurons have contrasting
responses to nicotine in vivo
WT mouse
10
Midbrain data show cell specificity of SePhaChARNS
Chronic nicotine does not change a4 levels in
dopaminergic neuron somata . . .
Substantia Nigra Pars Compacta ( VTA, not shown)
a4 intensity per TH neuron
. . . but does upregulate a4 levels in
GABAergic inhibitory neuron somata.
Substantia Nigra Pars Reticulata ( VTA, not
shown)
a4 intensity per GAD neuron
11
Test for functional a4 upregulation Electrophysi
ology in slices and intact anesthetized mice
Including studies with a4 knockout (KO) mice (J.
Drago)
ACh, nicotine puffs
(Tyrosine hydroxylase immunostain)
Cheng Xiao
12
Chronic nicotine modifies a4 currents in
substantia nigra neurons
SN pars compacta DA somata

13
Chronic nicotine cell-specifically up-regulates
functional a4 receptors Hyothesis for
circuit-based tolerance in midbrain (Nashmi et
al, 2007)
Chronic Saline
Endogenous ACh
VTA
NAc
LDT
DAergic
Cholinergic
GABAergic
Rahman et al, 2004
14
Chronic nicotine modifies firing rates in
substantia nigra neurons the role of a4 nAChRs
on GABAergic neurons
SN pars reticulata GABAergic neurons
SN pars compacta DA neurons
Also Tan . . . Laviolette Neuropharm 2009
vv
vv
a4 KO
a4 KO
a4 KO
15
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
16
We sought a4 nAChRs in striatal neurons, using
fluorescence and electrophysiology. We found
none. Therefore, we developed assays for the
a4 nAChRs on dopaminergic nerve terminals in
striatum . . .
17
a4ß2 nAChRs may modulate eEPSPs onto medium
spiny neurons
a4 KO
18
a4 nAChRs and dopamine D2/D3 receptors modulate
sEPSCs in MSNs

a4 KO
19
Chronic nicotine augments nicotinic modulation of
sEPSCs in MSNs
20
Chronic nicotine regulates the nigrostriatal
pathway via a4ß2 nAChR upregulation, with
cellular and subcellular selectivity
21
In the planning construction phases
Knock-in mice with fluorescent nAChR
subunits Monomeric GFP and Cherry for studies
on localization and on assembly (FRET) a3, a4,
a5, a6, a7, ß2, ß3, ß4
22
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
23
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
24
TM2 Pore-Lining Leu9 Residue Controls Receptor
Sensitivity

• Leu9 Lines the Ion Channel Pore
• Leu9 Mutations Shift Dose-Response Curve to Left
• Leu9 Mutations are Dominant Gain of Function

a4 data, not a6!
Fonck, et al. J. Neurosci. 2005
Miyazawa, Fujiyoshi, Unwin, Nature 2003
24
25
Recapitulation of Endogenous a6 Expression in Tg
Mice a6 is Expressed in DA Neurons but not GABA
Neurons But ?4 is expressed in both
26
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
27
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, a4 upregulation
explains enhanced LTP, via a direct presynaptic
mechanism. This is a simple model for cognitive
sensitization.

• 3. a. In midbrain, a4 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.

Behavior
Circuits
Synapses
Neurons
Subcell.
4. In striatal DA terminals, a4 upregulation
may increase the influence of cholinergic
interneurons on DA release.
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
28
Caltech
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
Univ. of Colorado, Denver
Robert Freedman, Sherry Leonard
Univ. Utah
J. Michael McIntosh
29
A carbon fiber electrode allows us to detect
dopamine electrochemically in striatal slices
carbon fiber