Marc G. Caron, Ph.D.

1
Phenotype-Driven Approaches to Understanding the
Neuronal Plasticity Associated with Drugs of
Abuse
Marc G. Caron, Ph.D.
Dept. Cell Biology Duke University
Medical Center
2
Addictive process is amenable to experimental
genetic approaches
Drug abuse/Addiction

• Addiction can be considered as a loss of control
  over the drug-taking or compulsive drug-seeking
  behavior, despite adverse physiological and
  devastation social consequences.
• (a failure in the neurobiology of
  decision making)

• Process can be modeled in animals because the
  causative agent is known and several behavioral
  paradigms have been established.
• These behavioral paradigms have been adapted to
  mice, which have the principal advantage of
  allowing precise genetic manipulation.

3
Behavioral Manifestations of the Addiction Process

• Behavioral Sensitization Repeated exposure to a
  drug leads to a progressive enhancement
  of the response (i.e. cocaine sensitization).
• Drug Tolerance Increasing doses of a drug
  become necessary to elicit an equivalent
  physiological response (i.e. morphine tolerance).
• Drug Dependence An adapted state of cells,
  circuits or organ systems unmasked by abrupt
  cessation of drug exposure (i.e. opiate
  withdrawal).
• Drug Craving Increased drug seeking behavior
  following abstinence usually occasioned by drug
  related cues.
• Compulsive Drug Taking Uncontrolled drug
  self-administration despite noxious behavioral
  consequences.
• Drug Relapse After the extinguishing of
  uncontrolled drug taking, reacquisition of the
  behavior following a conditioned cue

4
Dopamine System in Addiction

• All drugs of abuse affect the mesolimbic DA
  system leading to irreversible alterations in
  physiology/chemistry in the reward circuits

5
Approaches to Understanding the Molecular Basis
of Drug Abuse
Reverse Genetics
1) Manipulation of Candidate Genes
G protein-coupled receptor kinases (GRK
2-6) Arrestins (?arrestin 1 and ?arrestin 2)
Forward Genetics
1) Genome-wide Expression Profiling
Mice Genetically Sensitized to Cocaine
WT, WTcocaine, DAT-KO, NET-KO, VMAT2-HT
2) ENU Mutagenesis Screens in the Mouse
Dominant Modifiers of the DAT-KO Sensitized
Background
3) Phenotype-driven Neuroscience Screens
Northwestern Univ. Takahashi et. al.
6
Approaches to Understanding the Molecular Basis
of Drug Abuse
Reverse Genetics
1) Manipulation of Candidate Genes
G protein-coupled receptor kinases (GRK
2-6) Arrestins (?arrestin 1 and ?arrestin 2)
Forward Genetics
1) Genome-wide Expression Profiling
Mice Genetically Sensitized to Cocaine
WT, WTcocaine, DAT-KO, NET-KO, VMAT2-HT
2) ENU Mutagenesis Screens in the Mouse
Dominant Modifiers of the DAT-KO Sensitized
Background
3) Phenotype-driven Neuroscience Screens
Recessive Mutation screen Northwestern Univ.
Takahashi et. al.
7
Psychostimulants such as cocaine and amphetamine
produce their behavioral effects by raising the
levels of extracellular dopamine.
Behavioral Sensitization
Increased behavioral responses to chronic
intermittent exposure to drugs. Believed to
model in animals the initial stage of the drug
abuse processin humans.
Involves long term signaling plasticity resulting
in supersensitivity of dopamine receptors.
The enduring neuronal plasticity that underlies
the drugs of abuse phenotype might resemble
processes of learning and memory
8
VMAT /- Mice Decreased DA, NE, 5HT Mimics
reserpine
DAT-KO Mice Increased DAex Mimics
psychostimulants
NET-KO Mice Increased NEex Mimics antidepressant
s
All 3 KO mice show enhanced DA-mediated behaviors
9
Phenotype of pharmacogically and genetically
sensitized mice
NET KO
DAT KO
VMAT2 HT
10
Strategy for Isolation of Tissue mRNA
and Microarray Analysis
Striatum/NAc
11
Genes commonly affected between genetically
and pharmacologically sensitized mice
12
Genes Commonly Affected in the Striatum of
DAT-/-, NET-/-, VMAT2/- and Cocaine-Treated Mice
Identified by Microarrays
13
Genes Commonly Affected in the Striatum of
DAT-/-, NET-/-, VMAT2/- and Cocaine-Treated Mice
Identified by Microarrays
14
PSD-95 is one of the commonly affected gene
Abundant scaffolding protein at PSD of
excitatory synapses Three PDZ domains for
clustering PDZ domain containing proteins
SH3 domain GK domain Guanylate kinase-homology
domain Interacts with more than 50 proteins
including ion channels, receptors, kinases, etc
forming an intricate signaling complex
LTP and learning (Migaud et al., Nature,
1998) Synaptic maturation (El-Husseini et al.,
Science 1999) Synaptic strength (El-Husseini
et al., Cell, 2002) Chronic pain (Carry et al.,
Current Biology, 2003)
15
PSD-95 transcript is decreased in the basal
ganglia of behaviorally sensitized
mice
16
PSD-95 protein expression is decreased in the
striatum
17
Decreased in PSD-95 is selective to the striatum
in cocaine sensitized mice
18
Pre- and Post-synaptic markers
Selectivity of changes in the expression of
PSD-95
19
Persistent PSD-95 Down-regulation in
Cocaine-Sensitized Mice
20
Cellular Phenotype Enhanced LTP in Nucleus
Accumbens
21
PSD-95 KO mice are impaired in learning
Hippocampus
22
PSD-95 KO mice display NAc LTP similar to
sensitized mice
23
PSD-95 KO mice are more responsive to
psychostimulants
24
PSD-95 KO mice cannot be further sensitized
by chronic cocaine
25
Conclusions

• Phenotype-driven approach yielded 6 commonly
  identified genes
• Identification of previously recognized genes
  validates approach

• PSD-95 appears to be a common target of
• hyperdopaminergic responsiveness

• Changes in PSD-95 correlate with the
  development and
• persistence of psychostimulant sensitization

• Enhanced NAc LTP may be a cellular correlate of
• psychostimulant sensitization

• Functional inactivation of PSD-95(KO mice)
  leads to
• enhanced NAc LTP and responses to
  psychostimulants

• PSD-95 provides a molecular and cellular link
  shared between
• psychostimulant-related plasticity and learning

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Roles of PSD-95 in Synaptic Transmission and
Plasticity

• Serves as scaffold for NMDA receptors.
• Sheng Kim, Science 298,776 (2002)
• Serves as indirect docking sites for synaptic
  AMPA receptors
• Schnell et al., PNAS, 2002
• Controls bidirectional synaptic plasticity
  facilitate LTD and inhibits LTP
• Migaud et al., Nature, 1998 (Hippocampus)
• Stein et al., J. Neurosci. 2003 (Hippocampus)
• Beique and Andrade, J. Neurophys. 2003 (Cortex)
• Basal ganglia? Yao et al., Neuron 2004

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Cocaine Usurps the Cortico-Accumbal Glutamate
System by Altering the Bi-directional Synaptic
Plasticity

• Cocaine depresses cortical glutamate afferent
  (White el al., J. Pharmacol. Exp. Ther. 1995)
• Cocaine reduces AMPA currents (Thomas et al.,
  Nature Neurosci. 2001)
• Cocaine reduces LTD (Thomas et al., Nature
  Neurosci. 2001)
• Cocaine enhances LTP (This study)
• Reduction of PSD-95 may provide
• a consistent explanation for these
• phenomena

SNr/VTA
Cortex
GABA
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Open Questions Relating to PSD-95

• How does PSD-95 mediates the interplay between
  the converging DA and Glu systems in the
  striatum?
• DA release from dopaminergic terminals?
• DA receptor signaling?
• Enhanced modulation of glutamate transmission by
  DA?
• Mechanisms leading to PSD-95 reduction
• (transcriptional and posttranslational)
• Role of PSD-95 in reward and habit learning
• CPP, self administration and specificity for
  other drugs.
• How does PSD-95 regulate LTP and LTD in the basal
  ganglia?

29
Approaches to Understanding the Molecular Basis
of Drug Abuse
Reverse Genetics
1) Manipulation of Candidate Genes
G protein-coupled receptor kinases (GRK
2-6) Arrestins (?arrestin 1 and ?arrestin 2)
Forward Genetics
1) Genome-wide Expression Profiling
Mice Genetically Sensitized to Cocaine
WT, WTcocaine, DAT-KO, NET-KO, VMAT2-HT
2) ENU Mutagenesis Screens in the Mouse
Dominant Modifiers of the DAT-KO Sensitized
Background
3) Phenotype-driven Neuroscience Screens
Recessive Mutation screen Northwestern Univ.
Takahashi et. al.
30
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31
Phenotypic Screen Locomotor Activity in Novel
Environment
1. Use a two standard deviation cutoff for
identification of outliers Dat /- Outliers
6500 cm/ 2h Dat -/- Outliers 32000 cm or
6500 cm 2. Retest outliers Dat /- Outliers
4500 cm/ 2h Dat -/- Outlier 32000cm or cm
32
A Genetically Sensitized Background For Dopamine
Transmission
DAT KO mice cannot actively clear
synaptic dopamine. Extracellular dopamine
levels are 5 times higher than wildtype. DAT
HET mice have intermediate neurochemical phenotype
(2 times more extracellular DA). Numerous
neurochemical and behavioral consequences to
persistent high levels of DA including
hyperactivity in novel environment. Chronic
hyperdopaminergia provides a sensitized
background to uncover second site modifiers.
33
G2 Dominant Modifier Screen
X
ENU
G0
C57 Dat /-
C57Bl/6J
G1
Dat /-, /M
G2
Dat /-, /
Dat /-, /M
Dat -/-, /M
Dat /-, /
Dat /-, /M
Dat -/-, /
Dat /, /M
Dat /, /
34
G1 screens
X
ENU
G0
C57 Dat /-
C57 Dat /-
G1
Dat /-, /M
Dat /-, /M
Dat /-, /M
Dat -/-, /M
Dat /-, /M
Dat -/-, /M
392 Dat /- screened, 11 outliers with enhancer
phenotype in testcross (0.5X coverage) 135 Dat
-/- screened, 4 enhancer, 5 suppressor
phenotypes in testcross (0.2X coverage)
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Enhancer Phenotype Maintained on B6D2 F1
Background
37
Identification of the Akt/GSK-3 Cascade as a
Signaling Pathway Mediating the Behavioral
Actions of Dopamine

• Actions of dopamine are mediated via 5 distinct
• GPCRs (D1-like D1, D5 and D2-like D2, D3, D4)

• Classically most biochemical
• and behavioral actions of
• dopamine have been attributed
• to modulation of the cAMP
• signal transduction pathway

Adapted from Greengard et al., 2001
38
Lithium ions can antagonize the behavioral
actions of dopamine agonists in vivo
Li antagonizes the effect of psychostimulants
(Cox C et al., 1971 Nature 232336-8 many others
reviewed in Einat et al., 2000 in Contemporary
issues in modeling psychopathology). Li
antagonizes the effect of apomorphine locomotor
behavior and sniffing behavior (Fazli-Tabaei et
al., 2002 Pharmacol Toxicol 1998 91 135-139,
Dehpour et al., 1998 Pharmacol Toxicol 1998 82
147-52 Dehpour et al., 1995 Gen Pharmacol
261015-20). Li antagonizes the effect of brain
injection of dopamine agonists (Barnes et
al.,1986, Psychopharmacology 89311-6).
Lithium does not directly inhibit DA receptors,
nor does it affect the cAMP signaling pathway.
Lithium interferes with cellular signaling
-Inositol depletion hypothesis Lithium
inhibits inositol monophosphatase
(Berridge and Irvine 1989 Nature
341 197-205) -Glycogen Synthase Kinase 3
(GSK-3) Hypothesis Lithium is a direct inhibitor
of GSK-3 (Klein and
Melton 1996, PNAS 93 8455-59)
39

_________
()
()
(-)

()

• In the DAT-KO Lithium inhibits the hyperactivity
• phenotype associated with high extracellular
  DA
• Lithium enhances the phosphorylation of both
• Akt and GSK-3? ? in DAT-KO
• No effects on DARPP-32 phosphorylation and
• cAMP signaling pathway
• In DAT-KO mice both Akt(thr308) and GSK-3? ?
• are dephosphorylated
• In vitro GSK-3 assay reveals increased
• activity of enzyme in DAT-KO

(-) ____
(-)
()

(-)

Beaulieu et al, PNAS 101, 5109 (2004)
40
Inhibitors of GSK-3 reduce the hyperactivity
of
DAT-KO mice
41
GSK-3 /- mice show a diminished response to the
psychostimulant
Amphetamine
42
Potential Signaling pathways mediating
the actions of dopamine
43
Conclusions
Actions of dopamine can be mediated through both
cAMP-dependent and independent mechanisms. The
Akt/GSK-3 pathway may be a mediator of dopamine
actions under acute and chronic hyperdopaminergic
conditions. Akt/GSK-3?? pathway may provide new
targets for understanding aberrant DA-associated
behaviors.
Implications
Akt and GSK-3 genes might be candidate genes for
modulating the responsiveness to drugs of
abuse Inhibitors of the Akt/GSK-3 signaling
pathway might be represent novel pharmacological
targets for the treatment of drug abuse symptoms

44
Collaborators
HHMI-DUMC Wei-Dong Yao Amy Mohn Martin
Beaulieu Raul Gainetdinov Tatyana Sotnikova
Michel Cyr Gonzalo Torres Aki Laakso Bruno
Giros Mohamed Jaber Sara Jones Fei Xu Yan-Min Wang
UNIVERSITY OF EDINBURGH/ SANGER CENTER,
CAMBRIDGE Seth Grant Margaret McLean Arbuckle
DUMC/ Microarray Facility Holly Dressman
DUMC/Transgenic Facility Cheryl Bock
Ontario Cancer Institute (Univ.Toronto) (GSK-3?/-
mice) Lisa Kockeritz James R. Woodgett
Support NIDA, Zaffaroni Foundation
45
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46
Cellular Mechanism Enhanced LTP in Nucleus
Accumbens
47
WT
DAT-/-
VMAT2-/
NET-/-
Cocaine
Saline
48
Brain Reward Pathways and Drugs of abuse
Cocaine/ Amphetamine DAT/DAR
PCP NMDAR
Opiates muR
Nicotine nAchRs
Cannabinoids CB1CB2R
Ethanol GABAA/NMDARs
Sex/Food
49
PSD-95 is one of the commonly affected gene
PSD-95
Sheng Kim, Science 298,776 (2002)
50
QuickTime and a TIFF (Uncompressed) decompressor
are needed to see this picture.
51
Physiological Functions of Dopamine

• Brain Motor control
• Cognition
• Emotion/affect
• Reward mechanisms
• Retina Light/dark adaptation
• Pituitary Gland Hormone secretion

• Dysfunction of the Dopaminergic System Associated
  with
• Parkinsons Disease
• Schizophrenia and Affective Disorders
• Attention Deficit Hyperactivity Disorder
• Drugs of Abuse

52
Psychostimulants such as cocaine and amphetamine
produce their behavioral effects by raising the
levels of extracellular dopamine.
Behavioral Sensitization Increased behavioral
responses to chronic intermittent exposure to
drugs. Believed to model in animals the initial
stage of the drug abuse process in humans.
Involves long term signaling plasticity
resulting in supersensitivity of dopamine
receptors.
Components of GPCR desensitization are good
candidates!!
53
Possible Mechanisms of Drug-Induced Changes in
GPCR Sensitivity
GRKs and Arrestins 7 GRKs (2 visual) 4 Arrestins
(2 visual)
Nestler Aghajanian, Science 278, 58-63, 1997
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GRK6 Expression in Dopaminoceptive Medium Spiny
Striatal Neurons
55
Behavioral Responses to Cocaine in GRK6-KO Mice
Locomotor Activity
Sensitization
Day 1
Day 7
Dose Response
Total Distance (cm in 60 min)
56
Acute Effects of Cocaine in Various GRK and
Arrestin KO Mice
57
Influence of modifier genes on the DAT KO
Phenotype
Morice E, Denis C, Giros B, Nosten-Bertrand M.
Phenotypic expression of the targeted
null-mutation in the dopamine transporter
gene varies as a function of the genetic
background. Eur J Neurosci. 2004 Jul20(1)120-6.
58
Functional Polymorphism in TPH2
C57BL/6 and 129Xl/SvJ carry 1473 C allele
(Pro) DBA/2 and BALB/cJ carry 1473 G allele (Arg)
Zhang X, Beaulieu JM, Sotnikova TD, Gainetdinov
RR, Caron MG. Tryptophan hydroxylase-2 controls
brain serotonin synthesis. Science. 2004 Jul
9305(5681)217.
59
Inactivation of DARPP-32 in mice

• Inhibits the DA-dependent
• biochemical effects of DARPP-32
• on its target proteins

• Has partial effects on many
• behaviors elicited by
• direct or indirect DA
• agonists

Fienberg et al, 1998 (Science)
Adapted from Greengard et al., 2001, and
Waddington and Greengard et al 2003
Apomorphine mg/kg
60
Conclusions
Actions of dopamine can be mediated through both
cAMP-dependent and independent mechanisms. The
Akt/GSK-3 pathway may be a mediator of dopamine
actions under acute and chronic hyperdopaminergic
conditions. Akt/GSK-3?? pathway may provide new
targets for understanding aberrant DA-associated
behaviors.
Questions
What is the mechanism by which D2-like receptors
modulate Akt phosphorylation? What are the
substrates of GSK-3 contributing to the actions
of dopamine? What is the role of the Akt/GSK-3
pathway in manifestations of hyperdopaminergic
functions. sensorimotor gating, drug reward and
neuronal cell loss? Are there differential
behavioral effects of cAMP-dependent and
independent pathways?
61
Phenotypic Screen Locomotor Activity in Novel
Environment
1. Use a two standard deviation cutoff for
identification of outliers Dat /- Outliers
6500 cm/ 2h Dat -/- Outliers 32000 cm or
6500 cm 2. Retest outliers Dat /- Outliers
4500 cm/ 2h Dat -/- Outlier 32000cm or cm
62

Lithium inhibits behavioral activity without
affecting extracellular dopamine levels in
DATKO mice
Acute dose

• No effects on DARPP-32 phosphorylation and
• and cAMP signaling pathway

63
Attractive potential mechanism
Akt/GSK-3 Signaling Pathway
Can we find biochemical corollaries in this
pathway for the effects of Li in DAT KO mice?

• Insulin signaling in
• glycogen synthesis
• Wnt signaling in
• cell fate proliferation

64
Status of Akt/GSK3 in DAT-KO and in response to Li

• Lithium enhances the phosphorylation of
  both
• Akt and GSK-3? ? in DAT-KO

• In DAT-KO mice both Akt(thr308) and
  GSK-3? ?
• are dephosphorylated

• In vitro GSK-3 assay reveals increased
• activity of enzyme in DAT-KO

65
Amphetamine
aMPT
Normal Neurotransmission
DA
TH
Lack of Dopamine Transporter the DAT-KO mice
66
Elimination of DA tone reverses Akt/GSK-3 state
of DAT-KO
Increasing DA tone in WT mice reproduces DAT-KO
Akt/GSK-3 state

• D2 but not D1 class DA receptors mediate changes
• in Akt and GSK-3

67

_________
()
()
(-)

()

(-) ____
(-)
()

(-)

68
GSK-3 inhibitors
SB216763 Glaxo Smith Kline (ATP binding)
Valproic acid (mechanism?)
Indirubines Qing Dai, Chinese medicine(ATP
binding)
Paullones (ATP binding)
Thiadiazolidinones TDZD (Substrate binding)
69
Alsterpaullone reduces hyperactivity in DAT-KO
mice
70
Animal models of GSK-3 dysfunction
GSK-3? knockout is lethal due to a disruption of
NF-k-B. (Hoeflich et al., 2000 Nature
40686-90) GSK-3?/- develop normally and do not
display overt phenotype. (Hoeflich et al.,
2000) GSK-3? Transgenic exhibit developmental
deficits and neuropathies associated with Tau
hyperphosphorylation. (Spittaels et al.,2002
Neuroscience 113797-808, Lucas et al., 2001,
EMBO 20 27-39 Brownlees J et al.,1997
Neuroreport 3251)