almost htere

1. Marc G. Caron, Ph.D.
Dept. Cell Biology
Duke University Medical Center
Phenotype-Driven Approaches to
Understanding the Neuronal Plasticity
Associated with Drugs of Abuse

2. Addictive process is amenable to experimental genetic
approaches
• 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.
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)

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
cocaine
amphetamine
opiates nicotine
heroin
sex
food
ethanol
•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)
2) ENU Mutagenesis Screens in the Mouse
Dominant Modifiers of the DAT-KO
Sensitized Background
1) Genome-wide Expression Profiling
Mice Genetically Sensitized to Cocaine
WT, WT+cocaine, DAT-KO,
NET-KO,& VMAT2-HT
3) Phenotype-driven Neuroscience Screens
Northwestern Univ. Takahashi et. al.
Forward Genetics

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)
2) ENU Mutagenesis Screens in the Mouse
Dominant Modifiers of the DAT-KO
Sensitized Background
1) Genome-wide Expression Profiling
Mice Genetically Sensitized to Cocaine
WT, WT+cocaine, DAT-KO,
NET-KO,& VMAT2-HT
3) Phenotype-driven Neuroscience Screens
Recessive Mutation screen
Northwestern Univ. Takahashi et. al.
Forward Genetics

7. Psychostimulants such as cocaine and amphetamine produce their
behavioral effects by raising the levels of extracellular dopamine.
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.
0
500
1000
1500
2000
2500
Day 1 Day 7
**
COCAINE INJ
1 2 3 4 5 6 7 days
Activity
Monitor
Activity
Monitor
Distance
Traveled
(cm/15
min)
Behavioral Sensitization
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 [DA]ex
Mimics psychostimulants
NET-KO Mice
Increased [NE]ex
Mimics
antidepressants
All 3 KO mice show enhanced DA-mediated behaviors

9. Phenotype of pharmacogically and genetically “sensitized” mice
NET KO VMAT2 HT
DAT KO

10. Striatum/NAc
Berke and Hyman 2000
3x5 WT
3x5 WT+C
3x5 DAT-/-
3x5 NET-/-
3x5 VMAT+/- mRNA isolation
IVT
cRNA Labeling
Hybridization to Affymetrix Mu74
Wash and stain
Scan
Data analysis
Strategy for Isolation
of Tissue mRNA and
Microarray Analysis
PFC
CPU
NAc
Hip
Amg
VTA
SNr

11. Genes commonly affected between genetically
and pharmacologically sensitized mice
0.0 2.5 5.0
0.5
1.0
1.5
2.0 104175_at
97560_at
160184_at
109710_at
116691_at
113672_at

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. Selectivity of changes in the
expression of PSD-95
Pre- and Post-synaptic markers

19. Persistent PSD-95 Down-regulation in
Cocaine-Sensitized Mice

20. Cellular Phenotype:
Enhanced LTP in
Nucleus Accumbens

21. Nature 396 433-439, 1998
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
• 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
• Phenotype-driven approach yielded 6 commonly identified genes
• Identification of previously recognized genes validates approach
• PSD-95 provides a molecular and cellular link shared between
psychostimulant-related plasticity and learning

26. 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

27. 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
Glu
DA
Striatal
MSN
SNr/VTA Cortex
GABA

28. 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)
2) ENU Mutagenesis Screens in the Mouse
Dominant Modifiers of the DAT-KO
Sensitized Background
1) Genome-wide Expression Profiling
Mice Genetically Sensitized to Cocaine
WT, WT+cocaine, DAT-KO,
NET-KO,& VMAT2-HT
3) Phenotype-driven Neuroscience Screens
Recessive Mutation screen
Northwestern Univ. Takahashi et. al.
Forward Genetics

31. 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 < 6500 cm
Phenotypic Screen: Locomotor Activity in Novel Environment

32. WT Nr1 +/- Dat -/- Dat -/- Nr1 +/-
0
5000
10000
15000
WT Dat +/- Dat +/- D3 +/-
0
1000
2000
*
*
D3 and Nr1 mutations ar
e dominant modifiers
of the DAT knockout phenotype
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
C57 Dat +/- C57Bl/6J
G0
G1
G2
Dat +/-, +/M
Dat +/-, +/+
Dat +/-, +/+
Dat +/+, +/M
Dat +/-, +/M
Dat +/-, +/M
Dat +/+, +/+
Dat -/-, +/+
Dat -/-, +/M
ENU
X

34. C57 Dat +/- C57 Dat +/-
G0
G1
Dat +/-, +/M
ENU
X
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)
G1 screens

36. Enhancer Phenotype Maintained
on B6D2 F1 Background
Enhancer phenotype
maintained on B6D2 F1
background
C57/DBA F1
0
2500
5000
7500
Distance
Traveled
in
cm/2
hr

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)
Dopamine-dependent behaviors
• 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
232:336-8; 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
26:1015-20).
Li antagonizes the effect of brain injection of dopamine agonists : (Barnes et
al.,1986, Psychopharmacology 89:311-6).
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
•D2 but not D1 class DA receptors mediate changes
in Akt and GSK-3
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. -Catenin
Inhibitor-2
Transcription
Factors
Receptors
PI3 kinase
Phosphatases
Calcium
Potential Signaling pathways mediating
the actions of dopamine

43. 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.
Conclusions
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
Implications

44. 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
Collaborators
DUMC/ Microarray Facility
Holly Dressman
DUMC/Transgenic Facility
Cheryl Bock
Support: NIDA, Zaffaroni Foundation
Ontario Cancer Institute
(Univ.Toronto)
(GSK-3+/- mice)
Lisa Kockeritz
James R. Woodgett

46. Cellular Mechanism:
Enhanced LTP in
Nucleus Accumbens

47. WT DAT-/-
NET-/-
VMAT2-/+
Saline Cocaine

48. Q ui
ckTi
m e™ and a TI FF ( U
ncom pr essed) decom pr essor ar e needed t o see t hi
s pi
ct ur e.
Ethanol
GABAA/NMDARs
Nicotine
nAchRs
Cannabinoids
CB1&CB2R
PCP
NMDAR
Cocaine/
Amphetamine
DAT/DAR
Opiates
muR
Sex/Food
Brain Reward Pathways and Drugs of abuse

49. PSD-95 is one of the commonly affected gene
Sheng & Kim, Science 298,776 (2002)
PSD-95

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:
• Parkinson’s Disease
• Schizophrenia and Affective Disorders
• Attention Deficit Hyperactivity Disorder
• Drugs of Abuse

52. 0
500
1000
1500
2000
2500
Day 1 Day 7
**
COCAINE INJ
1 2 3 4 5 6 7 days
Activity
Monitor
Activity
Monitor
Distance
Traveled
(cm/15
min)
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. Nestler &Aghajanian, Science 278, 58-63, 1997
Possible Mechanisms of Drug-Induced Changes in GPCR Sensitivity
GRKs and Arrestins
7 GRKs (2 visual)
4 Arrestins (2 visual)

54. GRK6 Expression in Dopaminoceptive
Medium Spiny Striatal Neurons

55. Locomotor Activity Sensitization
Dose Response
Day 1 Day 7
Behavioral Responses to Cocaine in GRK6-KO Mice
0 30 60 90 120
0
500
1000
1500
Cocaine (20 mg/kg, i.p.)
WT
GRK6-HET
GRK6-KO
Time (min)
Horizontal
activity
(distance
in
cm/5min)
Horizontal
Activity
(distance
in
cm/5min)
0 10 20 30
0
5000
10000
15000
Dose (mg/kg, i.p.)
Total
distance
(cm
in
60
min)
Total
Distance
(cm
in
60
min)
0 30 60 90 120
0
500
1000
1500
2000
2500 Cocaine (20 mg/kg, i.p.)
KO
WT
Time (min)
0 30 60 90 120
0
500
1000
1500
2000
2500 Cocaine (20 mg/kg, i.p.)
KO
WT
Time (min)
Horizontal
Activity
(distance
in
cm/5min)
COCAINE INJ
1 2 3 4 5 6 7 days
Activity
Monitor
Activity
Monitor
0
5000
10000
15000
GRK6-KO
Day 1 Day 7
***
** **
WT
Horizontal
Activity
(distance
in
cm
in
90
min)

56. Acute Effects of Cocaine in Various GRK and Arrestin KO Mice
0 20 40 60 80 100 120 140 160 180
0
250
500
750
1000
WT (n=15)
arr2-KO (n=10)
Cocaine (20 mg/kg, i.p.)
Time (min)

57. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.
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 Jul;20(1):120-6.
Influence of modifier genes on the DAT KO Phenotype

58. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.
Zhang X, Beaulieu JM, Sotnikova TD, Gainetdinov RR, Caron MG.
Tryptophan hydroxylase-2 controls brain serotonin synthesis.
Science. 2004 Jul 9;305(5681):217.
Functional Polymorphism in TPH2
C57BL/6 and 129Xl/SvJ carry 1473 C allele (Pro)
DBA/2 and BALB/cJ carry 1473 G allele (Arg)

59. Adapted from Greengard et al., 2001,
and Waddington and Greengard et al 2003
QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.
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
Apomorphine mg/kg
Fienberg et al, 1998 (Science)

60. 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.
Conclusions
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?
Questions

61. 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 < 6500 cm
Phenotypic Screen: Locomotor Activity in Novel Environment

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
Substrates
Cohen and Frame 2001 Nat Rev Mol Cell Biol 2:769-76
Li
(+/-)
Li
(-)
P
PI3K
P
Can we find biochemical
corollaries in this pathway
for the effects of Li
in DAT KO mice?
Akt/GSK-3 Signaling Pathway
• Insulin signaling in
glycogen synthesis
• Wnt signaling in
cell fate & proliferation

64. • In vitro GSK-3 assay reveals increased
activity of enzyme in DAT-KO
• In DAT-KO mice both
Akt(thr308) and GSK-3 &
are dephosphorylated
• Lithium enhances the
phosphorylation of both
Akt and GSK-3 &  in
DAT-KO
Status of Akt/GSK3 in DAT-KO and in response to Li

65. DA
TH
DAT
DA
TH
Normal
Neurotransmission
Lack of Dopamine Transporter:
the DAT-KO mice
Amphetamine
MPT

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)
Indirubines
Qing Dai, Chinese
medicine(ATP binding)
Valproic acid
(mechanism?)
Paullones
(ATP binding)
Thiadiazolidinones
TDZD
(Substrate binding)
DA
AKT
GSK-3/
MPT
Raclopride
Hyperactivity?
Not responsive to:
- SCH23390
- 8-Br-cAMP
D2R
Chronic/
DAT-KO
Li
(+)
(-)
(?)

69. Alsterpaullone reduces
hyperactivity in DAT-KO mice

70. GSK-3 knockout is lethal due to a disruption of NF-k-B.
(Hoeflich et al., 2000 Nature 406:86-90)
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 113:797-808, Lucas et al., 2001, EMBO 20: 27-39;
Brownlees J et al.,1997 Neuroreport : 3251)
Animal models of GSK-3 dysfunction