1. Role of VTA Glutamate in Contextual Cue-Induced Relapse to Heroin-Seeking
Yavin M. Shaham, Ph.D.
Behavioral Neuroscience Branch
IRP/NIDA/NIH/DHHS, Baltimore
Outline
Role of VTA glutamate in context-induced relapse to heroin seeking
Role of central amygdala ERK signaling pathway and glutamate in incubation of cocaine craving

2. The reinstatement model of drug relapse
Drug is available
Drug is NOT available
Drug priming
Drug cues
Stress
Responses
Self-administration training
Extinction
TESTING
Reinstatement studies
( ) 3 4 10 13 20 93
265 50
100
150
200
250
300 70 75 80 85 90 95 00 04
Year
cumulative #
Inactive lever
Active lever

3. Role of VTA glutamate in context-induced relapse to heroin seeking
Dr. Jennifer Bossert
Systemic injections of mGluR2/3 agonists attenuate:
Opiate withdrawal symptoms (Vandergriff and Rasmussen, 1999)
Amphetamine-induced locomotor sensitization (Vezina, 2004)
Discriminative cue-induced reinstatement of cocaine seeking (Baptista et al., 2004)
From Schoepp. JPET 2001
LY379268
a selective agonist of mGluR2/3 receptors
Intra-VTA injections of ionotropic glutamate receptor antagonists attenuate:
Morphine and cocaine place preference (Harris et al. 2003; Byrne et al. 2003)
Reinstatement of cocaine seeking induced by ventral subiculum stimulation (Vorel et al. 2001) or stress (Wang et al. SFN. 2004)
Also: Over-expression of VTA GluR1 enhances morphine place preference (Carlezon et al. 1997)

4. Types of relapse-provoking drug cues in humans and laboratory animals
Humans: Two major types of cues that provoke relapse after abstinence:
Discrete cues (e.g., drug paraphernalia) that predict drug effects
Contextual cues (e.g., street corner, bar) that predict drug availability
Laboratory animals: Two types of cues that provoke relapse to drug seeking after extinction of the operant responding in their absence:
Discrete cues (e.g., tone, light) paired with drug injections
Discriminative cues (e.g., specific odors) that become predictors of drug availability after discrimination training
Research question: Can we study the role of the drug context in reinstatement of drug seeking after extinction of the operant responding in the presence of the discrete cues (an animal model of the cue exposure treatment method)?
This issue can be addressed using a “renewal” procedure, commonly used to study the role of the environmental context in the resumption of conditioned fear responses to discrete cues after extinction (Bouton & Bolles 1979)

5. A “renewal” procedure to study the effect of contextual drug cues on drug seeking
Training
(10 d)
Extinction
(20 d)
Test
(1 d)
Differences between contexts A and B:
--type of grid floor
--background noise
--illumination level
--type of odor
Control 1
Context A
Context A
Context A
Control 2
Context A
Context B
Context B
Novel
Context A
Context A
Context B
Renewal
Context A
Context B
Context A
Context A
Novel
Control
Renewal 25 50 75
100
125
Extinction 5 10 15 20
Session 1
Responses (2 h)
Reinstatement
test
Renewal
Novel
Control (1+2) *
Crombag & Shaham. Behavioral Neuroscience, 2002

6. Reinstatement of heroin seeking by contextual drug cues
Training
(12 d)
Extinction
(12-20 d)
Test
(1 d)
Control 1
Context A
Context A
Context A
Differences between contexts A and B:
--Time of day (circadian cues)
--Type of grid floor (tactile)
--Background noise (auditory)
--Distinct chamber cues
--Illumination level (visual cues)
Control 2
Context A
Context B
Context B
Novel
Context A
Context A
Context B
Renewal
Context A
Context B
Context A
No Extinction
Context A
Context A 15 30 45 60 75 1 2 3 4 5 6 7 8 9 10 11 12
Extinction day
Responses (1 h)
Same context
Different context
Extinction *
Control
(1&2)
Novel
Renewal
No Extinction
Reinstatement test

7. Effects of systemic or intra-VTA injections of an mGluR2/3 agonist on context-induced reinstatement of heroin seeking
Systemic LY379268 15 30 45 60 75
Extinction
(Context B)
Training
(Context A)
Responses (3 h)
Vehicle
1 mg/kg
3 mg/kg * *
Intra-VTA LY379268
Vehicle
0.3 µg
1.0 µg
Extinction
(Context B)
Training
(Context A)
VTA
Bossert et al. The Journal of Neuroscience, in press

8. Intra-substantia nigra LY379268
Control experiments
Intra-substantia nigra LY379268
Lever presses for a 5% sucrose solution 75
100
Vehicle 60
1.0 µg 75 45
Responses (3 h)
Rewards (30 min) 50 30 25 15 * SN 1 3 6
Extinction
(Context B)
Training
(Context A)
LY dose
(mg/kg, i.p)

9. Conclusions and implications
As in the case of relapse to anxiety-related disorders (Bouton 2002), the role of the environmental context in drug relapse can be studied in laboratory rats using the renewal procedure
Glutamate transmission in the VTA plays an important role in context-induced relapse to heroin seeking
The present data and those from previous studies (see Baptista et al. 2004) suggest that mGluR2/3 agonists and other drugs that target metabotropic glutamate receptors (see Kenny & Markou 2004) should be considered in the treatment of drug relapse

10. (extracellular signal-regulated kinase)
Role of central amygdala ERK and glutamate in incubation of cocaine craving Dr. Lin Lu Collaborator: Dr. Bruce Hope
A key regulator of synaptic plasticity and learning and memory (Sweatt 2001)
Amygdala ERK is involved in conditioned fear responses (Schaffe et al. 2000)
Mesolimbic ERK is activated by cocaine (Licata and Pierce 2003; Valjent et al. 2000)
ERK
MEK
Raf
U0126
NMDA
receptor
NMDA/AP-5
From Thomas and Huganir.
Nature Neuroscience, 2004
(extracellular signal-regulated kinase)

11. (drug is NOT available)
Time-dependent changes in extinction responding and cue-induced reinstatement of cocaine seeking after withdrawal
Training phase
Withdrawal
phase
Test phase
(drug is NOT available) 80
1 day
2 days
4 days
7 days
15 days
29 days
60 days 60
No T+L
Tone+light cue
Infusions (6 h) 40
6-8
60-min
sessions
Tone+light cue 20
(n=67) 1 2 3 4 5 6 7 8 9 10
“Extinction” + Testing (same day)
Day 60
120
180
240
300 1 2 4 7 15 29
Withdrawal day
Responses (6 h) *
Extinction responding * 20 40 60 80
100 1 2 4 7 15 29
Withdrawal day
Responses (1 h)
Cue-induced reinstatement
Cue available
Last extinction
session (no T+L)
Grimm et al. Incubation of cocaine craving after withdrawal. Nature, 2001

12. Incubation of reward craving after withdrawal from drug and non-drug reinforcers
Cocaine (2)
Neisewander et al. 2000
Priming 1 21
Cue
Extinction
Cocaine (1)
Tran-Nguyen et al. (1998)
Control
1 day
7 day
1 month
Withdrawal day 1 6 12 25 66 *
100
200
300
400
Withdrawal day
Responses (5 h)
Heroin
Shalev et al. 2001 1 30 90
180
Cocaine (4)
Lu et al. 2004 * 2 4 7 15 29 60
Withdrawal day
Cocaine (3)
Grimm et al. 2001 75
150
225
300
Responses (6 h) 7 60
180 *
Withdrawal day 30 90 1 50
100
150
200
Responses (6 h)
Sucrose
Lu et al. 2004
Methamphetamine
Shepard et al. 2004 30 60 90
120
150 1 21 51
Withdrawal day *
Responses (3 h)
Alcohol
Bienkowski et al. (2004)
Withdrawal period in
home cage prior
to extinction tests

13. Experiment 1: activation of amygdala ERK by cocaine cues
Withdrawal day 75
150
225
300 1 30 90
180
Responses (6 h) *
Training condition: saline versus cocaine
Withdrawal period: day 1 versus day 30
Test condition: no cue versus cue exposure (30 min extinction test)
Cocaine/saline SA training (10 Days)
Withdrawal period (1 or 30 Days)
Extinction test
Day 1
Day 30
4 groups
Cocaine/saline SA training (10 Days)
Withdrawal period (1 or 30 Days)
No test
Day 1
Day 30
4 groups

14. Behavioral data: Training and extinction test
Cocaine/saline SA training (10 Days)
Withdrawal period (1 or 30 Days)
Extinction test
Day 1
Day 30
Training day 20 40 60 80
100 1 2 3 4 5 6 7 8 9 10
Infusions (6 h)
Cocaine SA (n=34)
Saline SA (n=34)
Training
Withdrawal day
Responses (30 min)
Extinction test 15 30 45 60 1 *
Cocaine-trained rats
Saline-trained rats
Lu, Hope et al. Submitted

15. Exposure to cocaine cues increases ERK phosphorylation in the central amygdala after 30 days of withdrawal
DAY 30
Central amygdala 1
Saline SA
Cocaine SA
Extinction test
No test 50
100
150
200
% of naive rats *
pERK1
pERK2
Basolateral amygdala
DAY 1 30
Saline SA
Cocaine SA *
CeA
BLA

16. Inhibition of ERK phosphorylation in the central amygdala attenuates cocaine seeking after 30 days of withdrawal 50
100
150
Central
amygdala
% of vehicle values
Basolateral
Vehicle
U0126 *
Phosphorylated ERK 20 40 60 80
Vehicle
U0126
Responses (30 min)
Inactive lever
Active lever *
Extinction responding
U0126
ERK
MEK
Raf
NMDA
receptor
CeA

17. Inhibition of ERK phosphorylation in the basolateral amygdala has no effect on cocaine seeking after 30 days of withdrawal
% of vehicle values 50
100
150 *
Phosphorylated ERK
Central
amygdala
Basolateral
Vehicle
U0126 20 40 60 80
Vehicle
U0126
Responses (30 min)
Extinction responding
Inactive lever
Active lever
U0126
ERK
MEK
Raf
NMDA
receptor
BLA

18. Induction of ERK phosphorylation in the central amygdala restores cocaine seeking after 1 day of withdrawal
Phosphorylated ERK 50
100
150
200 *
Central
amygdala
% of vehicle values
Basolateral
Vehicle
NMDA (25 ng)
NMDA (250 ng)
Extinction responding
Inactive lever
Active lever 25
250
NMDA dose (ng/site) 20 40 60 80
Responses (30 min) *
NMDA
receptor
CeA
Raf
MEK
ERK

19. Extinction responding
Inhibition of ERK phosphorylation in the central amygdala reverses the effect of NMDA on cocaine seeking after 1 day of withdrawal
Phosphorylated ERK 50
100
150
200
% of vehicle values
Veh
NMDA
U0126
VEH *
* #
Extinction responding
Inactive lever
Active lever 20 40 60 80
Veh
NMDA
U0126
VEH
Responses (30 min) *
NMDA
U0126
ERK
MEK
Raf
NMDA
receptor
CeA

20. Blockade of NMDA receptors in the central amygdala attenuates cocaine seeking after 30 days of withdrawal
Phosphorylated ERK 50
100
150
200
Central
amygdala
Basolateral
% of vehicle values
Vehicle
AP-5 (3 µg) *
Extinction responding
AP-5 dose (ug/site)
Inactive lever
Active lever 20 40 60 80
3.0
Responses (30 min) *
AP-5
NMDA
receptor
CeA
Raf
MEK
ERK

21. Cocaine self-administration Food self-administration
Control experiment Inhibition of ERK phosphorylation in the central amygdala has no effect on cocaine or high-fat food self-administration
Cocaine self-administration
Food self-administration 40
160
Vehicle 30
U0126
120
Lever responses 20 80 10 40 1 2 3 4 5 6 1 2 3 4 5 6
hour
hour
Bregma -2.3
CeA

22. Conclusions and implications
The phenomenon of incubation of reward craving has important implications for the treatment of relapse to a range of addictive disorders (drug addiction, excessive eating, gambling?)
Our results suggest that time-dependent increases in the responsiveness of central amygdala ERK signaling pathway to cocaine cues mediate the incubation of cocaine craving
Our results also suggest a novel function of activation of the ERK pathway in the amygdala in associative learning: enhancement of the motivational impact of learned reward cues
Lu L, Grimm JW, Hope BT, Shaham Y (2004)
Incubation of cocaine craving after withdrawal: a review of preclinical data. Neuropharmacology 47S1: (NIDA special issue)

23. Animal models and FACE validity
Courtesy of Taco de Vries

24. Acknowledgements Former post-baccalaureate students Shirley Liu Jack Dempsey Present post-baccalaureate students Sarah Gray Robert Busch Deepti Nagarkar