Yavin M. Shaham, Ph.D. Behavioral Neuroscience Branch IRP/NIDA/NIH/DHHS, Baltimore

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 Reinstatement studies (1971-2004) 300 265 250 200 150 cumulative # 93 100 20 50 13 10 4 3 0 70 75 80 85 90 95 00 04 Year Drug is available Drug is NOT available Drug priming Drug cues Stress Responses Self-administration training Extinction TESTING Inactive lever Active lever

3. LY379268 a selective agonist of mGluR2/3 receptors From Schoepp. JPET 2001 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) 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. Extinction Reinstatement test 125 Control (1+2) * 100 Renewal Control Novel 75 Novel Responses (2 h) 50 Renewal 25 0 1 5 10 15 20 Session A “renewal” procedure to study the effect of contextual drug cueson 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 ContextA Control 1 Context A Context A Control 2 Context B Context B Context A Novel Context B ContextA Context A Renewal ContextA Context B Context A ContextA Crombag & Shaham. Behavioral Neuroscience, 2002

6. Reinstatement test Extinction 75 * 60 Same context * Different context 45 Responses (1 h) 30 15 0 Control (1&2) Renewal 1 2 3 4 5 6 7 8 9 10 11 12 Novel No Extinction Extinction day Reinstatement of heroin seeking by contextual drug cues Training (12 d) Extinction (12-20 d) Test (1 d) 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) ContextA Control 1 Context A Context A Control 2 Context B Context B Context A Novel Context B ContextA Context A Renewal ContextA Context B Context A No Extinction ContextA Context A

7. Systemic LY379268 Intra-VTA LY379268 75 Vehicle Vehicle 1 mg/kg 60 0.3 µg 3 mg/kg 1.0 µg * 45 * * Responses (3 h) 30 * VTA 15 0 Extinction (Context B) Training (Context A) Extinction (Context B) Training (Context A) Effects of systemic or intra-VTA injections of an mGluR2/3 agonist on context-induced reinstatement of heroin seeking Bossert et al. The Journal of Neuroscience, in press

8. Control experiments Lever presses for a 5% sucrose solution Intra-substantia nigra LY379268 100 75 Vehicle 60 75 1.0 µg 45 Rewards (30 min) 50 Responses (3 h) 30 25 * 15 SN 0 0 0 1 3 6 Training (Context A) Extinction (Context B) LY379268dose (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. NMDA/AP-5 NMDA receptor ERK (extracellular signal-regulated kinase) 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) Raf MEK ERK U0126 From Thomas and Huganir. Nature Neuroscience, 2004 Role of central amygdala ERK and glutamate in incubation of cocaine cravingDr. Lin LuCollaborator: Dr. Bruce Hope

11. Time-dependent changes in extinction responding and cue-induced reinstatement of cocaine seeking after withdrawal Extinction responding Cue-induced reinstatement * * 100 300 * Last extinction session (no T+L) 80 240 * Cue available * * 180 60 Responses (6 h) Responses (1 h) * * 120 40 60 20 1 2 4 7 15 29 60 1 2 4 7 15 29 60 0 0 Withdrawal day Withdrawal day 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 No T+L 60 Tone+light cue 6-8 60-min sessions 40 Infusions (6 h) Tone+light cue 20 (n=67) 0 “Extinction” + Testing (same day) 1 2 3 4 5 6 7 8 9 10 Day 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 (1) Tran-Nguyen et al. (1998) Heroin Shalev et al. 2001 Cocaine (2) Neisewander et al. 2000 Cocaine (4) Lu et al. 2004 Cocaine (3) Grimm et al. 2001 * * * 400 300 Control 1 day 7 day 1 month * * 300 225 * * * * Responses (5 h) 200 150 Responses (6 h) * 75 100 Priming Extinction Cue 0 0 1 21 1 6 12 25 66 1 30 90 180 1 2 4 7 15 29 60 Withdrawal day Withdrawal day Withdrawal day Methamphetamine Shepard et al. 2004 Sucrose Lu et al. 2004 Alcohol Bienkowski et al. (2004) * 200 150 * 120 * 150 90 Responses (6 h) Responses (3 h) 100 60 50 30 0 0 1 21 51 1 7 30 60 90 180 Withdrawal day Withdrawal day Withdrawal period in home cage prior to extinction tests

13. 300 * * 225 150 Responses (6 h) * 75 0 1 30 90 180 Withdrawal day Cocaine/saline SA training (10 Days) Cocaine/saline SA training (10 Days) Withdrawal period (1 or 30 Days) Withdrawal period (1 or 30 Days) 4 groups Extinction test Day 1 No test Day 1 Extinction test Day 30 No test Day 30 4 groups Experiment 1: activation of amygdala ERK by cocaine cues Training condition: saline versus cocaine Withdrawal period: day 1 versus day 30 Test condition: no cue versus cue exposure (30 min extinction test)

14. Cocaine/saline SA training (10 Days) Withdrawal period (1 or 30 Days) Extinction test Day 1 Extinction test Day 30 Training Extinction test 60 100 * 80 45 60 Responses (30 min) 30 Cocaine SA (n=34) Infusions (6 h) 40 Saline SA (n=34) 15 20 0 0 1 2 3 4 5 6 7 8 9 10 Withdrawal day Training day 1 30 Saline-trained rats Cocaine-trained rats Behavioral data: Training and extinction test Lu, Hope et al. Submitted

15. Central amygdala Basolateral amygdala 200 * * No test 150 Extinction test 100 % of naive rats 50 0 DAY 1 DAY 30 DAY 30 DAY 30 DAY 1 DAY 30 DAY 1 DAY 1 Saline SA Cocaine SA Saline SA Cocaine SA pERK1 pERK2 BLA CeA Exposure to cocaine cues increases ERK phosphorylation in the central amygdala after 30 days of withdrawal

16. Extinction responding Phosphorylated ERK 80 150 Vehicle Inactive lever U0126 Active lever 60 100 * Responses (30 min) 40 % of vehicle values * 50 20 0 0 Basolateral amygdala Central amygdala Vehicle U0126 Raf MEK CeA ERK U0126 Inhibition of ERK phosphorylation in the central amygdala attenuates cocaine seeking after 30 days of withdrawal NMDA receptor

17. Extinction responding Phosphorylated ERK 80 150 60 100 * % of vehicle values 40 Responses (30 min) 50 20 0 0 Basolateral amygdala Central amygdala U0126 Vehicle Inactive lever Vehicle Active lever U0126 Raf MEK BLA ERK U0126 Inhibition of ERK phosphorylation in the basolateral amygdala has no effect on cocaine seeking after 30 days of withdrawal NMDA receptor

18. Phosphorylated ERK Extinction responding 200 80 Inactive lever * 150 Active lever 60 NMDA 100 * % of vehicle values Responses (30 min) 40 50 20 0 0 Basolateral amygdala Central amygdala 0 25 250 NMDA dose (ng/site) NMDA receptor Vehicle NMDA (25 ng) NMDA (250 ng) CeA Induction of ERK phosphorylation in the central amygdala restores cocaine seeking after 1 day of withdrawal Raf MEK ERK

19. Phosphorylated ERK Extinction responding 200 80 150 60 * * NMDA 100 * # % of vehicle values 40 Responses (30 min) * 50 20 * * 0 0 Veh Veh U0126 U0126 Veh Veh U0126 U0126 Veh NMDA VEH NMDA Veh NMDA VEH NMDA Inactive lever Active lever Raf MEK CeA ERK U0126 Inhibition of ERK phosphorylation in the central amygdala reverses the effect of NMDA on cocaine seeking after 1 day of withdrawal NMDA receptor

20. Phosphorylated ERK Extinction responding 200 80 Vehicle AP-5 (3 µg) 60 150 % of vehicle values Responses (30 min) 100 40 * * 20 50 0 0 0 3.0 Central amygdala Basolateral amygdala AP-5 dose (ug/site) Inactive lever Active lever CeA Blockade of NMDA receptors in the central amygdala attenuates cocaine seeking after 30 days of withdrawal AP-5 NMDA receptor Raf MEK ERK

21. 1 2 3 4 5 6 Bregma -2.3 CeA Control experimentInhibition of ERK phosphorylation in the central amygdalahas no effect on cocaine or high-fat food self-administration Food self-administration Cocaine self-administration 160 40 Vehicle 30 120 U0126 Lever responses 20 80 10 40 0 0 1 2 3 4 5 6 hour hour

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: 214-227 (NIDA special issue)

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

24. AcknowledgementsFormer post-baccalaureate studentsShirley LiuJack DempseyPresent post-baccalaureate studentsSarah GrayRobert BuschDeepti Nagarkar