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

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 (1971-2004) 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

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)

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)

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

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

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

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) LY379268 dose (mg/kg, i.p)

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

(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)

(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

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

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

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

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

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

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

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

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

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

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

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)

Animal models and FACE validity Courtesy of Taco de Vries

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