1. Yavin Shaham Behavioral Neuroscience Branch IRP/NIDA/NIH/DHHS, Baltimore Incubation of cocaine craving: behavioral and neuronal mechanisms Special thanks: The Waletzky family SfN committee Jane Stewart Roy Wise Lin Lu Bruce Hope

2. Ongoing research projects 1. Stress-induced reinstatement of drug and palatable food seeking (Sunila Nair, Udi Ghitza) 2. Context-induced reinstatement of heroin seeking (Jennifer Bossert) 3. Developing a new model to assess relapse to cocaine seeking (Abraham Zangen, Weizmann Institute, Israel) 4. Time-dependent increases in cocaine seeking after withdrawal (incubation of cocaine craving)

3. 1. The clinical problem 2. Behavioral studies 3. Neurobiological studies 4. Conclusions and a brief summary of recent results on incubation of craving 5. Acknowledgements Outline

4. The clinical problem: Relapse to cocaine use after prolonged abstinence Research question: how do we study this “incubation” phenomenon in the laboratory rat? Based on anecdotal evidence, Gawin and Kleber (1986) suggested that cue-induced cocaine craving increases over the first several weeks of abstinence and remains high over extended abstinence periods This relapse can be provoked by re-exposure to cocaine-associated cues that also induce drug craving Background

6. Withdrawal (abstinence) period: 1 to 180 days Cocaine self- administration: 10 days 12471530180 Extinction tests for cocaine seeking in a drug-free state 60 90 X An experimental procedure to study time-dependent changes in cue-induced cocaine seeking

7. Grimm et al. Incubation of cocaine craving after withdrawal. Nature, 2001 Time-dependent increases in cue-induced cocaine-seeking after withdrawal from drug self-administration 60 days in rats ~ 5-6 years in humans 180 days in rats ~ 15-18 years in humans 1247153060 Withdrawal day Lever presses (6 h) 0 75 150 225 300 * * * * Withdrawal day 13090180 0 75 150 225 300 Lever presses (6 h) * * * Withdrawal period (1 to 180 days) Cocaine self- administration (10 days) 12471530180 Extinction tests for cocaine seeking in a drug-free state 60 90

8. Generality of the incubation phenomenon to other drug and non-drug rewards 16122566 * * * 0 100 200 300 400 Withdrawal day Lever presses (5 h) Heroin 760180 * Withdrawal day 30901 0 50 100 150 200 Lever presses (6 h) Sucrose Methamphetamine 0 30 60 90 120 150 12151 Withdrawal day * * Lever presses (3 h) Shalev et al. Psychopharmacology, 2001 Lu et al. Neuropharmacology, 2004 Shepard et al. Biol Psychiatry, 2004 Withdrawal period (1 to 180 days) Reward self- administration (10 days) Extinction tests

9. Cocaine 1 Neisewander et al. 2000 1 21 Withdrawal day More evidence for incubation of reward craving Cocaine 2 Conrad, Marinelli, Wolf, 2005 0 10 20 30 40 145 Withdrawal day Nose pokes (30 min) * Cocaine 3 Sorge and Stewart, 2005 0 50 100 150 200 11060 Withdrawal day Lever presses * * Alcohol Bienkowski et al. 2004 0 15 30 45 60 12856 Withdrawal day Lever presses (20 min) * Sucrose Di Ciano and Everitt 2004 10 30 50 70 1231017243159 Withdrawal day * * * Lever presses (30 min) Heroin Di Ciano and Everitt 2004 20 40 60 80 100 120 1231017243159 Withdrawal day 0 * * * * Lever presses (30 min) * * 0 20 40 60

10. Summary of behavioral findings Incubation of reward craving: Long-lasting, but not permanent Observed with several drugs of abuse Observed with non-drug reinforcers Not evident after acute re-exposure to cocaine priming injections 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)

11. The neuronal mechanisms of incubation of craving

12. Initial molecular findings The neuroadaptation hypothesis of addiction Chronic drug exposure causes long-lasting molecular, cellular, and neurochemical adaptations in the mesocorticolimbic dopamine system that underlie compulsive drug use and prolonged relapse vulnerability during abstinence (Karler et al., 1989; Wolf and Khansa, 1991 ; Nestler et al. 1990; Kalivas and Stewart 1991) Amygdala Ventral tegmental area (VTA) Nucleus accumbens Cortex The mesocorticolimbic dopamine system From Nestler. Nat. Rev. Neurosci. 2001

13. Associated with increases in peptide levels of brain-derived neurotrophic factor (BDNF) in VTA, accumbens and amygdala Not associated with increases in protein levels of AMPA and NMDA glutamate receptor subunits in VTA, accumbens and amygdala Not associated with increases in protein levels of cyclin-dependent kinase 5 (cdk5) and tyrosine hydroxylase (TH) in VTA and accumbens Not associated with increases in the activity of cAMP-dependent protein kinase (PKA) and adenylate cyclase (AC) in VTA and accumbens Incubation of cocaine craving: Grimm et al. J Neurosci 2003 Lu et al. J Neurochem 2003, 2005 Lu et al. J Neurosci 2004 Summary of initial molecular findings

14. ERK MEK Raf U0126 NMDA receptor NMDA/AP-5 From Thomas and Huganir. Nature Neuroscience, 2004 Role of central amygdala ERK in incubation of cocaine craving Lin Lu Collaborator: 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 (extracellular signal-regulated kinase) Also: the human amygdala is activated by cocaine cues (Grant et al. 1996; Childress et al. 1999)

15. Cocaine/saline self- administration training (10 days) Withdrawal period (1 or 30 Days) Extinction test Day 1 Extinction test Day 30 Behavioral data: Training and extinction tests Withdrawal day Lever presses (30 min) Extinction test 0 15 30 45 60 130 * Cocaine-trained rats Saline-trained rats Lu, Hope et al. Nature Neuroscience, 2005

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

17. Inhibition of ERK phosphorylation in the central amygdala attenuates cocaine seeking after 30 days of withdrawal 0 20 40 60 80 VehicleU0126 Lever presses (30 min) * Extinction responding 0 50 100 150 Central amygdala % of vehicle values Basolateral amygdala Vehicle U0126 * Phosphorylated ERK U0126 ERK MEK Raf NMDA receptor CeA

18. 0 20 40 60 80 Vehicle U0126 Lever presses (30 min) Extinction responding % of vehicle values 0 50 100 150 * Phosphorylated ERK Central amygdala Basolateral amygdala Vehicle U0126 Inhibition of ERK phosphorylation in the basolateral amygdala has no effect on cocaine seeking after 30 days of withdrawal U0126 ERK MEK Raf NMDA receptor BLA

19. Extinction responding 025250 NMDA dose (ng/site) 0 20 40 60 80 Lever presses (30 min) * Phosphorylated ERK 0 50 100 150 200 * Central amygdala % of vehicle values Basolateral amygdala Vehicle NMDA (25 ng) NMDA (250 ng) ERK MEK Raf Induction of ERK phosphorylation in the central amygdala restores cocaine seeking after 1 day of withdrawal NMDA receptor CeA

20. Blockade of NMDA receptors in the central amygdala attenuates cocaine seeking after 30 days of withdrawal ERK MEK Raf AP-5 Phosphorylated ERK 0 50 100 150 200 Central amygdala Basolateral amygdala % of vehicle values Vehicle AP-5 * NMDA receptor CeA Extinction responding 0 20 40 60 80 Lever presses (30 min) * Vehicle AP-5

21. LY379268: a selective agonist of mGluR 2/3 receptors that decreases evoked glutamate release Lu et al. Biological Psychiatry 2006 Day 3 withdrawal Day 21 withdrawal Basolateral amygdala injections Systemic injections Central amygdala injections * LY379268 dose (mg/kg, i.p) 0 50 100 150 200 01.53.0 Lever presses (3 h) * LY379268 dose (µg/site) 00.51.0 LY379268 dose (µg/site) 01.0 Courtesy of Carl Lupica Glutamate GABA mGluR2 mGluR3 More evidence for a role of central amygdala glutamate in incubation of craving

22. Conclusions Incubation of reward craving is a general phenomenon that occurs with both drug and non-drug rewards Time-dependent increases in the responsiveness of central amygdala ERK and glutamate to cocaine cues mediate the incubation of craving Implications for treatment Drug addicts may be highly vulnerable to relapse at time periods that are far beyond the acute drug withdrawal phase Time away from drug (e.g., incarceration) is not a good method for relapse prevention Our studies suggest that mGluR 2/3 agonists should be considered in the treatment of relapse to cocaine and other drugs

23. Exposure to cocaine cues increases ERK phosphorylation in the medial prefrontal cortex after 30 days of withdrawal Koya, Uejima et al. SFN 2006 Extinction test 0 25 50 75 100 130 Withdrawal day Lever presses (30 min) * An update on incubation of cocaine craving (1) dmPFC vmPFC Dorsal mPFCVentral mPFC No test Extinction test 80 40 60 20 0 * 130 Withdrawal day p-ERK positive cells/mm 2 * 130 Withdrawal day

24. An update on incubation of cocaine craving (2) Incubation of cocaine craving: Enhanced in GluR1 knockout mice (Mead et al. Neuropsychopharmacol. 2006) Attenuated by inhibition of protein synthesis in the amygdala following cue exposure during early withdrawal (Lee et al. J. Neurosci. 2006) Associated with increases in neuronal activity in the accumbens (Hollander & Carelli. Neuropsychopharmacol. 2006) Associated with greater total production and surface expression of GluR1 in the accumbens (Conrad et al. SFN 2006) Incubation of cocaine craving in humans: Incubation of cocaine relapse during a disulfiram clinical trial (Kosten et al. CPDD 2005). “In support of human ‘incubation’, only 3% of cocaine dependent subjects who stopped cocaine use for at least 2 weeks relapsed before week 4, and relapse peaked after 6.6 weeks of abstinence” Withdrawal day Extinction tests 0 10 20 30 40 Nose pokes 1 45 GluR1 surface expression 0 100 200 300 % of control 1 45 * *

25. Acknowledgements Present post-doctoral fellows: Jennifer Bossert, Sunila Nair Present students: Jamie Uejima, Gabriela Poles, Sam Golden Former post-doctoral fellows: Yvona Buczek, David Highfield, Jeff Grimm, Hans Crombag, Uri Shalev, Lin Lu Former students: Shirley Leung, Alison Clements, Jasmine Yap, Kelly Badger, Polly Robarts, Shirley Liu, Jack Dempsey, David Chuang, Robert Busch, Deepti Nagarkar, Sean Sheffler-Collins, Sarah Gray Intramural collaborators: Bruce Hope, Marisela Morales, Eisuke Koya, Udi Ghitza, Jack Shepard, Tsung-Ping Su, Teruo Hayashi, Brandon Harvey, Michael Baumann, George Uhl, Tim Liu, Kenzie Preston, David Epstein, Zhi-Bing You, Roy Wise, Barry Hoffer Extramural collaborators: Jane Stewart, AD Le, Doug Funk, Geoff Schoenbaum, Taco de Vries, Paul Fletcher, Diane Lattemann, Gal Yadid, Abraham Zangen, Suzanne Erb, Micky Marinelli Final thanks: Pier-Vincenzo Piazza, Barry Everitt, Neil Grunberg, Klaus Miczek