Relapse to food seeking: Role of CRF, PYY3-36, and hypocretin Yavin Shaham Neurobiology of Relapse Section Behavioral Neuroscience Branch IRP/NIDA/NIH/DHHS, Baltimore Post-doctoral fellows: Sunila Nair, Udi Ghitza Students (NIH training program): Sarah Gray, Sam Golden, Tristan Adams-Deutsch CRF = Corticotropin-releasing factor PYY3-36 = Peptide YY3-36 Hypocretin = Orexin

Neurobiology of Relapse Section Shaham laboratory Staff scientist: Jennifer Bossert Post-doctoral fellows: Sunila Nair, Charles Pickens Post-baccalaureate students: Sam Golden, Kristina Wihbey, Tristan Adams-Deutsch Hope laboratory Staff scientist: Bruce Hope Post-doctoral fellow: Eisuke Koya Graduate student: Danielle Guez (Yale U) Post-baccalaureate student: Alex Berkow

Ongoing research projects 1. Relapse to food seeking 2. Context-induced reinstatement of heroin seeking 3. Incubation of cocaine craving (time-dependent increases in cue-induced cocaine seeking) 4. Incubation of conditioned fear (time-dependent increases in conditioned fear) 5. Neuronal mechanisms of context-dependent cocaine sensitization (Hope lab) Extramural collaborations: AD Le (Toronto U): Stress-induced relapse to alcohol (NIAAA) Geoff Schoenbaum (U of Maryland): Effect of cocaine self-administration on learning and memory (NIDA) Abraham Zangen (Weizmann Inst., Israel): Developing a novel conflict model of drug (NIDA) Marina Wolf and Micky Marinelli (Rosalind Franklin University): Accumbens synaptic plasticity and incubation of craving (NIDA)

Outline A brief description of a reinstatement model—an animal model of relapse to drug-taking behavior The adaptation of the reinstatement model to study: 1. Role of CRF in reinstatement of food seeking induced by: * A pharmacological stressor (yohimbine) * Acute re-exposure to food pellets (pellet priming) 2-3. Effect of PYY3-36 and hypocretin 1 receptor antagonist on reinstatement induced by: * Yohimbine * Pellet priming * Cue (a tone-light cue previously paired with pellet delivery)

X The reinstatement model of drug relapse Drug priming Drug cues Stress X Lever presses Self-administration training (Drug is available) Extinction (Drug is NOT available) TESTING Experimental day Reinstatement studies (1971-2007) Year 3 4 10 13 20 96 332 478 100 200 300 400 500 70 75 80 85 90 95 00 05 07 Cumulative # of publications

Study 1 The role of CRF in reinstatement of food seeking induced by the pharmacological stressor yohimbine Hypothalamic-pituitary- adrenal (HPA) axis Extra-hypothalamic CRF systems From Behan et al. 1996

Stress and relapse to maladaptive eating habits The clinical problem: Excessive eating of unhealthy food is a major health problem Stress can provoke relapse to unhealthy eating habits, and humans are particularly vulnerable to this effect when dieting The preclinical situation: This issue has not been explored in preclinical laboratory studies, which have primarily focused on the effect of stress on ongoing feeding behavior

Yohimbine dose (mg/kg, i.p.) Why yohimbine? Yohimbine (an alpha-2 adrenoceptor antagonist) increases brain noradrenaline release and induces stress-like responses in humans and laboratory animals Yohimbine induces heroin craving in human addicts Yohimbine reinstates drug seeking in monkeys and rats Methamphetamine Yohimbine dose (mg/kg, i.p.) 25 50 75 100 1.25 2.5 * Shepard et al. 2004 Biol. Psychiatry Alcohol Le et al. 2005 Psychopharmacology 15 30 45 60 20 40 80 Yap & Shaham. 2000 (unpublished) Lever presses Heroin 120 0.625 Le et al. 2007 (Unpublished) Nicotine

Why CRF? CRF injections into the ventricles and several extrahypothalamic sites (BNST, VTA, median raphe) reinstate drug seeking Non-selective and selective CRF1 receptor antagonists attenuate stress- induced reinstatement of drug seeking Heroin Cocaine Intermittent footshock No stress 15 30 CP-154,526 dose (mg/kg) 45 60 Lever presses * Shaham et al. Psychopharmacology, 1998 Alcohol 15 30 * Gehlert et al. J Neurosci, 2007 MTIP dose (mg/kg) CP-154,526 dose Le et al. Psychopharmacology, 1998

Experimental procedure Pellet self-administration Three 3-h sessions/d every other day (FR-1; 20 sec timeout) Extinction pellets NOT available Tests for reinstatement pellets NOT available Limited access to regular food: ~75% of daily intake Limited access to regular food: ~75% of daily intake (diet condition) 45 mg pellets: 25% fat 48% carbohydrate Exposure to: Yohimbine Non-contingent pellet (priming)

Changes in body weight during training, extinction, and reinstatement (n=35) Pellet self-administration Three 3-h sessions/d every other day (FR-1; 20 sec timeout) Extinction (pellets NOT available) Tests for reinstatement (pellets NOT available) Limited access to regular food: ~75% of daily intake Limited access to regular food: ~75% of daily intake (diet condition) Daily weight fluctuations Training Extinction & Reinstatement -20 -10 10 20 30 40 Day Weight change (g) No pellets Pellets available Ghitza et al. Neuropsychopharmacology, 2006

Development of “compulsive” food-taking behavior during training Pellet self-administration Three 3-h sessions/d every other day (FR-1; 20 sec timeout) Extinction Tests for reinstatement Limited access to regular food: ~75% of daily intake Limited access to regular food: ~75% % of daily intake (diet condition) Pellets 2 4 6 8 10 12 Training day Responses or pellets (9 h) 600 900 1200 300 Timeout responses

The CRF1 receptor antagonist antalarmin decreases yohimbine-induced reinstatement of food seeking Pellet self-administration Extinction (pellets NOT available) Tests for reinstatement (pellets NOT available) Limited access to regular food: ~75% of daily intake Limited access to regular food: ~75% of daily intake (diet condition) Extinction Reinstatement * 20 mg/kg 40 mg/kg Antalarmin dose Vehicle 2.0 Yohimbine dose (mg/kg, i.p.) 25 50 75 100 Lever presses (3 h) 150 300 450 600 1 2 3 4 5 6 7 8 9 10 Extinction day Lever presses 9 h/day 3 h/day Pellet priming Antalarmin was synthesized by Dr. Kenner Rice

Antalarmin decreases yohimbine-induced reinstatement of alcohol seeking 20 40 60 80 10 * Antalarmin dose (mg/kg, i.p.) Lever presses (1 h) Vehicle Yohimbine (1.25 mg/kg) Yohimbine-induced plasma corticosterone release 250 500 750 20 Vehicle Yohimbine (1.25 mg/kg) Antalarmin dose (mg/kg, i.p.) Corticosterone (ng/ml) Marinelli et al. Psychopharmacology, 2007

The CRF1 antagonist antalarmin reverses yohimbine’s effects on social and “antisocial” behaviors 2.0 Yohimbine dose (mg/kg, i.p.) 10 20 30 40 Antisocial bouts (10 min) * 25 50 75 100 Social bouts (10 min) Vehicle Antalarmin dose 20 mg/kg Social and “antisocial” behaviors were evaluated using a social interaction test: Social behavior: crawling together, grooming, sniffing, licking “Antisocial” behavior: wrestling, distress vocalization, confrontation

Conclusions: Study 1 As in the case of drug relapse, the reinstatement model can be used to study mechanisms underlying relapse to maladaptive eating habits The effect of antalarmin on yohimbine-induced reinstatement of food and alcohol seeking is likely mediated by extrahypothalamic CRF1 receptors

Project 2 Effect of PYY3-36 on yohimbine-, pellet-priming- and cue-induced reinstatement of high-fat food seeking Background The gut peptide PYY3-36 is an endogenous Y2 NPY receptor agonist that decreases home-cage feeding after systemic or arcuate nucleus injections PYY3-36 systemic effects are reversed by systemic or arcuate injections of the Y2 receptor antagonist BIIE0246.

Batterham + 9 authors (2002) Gut hormone PYY(3-36) physiologically inhibits food intake. Nature 418:650-654. Batterham + 7 authors (2003) Inhibition of food intake in obese subjects by peptide YY3-36. N Engl J Med 349:941-948. Pittner + 9 authors (2004) Effects of PYY(3-36) in rodent models of diabetes and obesity. Int J Obes Relat Metab Disord 28, 963-971 But…. Tschop + 42 authors (2004) Does gut hormone PYY3-36 decrease food intake in rodents? Nature 430:1 p following 165; discussion 162 p following 165. Boggiano + 36 authors (2005) PYY3-36 as an anti-obesity drug target. Obesity Reviews 6:307-322 * The effect of PYY(3-36) on operant food self-administration and reinstatement has not been assessed

X Experimental procedure Pellet self-administration Extinction Two 3-h sessions/d every other day (FR-1; 20 sec timeout) Extinction pellets NOT available Tests for reinstatement pellets NOT available Limited access to regular food: ~65% of daily intake Limited access to regular food: ~65% of daily intake (diet condition) (Tone-light cue available) (Tone-light cue NOT available) X Exposure to: Yohimbine Non-contingent pellet Cue 45 mg pellets: 35% fat 45% carbohydrate

PYY3-36 has a minimal effect on high-fat pellet self- administration two 3-h sessions/d every other day (FR-1; 20 sec timeout) Session 1: Total intake Session 2: Total intake Food pellets (3 h) 75 150 225 100 200 PYY3-36 dose (µg/kg, i.p.) Limited access to regular food: ~65% of daily intake Ghitza, Nair et al. The Journal of Neuroscience, 2007

PYY3-36 inhibits pellet-priming- and cue-induced reinstatement, but not yohimbine-induced reinstatement 25 50 75 100 Lever presses (3 h) 200 PYY3-36 dose (µg/kg, i.p.) No pellet Pellet * Pellet priming Cue 25 50 75 100 Lever presses (3 h) 200 PYY3-36 dose (µg/kg, i.p.) * No cue Yohimbine 50 100 150 200 250 PYY3-36 dose (µg/kg, i.p.) Vehicle Yohimbine (2.0 mg/kg) Lever presses (3 h)

Conclusions: PYY3-36 Systemic PYY3-36 decreased pellet-priming-induced reinstatement at doses that had minimal effect on ongoing high-fat food self-administration The systemic effect of PYY3-36 on reinstatement generalizes to cues- but not to yohimbine-induced reinstatement of food seeking The systemic effect of PYY3-36 is dependent on Y2 receptors Systemic PYY3-36 had no effect on reinstatement of heroin seeking, suggesting that its effects are specific to food relapse

Effect of the hypocretin 1 receptor antagonist Project 3 Effect of the hypocretin 1 receptor antagonist SB 334867 on high-fat food self-administration and relapse to food-seeking in rats The hypocretins (orexins) Prepro-hypocretin Hypocretin 1 Hypocretin 2 Hypocretin 1 receptor Hypocretin 2 receptor From Sakurai, 2006 and 2007

Why hypocretin 1 receptors? Systemic injections of SB 334,867 decrease home-cage food intake and reverse hypocretin 1-induced increases in food intake (Rodgers et al. 2002) Systemic injections of SB 334,867 decrease: Reinstatement of morphine conditioned place preference induced stimulation of lateral hypothalamus hypocretin neurons (Harris et al. 2005) Footshock stress-induced reinstatement of cocaine seeking (Boutrel et al. 2005) Cue-induced reinstatement of alcohol seeking (Lawrence et al. 2006)

SB 334,867 decreases high-fat pellet self-administration Pellet intake 100 200 300 400 10 20 SB 334867 dose (mg/kg, i.p.) Pellets (3 h) * Pellets Time course Vehicle 10 mg/kg 20 mg/kg Session minutes 10 20 30 40 50 60 90 120 150 180 SB 334867 dose (mg/kg i.p.) Timeout lever presses per pellet 2 4 6 10 20 Timeout responses/pellet Training 500 1000 1500 1 2 3 4 5 6 7 8 9 Training session Pellets or lever presses Timeout responses Pellets Nair et al. British Journal of Pharmacology, 2008

SB 334,867 has no effect on hypocretin 1-induced reinstatement of food seeking 25 50 75 100 3.0 6.0 Lever presses (3 h) * Hypocretin 1 dose (µg, icv) Vehicle 10 mg/kg 20 mg/kg Effect of SB 334,867 25 50 75 100 6.0 Hypocretin 1 dose (µg, icv) Lever presses (3 h)

SB 334,867 has no effect on pellet-priming-, pellet-cue- or yohimbine-induced reinstatement of food seeking Pellet priming 25 50 75 100 No pellet Pellet Lever presses (3 h) Vehicle 20 mg/kg Cue No cue Vehicle 20 mg/kg Yohimbine 2.0 Yohimbine (mg/kg, i.p.) Vehicle 20 mg/kg

Conclusions: hypocretin 1 receptor The hypocretin 1 receptor plays a role in the self-administration of high-fat pellets The hypocretin 1 receptor likely plays a minimal role in reinstatement induced acute re-exposure to hypocretin 1, food priming, cues and stress

Implications of the findings Our data suggest: 1. A dissociation between the neuronal mechanisms mediating stress-induced relapse to food seeking versus food priming- or cue-induced relapse 2. A potential dissociation between the neuronal mechanisms mediating food self-administration versus food priming- or cue-induced relapse From Shalev et al. Neurobiology of relapse to heroin and cocaine seeking: a review. Pharmacol Rev, 2002 “Taken together, it appears that multiple and dissociable brain systems are involved in relapse to heroin and cocaine seeking induced by drug priming, conditioned cues and stress. Somewhat surprisingly, it also appears that the neuronal events that mediate heroin- or cocaine-induced reinstatement are to some degree different from those involved in their reinforcing effects.”

Clinical implications CRF1 receptor antagonists (currently in clinical development for the treatment of anxiety, depression and drug abuse) should be considered as pharmacological adjuncts in dietary treatments of maladaptive or excessive eating habits PYY3-36 (currently in clinical development for the treatment of obesity) may be a more effective treatment for relapse prevention than for decreasing ongoing high-fat food intake Hypocretin 1 receptor antagonists may be a more effective treatment for decreasing ongoing high-fat food intake than for relapse prevention Acknowledgments Dr. Jennifer Bossert Jamie Uejima Kristina Wihbey Dr. Kenner Rice “The best material model for a cat is another, or preferably the same cat” Norbert Wiener, 1945