1. 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

2. 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

3. 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)

4. 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)

5. 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
( )
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

6. 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

7. 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

8. 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

9. 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

10. 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)

11. 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

12. 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

13. 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

14. Antalarmin decreases yohimbine-induced reinstatement of alcohol seeking20 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

15. 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

16. 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

17. 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.

18. Batterham + 9 authors (2002) Gut hormone PYY(3-36) physiologically inhibits food intake. Nature 418:
Batterham + 7 authors (2003) Inhibition of food intake in obese subjects by peptide YY N Engl J Med 349:
Pittner + 9 authors (2004) Effects of PYY(3-36) in rodent models of diabetes and obesity.
Int J Obes Relat Metab Disord 28,
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:
* The effect of PYY(3-36) on operant food self-administration and reinstatement has not been assessed

19. 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

20. 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

21. 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)

22. 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

23. Effect of the hypocretin 1 receptor antagonist
Project 3
Effect of the hypocretin 1 receptor antagonist
SB 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

24. 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)

25. SB 334,867 decreases high-fat pellet self-administration
Pellet intake
100
200
300
400 10 20
SB 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 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

26. SB 334,867 has no effect on hypocretin 1-induced reinstatement of food seeking25 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)

27. 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

28. 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

29. 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.”

30. 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