1. Orexin and binge-like consumption: Sucrose, Saccharin, Ethanol ANDY DEEMER

2. Bingeing Eating, Drinking, Drugs Orexin plays a role but… Caloric Status? (Non-)Caloric reward? Conditioning? Cue-induced reward seeking ♂ ♀ differences? Inconsistencies in several studies Methodology related? Gender related? Gender Differences Orexin system ♀ rat - higher Orexin A and OXR1 in HL than ♂ Responses to food restriction More studies = more differences revealed.

3. Economic, Social and Therapeutic Potential of Orexin Research Overconsumption of ______________ is a significant public health/economic issue. Potential applications in obesity, diabetes, drug abuse Understanding factors at play = better therapy Caloric restriction important? Gender diffs? Bingeing substance (caloric/non, hedonic)

4. Orexin system sexual dimorphism Eating disorders more common in women OX system gender differences may play a role Hedonic vs Homeostatic Saccharin vs sucrose Cason & Aston-Jones 2014 Role of orexin/hypocretin in conditioned sucrose-seeking in female rats

5. Methods Female Sprague-Dawley rats Two Feeding Treatments Ad libitum (AL) = free access Food-restricted (FR) = 1 daily feeding - maintained at 85% of AL body mass) Drug = SB-334867 (OXR1 antagonist) 3 doses used in this study 10, 20, and 30 mg/kg Intraperitoneal (4 mL/kg) "SpragueDawleyRat" by Jean-Etienne Minh-Duy Poirrier - http://www.flickr.com/photos/jepoirrier/422469518/in/set-72157594329856603/. Licensed under CC BY-SA 2.0 via Wikimedia Commons - http://commons.wikimedia.org/wiki/File:SpragueDawleyRat.jpg#/media/File:SpragueDawleyRat.jpg Cason & Aston-Jones 2014

6. Methods Training – self-administer sucrose pellets Press lever = get sucrose pellet Compound cue (light and sound) Fixed ratio (FR) 1 responding Until 10 sessions earning > 9 sucrose pellets Progressive ratio (PR) responding Using mice previously trained from FR experiment Find breakpoint for lever presses Images adapted from: http://rnpsychology.org/demo/index.htm Cason & Aston-Jones 2014

7. Fixed vs Progressive Ratio Schedule Progressive Ratio = 5e PelletNumber*0.2 – 5 Point at which no more rewards are earned over an hour = breakpoint. Cason & Aston-Jones 2014

8. Methods - Ratio Experiments Fixed Ratio: Train Vehicle/Drug injection Self-administration session Progressive Ratio: Use rats from fixed ratio experiment Train Injection Self-administration session Each rat was tested at multiple doses (2 doses) Vehicle/Drug injected 30 minutes prior to test sessions (all 3 of today’s papers) Cason & Aston-Jones 2014

9. Cue-induced reinstatement of sucrose-seeking Take mice used for FR1 (but not PR) Daily extinction sessions Lever presses = nothing happens Untraining Until 2 consecutive sessions with < 25 active lever presses Bring cues back (but no sucrose) Measure SB effect on presses 2 late extinction and 2 reinstatement sessions Cason & Aston-Jones 2014

10. Self-Admin Training # days to train similar for FR and AL rats Food-restricted More active and inactive presses + pellets earned Cason & Aston-Jones 2014

11. Fixed Ratio Experiment FR more active presses + pellets earned SD sig. effect on active presses only at high dose No sig. effect of SD on pellets earned Cason & Aston-Jones 2014

12. Progressive Ratio Experiment No effect of group or SB dose on breakpoint Cason & Aston-Jones 2014

13. Cue-induced reinstatement of sucrose-seeking Food group effect No sig. SB effect on CIRSS Contrary to their previous findings in ♂ rats AL and FR rats met extinction criteria < 5 extinction sessions SB attenuation of active presses - late extinction Cason & Aston-Jones 2014

14. Cason & Aston-Jones Take Homes Cue-induced reinstatement and SB-334867 Sex-dependent story ♀ - OXR1 unnecessary; important in ♂ Increased extinction responding Orexin role in learning/reward valuation in females? Future Research: Systems at play in ♀ cue-induced seeking (e.g. leptin, insulin, ghrelin, estrogen) Untangling ♂ ♀ circuitry differences SB-334867 effects on Fos expression in orexin targets Cason & Aston-Jones 2014

15. Cason & Aston-Jones Take Homes Agree with conclusion? “Our findings show that OxR1 regulates operant responding for sucrose reinforcement, but not motivation to work…” Fig 4, Panel 1: With OXR1 unblocked, try less = regulates motivation to work? Cason & Aston-Jones 2014

16. Objectives: SB-334867 effect on binge consumption of sucrose and saccharin in ad libitum-fed mice Effect of repetitive sucrose and saccharin bingeing on OX mRNA expr in LH Mirror chronic morphine, cocaine, ethanol? Binge-like consumption of caloric and non- caloric palatable substances in ad libitum- fed C57BL/6J mice: Pharmacological and molecular evidence of orexin involvement Alcaraz-Iborra et al. 2014

17. Methods Male C57BL/6J ( 8 wks old @ start ) Drug = SB-334867 (OXR1 antagonist) Intraperitoneal (10 mL/kg) Drinking-in-the-Dark (DID) procedure High voluntary bingeing on ethanol, sucrose, saccharin during early part of dark cycle Provide sucrose/saccharin ~ 3 hrs. into dark cycle Open-field activity monitoring qPCR Alcaraz-Iborra et al. 2014

18. Experiment 1: Effect of SB on sucrose and saccharin binge drinking Binge-training / screening 3 days of DID (2 hrs bottle access) ip injection w/ vehicle - habituation 4 th day = test day (4 hrs bottle access) Injection of SB (10, 20 or 30 mg/kg) or vehicle 30 min prior to test Measure: Liquid imbibed Calories consumed (chow included) Alcaraz-Iborra et al. 2014

19. Total calories consumed (kcal/g/4 h) on test day Sucrose group (kcal/g) Saccharin group (kcal/g) VEH3.47 ± 0.202.74 ± 0.42 SB (10 mg)2.24 ± 0.14**0.20 ± 0.09** SB (20 mg)1.11 ± 0.28**1.24 ± 0.49** SB (30 mg)0.97 ± 0.19**0.64 ± 0.39** * ip SB-334867 ↓ sucrose and saccharin bingeing Higher doses ↓ sucrose bingeing more than lowest dose Strange calories consumed data in saccharin group Alcaraz-Iborra et al. 2014

20. Experiment 2: Effect of SB on locomotor activity Open-field locomotor activity monitoring Days 1-3: Habituate to injection (vehicle) and activity chamber & record behavior Day 4: 30 mg/kg SB → activity chamber Evaluate difference Purpose: SB-334867 impact distance traveled and movement time? Explanation for reduced fluid/food intake? Results: No sig. diff. The means they present seem fairly different, but no sig. diff Alcaraz-Iborra et al. 2014

21. Experiment 3: Repeated sucrose/saccharin bingeing and mRNA expression in the HL Repeated bingeing (DID – i.e. voluntary) Four 2-hour daily binge sessions Sucrose, Saccharin, or Water group Brain dissection → LH removed → RNA extracted qPCR with GAPDH for comparison Alcaraz-Iborra et al. 2014

22. Consumption data (ml/kg/2 h) associated with the mRNA study. Water consumption (ml/kg/2 h) Sucrose consumption (ml/kg/2 h) Saccharin consumption (ml/kg/2 h) Day 134.40 ± 0.6472.42 ± 8.7867.70 ± 6.86 Day 223.42 ± 3.50117.72 ± 10.4694.41 ± 15.24 Day 322.14 ± 0.87125.80 ± 4.1677.88 ± 6.87 Day 421.12 ± 2.29103.74 ± 8.0786.69 ± 3.64 Repeated bingeing ↓ HL OX mRNA Sucrose group: 5x > water intake Alcaraz-Iborra et al. 2014

23. Alcaraz-Iborra et al. 2014: Take-homes OXR1 role in caloric bingeing in AL-fed animals SB-334867 ↓ caloric & non-caloric bingeing in AL-fed ♂ mice Unclear why in AL-fed mice (ip injection) Future Research: Site-directed SB studies 1 st evidence of OXR1 role in non-caloric bingeing/hedonic overconsumption Repeated daily bingeing ↓ HL OX expr (qPCR)

24. Alcaraz-Iborra et al. 2014: Take-homes SB effect on bingeing not caused by SB-altered locomotion. Why not show more data? DID potential issue: Energy status at time of DID Future Research: Alter energy status during DID

25. Binge-like consumption of Ethanol and Other Salient Reinforcers is Blocked by Orexin-1 Receptor Inhibition and Leads to a Reduction in Hypothalamic Orexin Immunoreactivity Olney et al. 2015 Illustration by Emily Coren.

26. Purpose Characterize OXR1 role in “binge” drinking Ethanol, sucrose, saccharin Binge-like EtOH and Sucrose drinking Effect on OX immunoreactivity in HL Effect of ip SB SB specificity for EtOH modulation Compare with saccharin bingeing Olney et al. 2015

27. Methods C57BL/6J male mice ( 7-9 wks old ) SB-334867 (0, 5, 10 mg/kg) Bingeing cycles: 1 or 3 cycles; EtOH or Sucrose DID (modified 2 hrs not 4 – short drug action) Except experiment 1 – used 4 hrs. Blood alcohol content Brain dissection Orexin A immunoreacitivy (LH and PFA) Olney et al. 2015

28. Methods SB ip injection Reduced EtOH consumption? Saccharin? Each mouse, all doses 4 day cycle with 3 days rest between doses Locomotion Open field test with Saccharin group mice 2 hours 0 or 10 mg/kg SB Olney et al. 2015

29. No cycle effect on: EtOH Consump. Sucrose Consump BEC EtOH bingeing ↓ LH OX levels

30. Olney et al. 2015 EtOH Bingeing reduced HL OX

31. Olney et al. 2015 SB ↓ EtOH consumption Short-lived (Hr 1 vs. Hr 2) BEC levels parallel total consumption SB ↓ Saccharin consumption Only signif. largest dose over 2 hrs. Take Home: SB effect on ↓ bingeing not specific to EtOH or caloric foods

32. Olney et al. 2015 Locomotion Like Alcaraz-Iborra: SB-induced general lethary cause of reduced bingeing? Open field test: No impairment of locomoter activity

33. Olney et al. 2015: Take-homes OXR1 role in caloric and non-caloric bingeing Not EtOH specific Bingeing ↓ HL OXR1 SB effects not due to altered locomotion DID 4hr vs 2hr Duration of drug effect may be less than 4 hrs Inconsistent dose effects between researchers SB variability: Supplier effects, batch effects Future Research: Mechanisms responsible for post-binge OX and OXR1 levels Interplay of dynorphin and OX w/in VTA

34. Overall Take-homes Site-specific studies necessary (not ip injection) - ID OX circuits underlying phenomena - VTA likely involved

35. Thank You!