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

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.

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)

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

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 (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 - Licensed under CC BY-SA 2.0 via Wikimedia Commons - Cason & Aston-Jones 2014

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: Cason & Aston-Jones 2014

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

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

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

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

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

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

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

Cason & Aston-Jones Take Homes Cue-induced reinstatement and SB 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 effects on Fos expression in orexin targets Cason & Aston-Jones 2014

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

Objectives: SB 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

Methods Male C57BL/6J ( 8 wks start ) Drug = SB (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

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

Total calories consumed (kcal/g/4 h) on test day Sucrose group (kcal/g) Saccharin group (kcal/g) VEH3.47 ± ± 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 ↓ sucrose and saccharin bingeing Higher doses ↓ sucrose bingeing more than lowest dose Strange calories consumed data in saccharin group Alcaraz-Iborra et al. 2014

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 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

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

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 ± ± ± 6.86 Day ± ± ± Day ± ± ± 6.87 Day ± ± ± 3.64 Repeated bingeing ↓ HL OX mRNA Sucrose group: 5x > water intake Alcaraz-Iborra et al. 2014

Alcaraz-Iborra et al. 2014: Take-homes OXR1 role in caloric bingeing in AL-fed animals SB ↓ 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)

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

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 Illustration by Emily Coren.

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

Methods C57BL/6J male mice ( 7-9 wks old ) SB (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

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

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

Olney et al EtOH Bingeing reduced HL OX

Olney et al 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

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

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

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

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