Supplementary Information s79

Psych 2017-00078 R2

Supplementary Information

Blockade of alcohol escalation and “relapse” drinking by pharmacological FAAH inhibition in male and

female C57BL/6J mice

Yan Zhou 1*, Benjamin I Schwartz 2*, Joanna Giza 3, Steven S Gross 2, Francis S Lee 2,3, Mary Jeanne Kreek 1

1 Laboratory of the Biology of Addictive Diseases, The Rockefeller University, New York, NY, USA

2 Department of Pharmacology, Weill Cornell Medical College, New York, NY, USA.

3 Department of Psychiatry, Weill Cornell Medical College, New York, NY, USA.

Corresponding Author: Yan Zhou, MD, PhD

Laboratory of the Biology of Addictive Diseases, The Rockefeller University

1230 York Avenue, New York, NY 10065, USA.

Tel: (212) 327 8248; Fax: (212) 327-8574

E-mail: [email protected]

Contributors: *YZ and BIS contributed equally to this work.

Running title: FAAH inhibitor reduces alcohol escalation and “relapse”

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Results S1. No effect of single, acute administration of URB597 (0.25 or 0.5 mg/kg) on 15% alcohol intake after 3-week drinking-in-dark (DID) model. To assess the effect of URB597 on the short-access “binge” alcohol drinking, the mice (n = 6/per group) received URB597 at 0, 0.25 or 0.5 mg/kg after 3 weeks of DID. There was no effect on alcohol intake after 4 hours (control mice: 4.3 ± 0.27 g; 0.25 mg/kg URB597 mice: 3.7 ± 0.39 g; 0.5 mg/kg

URB597 mice: 3.9 ± 0.44 g).

Table S1 No effects of single, acute administration of URB 597 (URB, 0.5mg/kg) on total fluid intake in male mice after 1 day of withdrawal from 3-week chronic intermittent access (IA) alcohol drinking.

Total fluid intake, ml Vehicle (n = 7) 0.5 mg/kg URB (n = 6) 4h 1.7 ± 0.2 1.6 ± 0.1 8h 1.1 ± 0.3 1.3 ± 0.5 24h 2.9 ± 0.3 2.8 ± 0.3

Table S2 Effects of single, acute administration of URB597 (URB, 0.5 mg/kg) on 15% alcohol intake and preference ratio in both male (presented in Figure 1) and female (presented in Figure S1) mice after 1 day of withdrawal from 3-week chronic intermittent access (IA) alcohol drinking at 4 hours. Statistical analysis was run for both male and female mice together. For intake, two-way ANOVA revealed a significant effect of URB597 treatment [F (1, 27) = 24.5, p<0.00005] and a significant effect of sex [F (1, 27) = 9.4, p<0.01], with no significant interaction between URB597 treatment and sex. Post hoc analysis showed that: (1) the vehicle-treated females had more intake than the vehicle-treated males (p<0.05); and (2) both the URB597-treated males and females had less intake than the vehicle-treated ones at 4 hours [p<0.05 and p<0.01, respectively]. For preference ratio, two two-way

ANOVA revealed a significant effect of URB597 treatment [F (1, 27) = 18, p<0.0005], with no significant interaction between URB597 treatment and sex. Post hoc analysis showed that both the URB597-treated males and females had less preference than the vehicle-treated ones at 4 hours [p<0.05 for both]. * p<0.05 and **p<0.01 vs. vehicle control in the same sex; + p<0.05 vs. male.

male female Vehicle 0.5 mg/kg URB Vehicle 0.5 mg/kg URB (n = 8) (n = 7) (n = 6) (n = 6) Intake, g/kg 6.0± 0.35 2.8 ± 0.40 * 9.7 ± 1.1 + 5.6 ± 1.0 ** Preference ratio 0.74 ± 0.06 0.48 ± 0.10 * 0.84 ± 0.04 0.56 ± 0.05 *

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Figure S1. Effects of 1-day withdrawal from 3-week chronic intermittent access (IA) alcohol drinking on brain

NAE abundances. Test male mice (labelled Alcohol, n = 6, each n is combined brain regions from 2 mice) were exposed to chronic IA alcohol (15%) drinking for 3 weeks and then 1 day of withdrawal. Control mice (labelled

Water, n = 6, each n is combined brain regions from 2 mice) were given only water. NAEs were then extracted and quantified from four brain regions on an LC-MS system. The family of NAEs is generally elevated globally after 1 day alcohol withdrawal, as shown by AEA (shown in Figure 6), palmitoyl ethanolamide (PEA) and oleoyl ethanolamide (OEA) abundances in the basolateral amygdala (shown in Figure 6), nucleus accumbens, cerebellum, and prefrontal cortex. Normalized, relative abundances are shown in all graphs. *p<0.05 **p<0.001 and

***p<0.0001 vs. control, error bars indicate standard error of the mean.

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Figure S2. No NAE changes after chronic IA or long-term withdrawal. Test male mice (n = 6, each n is combined brain regions from 2 mice) were exposed to chronic IA alcohol (15%) drinking for 3 weeks and then 1 week or 2 weeks of alcohol withdrawal. Control mice (labelled Water, n = 12, each n is combined brain regions from 2 mice) were given only water. NAEs were then extracted from the basolateral amygdala and quantified on an LC-MS system. Palmitoyl ethanolamide (PEA) and oleoyl ethanolamide (OEA) abundances were unchanged after chronic

IA, 1-week or 2-week withdrawal. Normalized, relative abundances are shown in all graphs. Error bars indicate standard error of the mean.

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