bioRxiv preprint doi: https://doi.org/10.1101/159871; this version posted July 5, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Kimbrough and George 1 1 CRF1 receptor-dependent increases in irritability-like behavior during abstinence from 2 chronic intermittent ethanol vapor exposure 3 4 Adam Kimbrough, Ph.D., Giordano de Guglielmo, Ph.D., Jenni Kononoff, Ph.D., Marsida 5 Kallupi, Ph.D., Eric P. Zorrilla, Ph.D., Olivier George, Ph.D. 6 7 Department of Neuroscience, The Scripps Research Institute, La Jolla, California 92037 8 9 *Correspondence should be addressed to 10 Dr. Olivier George, 1Department of Neuroscience, The Scripps Research Institute, 10550 North 11 Torrey Pines Road, SP30-2400, La Jolla, CA 92037. E-mail: [email protected]. Phone: +1- 12 858-784-9039 ; Fax: 858-784-7405 13 14 This work was supported by National Institutes of Health grants AA006420, AA020608, 15 AA022977, and AA007456 and the Pearson Center for Alcoholism and Addiction Research. The 16 content is solely the responsibility of the authors and does not necessarily represent the official 17 views of the National Institutes of Health. 18 19 20 bioRxiv preprint doi: https://doi.org/10.1101/159871; this version posted July 5, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Kimbrough and George 2 21 Abstract 22 Background: In humans, emotional and physical signs of withdrawal from ethanol are 23 commonly seen. Many of these symptoms, including anxiety-like and depression-like behavior, 24 have been characterized in animal models of ethanol dependence. One issue with several current 25 behavioral tests measuring withdrawal in animal models is they are often not repeatable within 26 subjects over time. Additionally, irritability, one of the most common symptoms of ethanol 27 withdrawal in humans, has not been well characterized in animal models. The corticotropin- 28 releasing factor (CRF)-CRF1 receptor system has been suggested to be critical for the emergence 29 of anxiety-like behavior in ethanol dependence, but the role of this system in irritability-like 30 behavior has not been characterized. Methods: The present study compared the effects of 31 chronic intermittent ethanol vapor exposure (CIE)-induced ethanol dependence on irritability- 32 like behavior in rats using the bottle-brush test during acute withdrawal and protracted 33 abstinence. Rats were trained to self-administer ethanol in operant chambers and then either left 34 in a nondependent state or made dependent via CIE. Naive, nondependent, and dependent rats 35 were tested for irritability-like behavior in the bottle-brush test 8 h and 2 weeks into abstinence 36 from ethanol. A separate cohort of dependent rats was used to examine the effect of the specific 37 CRF1 receptor antagonist R121919 on irritability-like behavior. Results: Dependent rats 38 exhibited escalated ethanol intake compared with their own pre-CIE baseline and nondependent 39 rats. At both time-points of abstinence, ethanol-dependent rats exhibited increased aggressive- 40 like responses compared with naive and nondependent rats. R121919 blocked the increased 41 irritability-like behavior in dependent rats. Conclusions: The effect of R121919 to block 42 increased irritability-like behavior suggests that CRF plays an important role in this behavior, 43 similar to other negative emotional withdrawal symptoms. Quantifying and understanding the bioRxiv preprint doi: https://doi.org/10.1101/159871; this version posted July 5, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Kimbrough and George 3 44 molecular basis of irritability-like behavior may yield new insights into withdrawal from ethanol 45 and other drugs of abuse. 46 47 Keywords: ethanol, dependence, chronic intermittent ethanol, irritability, corticotropin-releasing 48 factor 49 50 bioRxiv preprint doi: https://doi.org/10.1101/159871; this version posted July 5, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Kimbrough and George 4 51 Introduction 52 Alcoholism is a chronic relapsing disorder associated with compulsive drinking, the loss 53 of control over intake, and the emergence of a negative emotional state during abstinence (Koob 54 et al., 2004, Koob and Volkow, 2010). In humans, the emotional and physical signs of ethanol 55 withdrawal include anxiety, irritability, mood swings, insomnia, tremors, convulsions, higher 56 blood pressure, accelerated pulse, accelerated breathing, accelerated heart rate, dehydration, and 57 delirium tremens (Schuckit et al., 1995, Finn and Crabbe, 1997). The emotional symptoms of 58 ethanol dependence, including anxiety-like behavior and depression-like behavior, can be 59 modeled in animals during ethanol dependence (Pleil et al., 2015, Marcinkiewcz et al., 2015, 60 Thorsell et al., 2007, Varlinskaya et al., 2017, Kallupi et al., 2014, Gilpin et al., 2012, Gilpin et 61 al., 2015, Heilig et al., 2010, Kliethermes et al., 2004, Pandey et al., 2015, Pandey et al., 2003, 62 Valdez et al., 2004, Valdez et al., 2002a, Ehlers et al., 2013, Vetreno et al., 2016, Rylkova et al., 63 2009, McClintick and Grant, 2016, Egli et al., 2012, Buck et al., 2014). An important challenge 64 for preclinical researchers is the fact that these tests are usually not repeatable within subjects 65 over time, which prevents conducting reliable longitudinal studies. Moreover, increased 66 irritability is a key negative emotional symptom that has been largely neglected. 67 Irritability is one of the most common withdrawal symptoms in humans (Lubman et al., 68 1983) and has been anecdotally noted during withdrawal in animal models but shown to be 69 difficult to quantify experimentally (Frye and Ellis, 1977, Riihioja et al., 1997, Riihioja et al., 70 1999, Woldbye et al., 2002, Becker, 2000). Thus, it is critical to characterize irritability-like 71 behavior during withdrawal and protracted abstinence from ethanol in dependent rats. 72 A commonly used method to study ethanol dependence and withdrawal is the chronic 73 intermittent ethanol (CIE) vapor exposure model (Kissler et al., 2014, Gilpin et al., 2008c, Gilpin bioRxiv preprint doi: https://doi.org/10.1101/159871; this version posted July 5, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Kimbrough and George 5 74 et al., 2008b, Staples et al., 2015, Leao et al., 2015, de Guglielmo et al., 2016, Kimbrough et al., 75 2017). Rats that are made dependent with CIE exhibit clinically-relevant blood ethanol levels 76 (BELs; 150-250 mg%), an increase in ethanol drinking when tested during early and protracted 77 withdrawal, and compulsive-like ethanol drinking (i.e., responding despite adverse 78 consequences; (Rogers et al., 1979, Roberts et al., 1996, Roberts et al., 2000, O'Dell et al., 2004, 79 Schulteis et al., 1995, Vendruscolo et al., 2012, Seif et al., 2013, Leao et al., 2015, Kimbrough et 80 al., 2017). Ethanol dependence that is induced by ethanol vapor results in withdrawal symptoms 81 during both acute withdrawal (somatic and motivational) and protracted abstinence 82 (motivational; (Macey et al., 1996, Sommer et al., 2008, Schulteis et al., 1995, Williams et al., 83 2012, Kallupi et al., 2014, Valdez et al., 2002b, Zhao et al., 2007, Vendruscolo and Roberts, 84 2014, de Guglielmo et al., 2017), but the effects of abstinence from CIE on irritability-like 85 behavior during withdrawal and after protracted abstinence has not yet been reported. 86 Converging lines of evidence suggest that recruitment of the brain corticotropin-releasing 87 factor (CRF)-CRF1 receptor system during withdrawal from CIE is critical for the emergence of 88 anxiety-like behavior (Valdez et al., 2002a, Sabino et al., 2006, Finn et al., 2007, Gilpin et al., 89 2008b, Richardson et al., 2008b, Richardson et al., 2008a), but the role of the CRF-CRF1 system 90 in irritability-like behavior during withdrawal remains to be demonstrated. In the present study, 91 we hypothesized that (1) abstinence from ethanol after the escalation of ethanol drinking using 92 the CIE model would increase irritability-like behavior in the bottle-brush test 8 h into 93 withdrawal and after 2 weeks of protracted abstinence (Riittinen et al., 1986, Lagerspetz and 94 Portin, 1968) and (2) the CRF1 receptor antagonist R121919 would reduce irritability-like 95 behavior during withdrawal. 96 bioRxiv preprint doi: https://doi.org/10.1101/159871; this version posted July 5, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Kimbrough and George 6 97 Materials and Methods 98 Animals 99 Adult male Wistar rats (Charles River), weighing 250-300 g at the beginning of the 100 experiments, were used. The rats were group housed, two per cage, in a temperature-controlled 101 (22°C) vivarium on a 12 h/12 h light/dark cycle (lights on at 10:00 PM) with ad libitum access to 102 food and water. All of the procedures were conducted in strict adherence to the National 103 Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by The 104 Scripps Research Institute Institutional Animal Care and Use Committee. 105 106 Operant ethanol self-administration 107 Two cohorts of rats were used in this experiment. One cohort of rats (n = 24) and an 108 additional cohort of rats (n = 16) were trained to self-administer ethanol. Both cohorts self- 109 administered 10% (w/v) ethanol during daily sessions in standard operant conditioning chambers 110 (Med Associates) until stable responding was maintained as previously described (de Guglielmo 111 et al., 2016, Leao et al., 2015, Kimbrough et al., 2017).