bioRxiv preprint doi: https://doi.org/10.1101/2020.01.22.915728; this version posted April 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Ketamine blocks morphine-induced conditioned place preference and anxiety-like behaviors in mice. Authors: Greer McKendrick1,3, Hannah Garrett3,6, Holly E. Jones2,3,6, Dillon S. McDevitt2,3, Sonakshi Sharma3, Yuval Silberman4, Nicholas M. Graziane5* Author Affiliations: 1 Neuroscience graduate program, Penn State College of Medicine, Hershey, PA 17033, USA 2 Summer Undergraduate Research Internship Program, Penn State College of Medicine 3Department of Anesthesiology and Perioperative Medicine, Penn State College of Medicine, Hershey, PA 17033, USA 4Department of Neural and Behavioral Sciences, Penn State College of Medicine, Hershey, PA 17033, USA 5Departments of Anesthesiology and Perioperative Medicine and Pharmacology, Penn State College of Medicine, Hershey, PA 17033, USA 6These authors contributed equally: Hannah Garrett and Holly E. Jones. * Correspondence: Nicholas Graziane, Ph.D. E-mail: [email protected] Address: Department of Anesthesiology and Perioperative Medicine, Mail Code H187 Pennsylvania State College of Medicine 500 University Drive Hershey, PA 17033 Telephone: 717-531-0003 X-329159 Fax: 717-531-6221 Running title: Ketamine effects on morphine-induced CPP bioRxiv preprint doi: https://doi.org/10.1101/2020.01.22.915728; this version posted April 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Abstract Patients suffering from opioid use disorder often relapse during periods of abstinence, which is posited to be caused by negative affective states that drive motivated behaviors. Here, we explored whether conditioning mice with morphine in a CPP training paradigm evoked anxiety- like behavior during morphine abstinence. To do this, mice were conditioned with morphine (10 mg/kg, i.p.) for five days. 24 h following conditioning, anxiety levels were tested by measuring time in the open arms of the elevated plus maze. The next day, mice were placed in the three compartment chamber to measure morphine-induced conditioned place preference (CPP). Our results show that following morphine conditioning, mice spent significantly less time in the open arm of the elevated plus maze and expressed robust morphine CPP on CPP test day. Furthermore, we found that an acute treatment with (R,S)-ketamine (10 mg/kg, i.p.), a medication demonstrating promise for preventing anxiety-related phenotypes, 30 min. prior to testing on post conditioning day 1, increased time spent in the open arm of the elevated plus maze in saline- and morphine-conditioned mice. Additionally, we found that a second injection of ketamine 30 min. prior to CPP tests on post conditioning day 2 prevented morphine-induced CPP, which lasted for up to 28 d post conditioning. Furthermore, we found that conditioning mice with 10% (w/v) sucrose using an oral self-administration procedure did not evoke anxiety- like behavior, but elicited robust CPP, which was attenuated by ketamine treatment 30 min. prior to CPP tests. Overall, our results suggest that the ketamine-induced block of morphine CPP may not be attributed solely to alleviating negative affective states, but potentially through impaired memory of morphine-context associations. Keywords: negative affect, morphine, conditioned place preference, anxiety, opioid use disorder, ketamine, psychedelics bioRxiv preprint doi: https://doi.org/10.1101/2020.01.22.915728; this version posted April 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Introduction 2 The motivation to continually seek and obtain addictive substances during periods of abstinence 3 or recovery is caused, in part, by the necessity to avoid aversive internal states (Solomon and 4 Corbit, 1978). Evidence for this comes from patients with substance use disorders who self- 5 report urges and intentions to take drugs to avoid drug-withdrawal symptoms (O'Brien, 6 1975;Baker et al., 2004;Wikler, 2013) or to cope with negative affect (Perkins and Grobe, 7 1992;Zinser et al., 1992;Wetter et al., 1994;Cooney et al., 1997;Conklin and Perkins, 2005;Fox 8 et al., 2007). For example, abstinence from morphine, a highly addictive opioid, facilitates 9 increases in anxiety (Gold et al., 1978;1979), which is a potential factor in continued drug use 10 (Martins et al., 2012). 11 In order to better understand the mechanisms mediating drug-craving and subsequent relapse, 12 preclinical models have been developed whereby drug-seeking behaviors are monitored in drug- 13 exposed rodents. In the conditioned place preference (CPP) paradigm, a drug is paired with a 14 context during conditioning. This is followed by a test day whereby the time spent in the drug- 15 paired context is measured. This behavioral paradigm is a form of Pavlovian learning whereby 16 an injection of a drug (i.e., unconditioned stimulus) elicits a hedonic feeling of pleasure (i.e., 17 unconditioned response), which, when paired with a context (neutral stimulus), invokes incentive 18 value to the context (i.e., now a conditioned stimulus), thus driving a behavioral response to 19 “seek” the context (conditioned response). This is similar to sign-tracking behaviors (Huston et 20 al., 2013), which refer to a behavior that is directed toward a stimulus as a result of that stimulus 21 becoming associated with a reward (Huys et al., 2014). Therefore, CPP provides a valuable tool 22 used to understand how drugs of abuse become associated with environmental contexts, which is 23 implicated in context-induced drug craving and relapse (O’Brien CP, 1986;O'Brien et al., 1992). 24 We have found that five days of morphine (10 mg/kg) conditioning elicits robust morphine CPP 25 (Graziane et al., 2016;McDevitt and Graziane, 2019). However, it is unclear whether this “drug 26 context-seeking” behavior is mediated by negative affective states. Additionally, it is unclear 27 whether a subanesthetic dose of ketamine, an anxiolytic agent (Engin et al., 2009b), blocks 28 morphine-induced CPP by mitigating morphine-induced negative affective states. 29 Here, we attempt to investigate whether morphine conditioning in our CPP paradigm generates 30 negative affect during morphine abstinence. Additionally, we investigate whether an acute, 31 subanesthetic dose of (R,S)-ketamine prior to testing is sufficient to disrupt morphine-induced 32 anxiety and/or morphine-induced CPP behaviors. Lastly, it has been shown that an acute 33 administration of (R,S)-ketamine is sufficient to block the expression of morphine CPP (Suzuki 34 et al., 2000). Here, we investigate whether this ketamine-induced block of morphine CPP, in our 35 behavioral training paradigm, is mediated by the impairment of drug-context associations or by 36 the attenuation of morphine-induced negative affective states. 37 Methods 38 Animals 39 All experiments were done in accordance with procedures approved by the Pennsylvania State 40 University College of Medicine Institutional Animal Care and Use Committee. Male C57BL/6J bioRxiv preprint doi: https://doi.org/10.1101/2020.01.22.915728; this version posted April 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 41 mice aged 5-8 weeks were purchased from Jackson Labs (stock #000664) (Bar Harbor, ME), 42 singly-housed, and maintained on a regular 12 hour light/dark cycle (lights on 07:00, lights off 43 19:00) with ad libitum food and water. Mice were singly housed for the following reasons. First, 44 we have reliably developed morphine conditioned place preference (CPP) in singly-housed mice 45 (Graziane et al., 2016;McDevitt and Graziane, 2019). Second, evidence suggests that socially 46 isolated rodents are more vulnerable to developing drug-context associations (Whitaker et al., 47 2013). In humans, social isolation increases vulnerability to substance use disorders (Newcomb 48 and Bentler, 1988;Sinha, 2008), which often are accompanied by the development of drug- 49 context associations (O’Brien CP, 1986;O'Brien et al., 1992;Xue et al., 2012). Therefore, our 50 studies are designed to model this patient population. 51 Drugs 52 (−)-morphine sulfate pentahydrate was provided by the National Institute on Drug Abuse Drug 53 Supply Program. Ketamine hydrochloride (racemic mixture of 50% R-ketamine and S-ketamine) 54 (Dechra Pharmaceuticals, Northwich, United Kingdom) was purchased from the Comparative 55 Medicine Department at the Pennsylvania State University College of Medicine. 56 Non-Contingent Conditioned Place Preference 57 Conditioned place preference (CPP) chambers (Med Associates) were located in the mouse 58 housing room and consisted of three distinct compartments separated by manual guillotine-style 59 doors. Each compartment had distinct contextual characteristics: the middle (neutral) 60 compartment (7.2 cm × 12.7 cm × 12.7 cm) had grey walls and grey plastic floor, while the 61 choice compartments (16.8 cm × 12.7 cm × 12.7 cm, each) had either white walls and stainless 62 steel mesh floor or black walls and stainless steel grid floor. All compartments were illuminated 63 with a dim light during use. Immediately following use the entire preference chamber was 64 cleaned thoroughly with a scent-free soap solution.