1. Anxiolytic Placebo Response in Outbred Mice Kelsey I. Landaverde, Stacey L. Kirkpatrick, Lisa R. Goldberg, Camron D. Bryant Laboratory of Addiction Genetics, Department of Pharmacology and Experimental Therapeutics and Psychiatry, Boston University School of Medicine  In assessing EPM percent open arm time, there was a significant effect of treatment (F3,39=6.05, P<0.01) but no dose dependency, therefore we will use the lowest effective dose (5mg/kg) for placebo conditioning.  The dose-response experiment for DZ indicated an effect of DZ on anxiety-like behaviors and other measures, including freezing episodes.  Here, for the first time we demonstrate evidence for DZ-conditioned anxiolysis in the EPM, providing evidence for a placebo response. However, conditioned anxiolysis was observed in both conditioned and unconditioned groups trained with DZ, indicating that mice did not distinguish DZ-predictive from non-predictive cues.  Future studies could involve additional and/or longer training sessions, changes in the EPM conditioning enclosure, or the use of additional cues and contexts that better distinguish the prediction of DZ versus vehicle treatment.  We did not observe any DZ conditioned reward relative to vehicle controls under these conditions. Background: A placebo is a biologically inert substance that can stimulate an effect resembling that of the active drug and is typically administered as a control treatment in clinical drug trials. Anxiety and depressive disorders can be attenuated by placebo response in human subjects (Stein, 2006), thus they have potential to initiate therapeutic purposes. Because of this, it is important to investigate the biological mechanism of placebo response to inform our understanding of the role of various pharmacological interventions in the treatment of these disorders. Here, we developed an animal model of conditioned placebo anxiolysis. The anxiolytic drug we used was Diazepam (DZ), a benzodiazepine that is commonly used to treat anxiety, muscle spasms and seizures. To determine the dose of DZ, we conducted a dose response experiment using the Elevated Plus Maze (EPM) (0, 5, 10 and 20mg/kg, i.p) based on the results we decided to use a 5 mg/kg dose. Methods: DZ was prepared by sonication in 5% Tween 80 made fresh each day prior to testing. Male and female CFW Mice (56 days old, Jackson Laboratory, Bar Harbor, ME) were used. We used a Pavlovian conditioning of DZ reward in mice by pairing DZ. Briefly, we utilized a 10 day protocol with 30 min training and testing sessions to assess both EPM placebo behaviors and DZ reward. To assess DZ reward, we used a conditioned place preference (CPP) apparatus, which consists of a Plexiglas box with two sides (differentiated by floor texture) with a divider that can allow for open access or confinement to one side. To assess anxioylsis we utilized an apparatus used to measure anxiety in mice the EPM a cross-shaped maze 24” off the floor with two open, exposed anxiogenic arms and two closed arms surrounded by opaque walls with a removable Plexiglas conditioning chamber in the center. On Day 1 (D1), all mice were given vehicle and immediately placed in the CPP, and allowed to explore both sides. Conditioned mice were confined to the DZ- paired side (D2, D4) or the vehicle-paired side (D3, D5). Immediately following the 30 min training session (at DZ peak effect), conditioned mice were confined to the conditioning chamber of the EPM (D2, D4), or placed back in their home cage (D3, D5). Increased time in the open arms of the EPM was interpreted as an anxiolytic response. Unconditioned mice were confined to the DZ-paired side (D2, D4) or the vehicle-paired side (D3, D5). Immediately following the 30 min training session, unconditioned mice were placed back in their home cage (D2, D4) or confined to the EPM (D3, D5). On D8, we assessed the placebo response by testing the mice on the EPM following vehicle treatment. On D9 and D10, we assessed conditioned DZ reward (D9) and state-dependent DZ reward (D10) by placing the mice in the CPP chamber with open access to both sides. We assessed time spent on the DZ-paired side as a measure of reward. Behavioral data was video tracked using Anymaze (Stoelting, Wheaton, IL) software. We used additional measures to phenotypically characterize the anxiolytic placebo response including locomotor activity, entries into open arms and zone-specific behaviors. Dose-response for DZ-induced anxiolysis BU Undergraduate Research Opportunity Program, R00DA029635, T32GM0086541, R03DA038287, R21DA038738, R01DA039168 Results and Future Directions Conditioned DZ placebo anxiolysis and reward DZ-induced open arm behaviors EPM conditioning chamber # Of Open Arm Sequences DZ dose (mg/kg, i.p.) Open arm sequences Dose response for DZ-induced freezing Funding Pavlovian conditioning paradigm for placebo anxiolysis DZ dose (mg/kg, i.p.) EPM CONDITIONED D1D2D3D4D5 D6 & D7 D8 D9 D10 D1 D2D3D4D5 D6 & D7 D8D9 D10 PLACEBO AND CPP TEST DAYS UNCONDITIONED PLACEBO AND CPP TEST DAYS DZ 0 0 0 CPP Initial assessment day 0 DZ 0 CPP assessment State Dependent CPP DZ0 0 0 CPP Initial assessment day 0 0 DZ CPP assessment State Dependent CPP Home Cage DZ dose (mg/kg, i.p.) Treatment %open arm time= open time open +closed time x 100%