Clinically Effective OCD Treatment Prevents 5-HT1B Receptor-Induced Repetitive Behavior

Clinically effective OCD treatment prevents 5-HT1B receptor-induced repetitive behavior and striatal activation

Emily V. Ho, B.A., Summer L. Thompson, M.S., William R. Katzka, Mitra F. Sharifi, B.A., James A. Knowles, M.D., Ph.D., Stephanie C. Dulawa, Ph.D.

Corresponding Author: Stephanie C. Dulawa, Ph.D.

Department of Psychiatry and Behavioral Neuroscience, Committee on Neurobiology, University of Chicago, Chicago, IL 60637

Email:

Chemicals

RU24969, WAY100635, and GR127935 were purchased from Tocris Bioscience (Minneapolis, MN). Fluoxetine was purchased from LKT Laboratories (St. Paul, MN), and desipraminefrom Sigma-Aldrich (St. Louis, MO).

Animals

Mice were housed in a temperature-controlled colony room on a 12-h light/dark schedule with food and water available ad libitum. Behavioral testing occurred during the light phase. Animal testing was conducted in accordance with the National Institutes of Health Laboratory Animal Care Guidelines and with the Institutional Animal Care and Use Committee approval.

Fos mRNA quantification

RNA was isolated using RNeasy Lipid Tissue Mini Kit (Qiagen, Germantown, MD) and cDNA was generated by reverse transcription (Life Technologies, Grand Island, NY). Quantitative real-time polymerase chain reaction was performed using an ABI PRISM 7900HT Sequence Detection System with TaqMan Gene Expression Assays for Fos (Mm00487425_m1) and eukaryotic 18S (Life Technologies, Grand Island, NY). Relative mRNA expression levels were quantified using the comparative Ct method, with samples run in triplicate and 18s ribosomal RNA providing an endogenous control. Sample triplicates with Ct values over 35, not within 0.5 CT cycles, or with a ∆Ct standard deviation over 0.25 were excluded to remove poor quality samples from analysis. RQ values were binary log transformed (log2(RQ)) for analysis.

Open Field Test

Mice were placed in a corner of the open field and monitored for 20 min. Locomotor activity was quantified using 42×42×30 cm Plexiglas activity chambers (Accuscan, Columbus, OH). Chambers were equipped with infrared beams (2.5 cm apart) along each wall. Paths taken by mice were recorded as x, y coordinate sequences. Dependent measures were total distance traveled, time and distance traveled in the center, number of rearings, time spent rearing, and the spatial scaling exponent “spatial d”. Spatial d quantifies the degree to which locomotor patterns are straight (d ~ 1) or circumscribed (d ~ 2), and are independent of the amount of locomotor activity (Paulus and Geyer 1991).

Dig Test

Immediately following open field test, mice were transferred to test cages with 1” of standard bedding and videotaped for 3 min (adapted from Deacon 2006). Videos were later scored for digging behavior, defined as the movement of bedding as a result of fast alternating movements of the forepaws (van Oortmerssen 1971).

Splash Test

Immediately after the dig test, mice continued acclimatization to test cages for 1 hr. Mice were then sprayed with a 10% sucrose solution, twice dorsally from approximately 5” away (Ducottet and Belzung 2004). Subsequent behavior was videotaped for 5 min. Videos were later scored for grooming behavior (Smolinsky et al. 2009).