Psychopharmacology (2012) 223:281–297 injected once a day during the 21 consecutive days of the experiment, either with saline or antidepressant. The doses of drugs were chosen based on our previous experience and literature data (Kuśmider et al. 2006, 2007; Finnegan et al. 1987; Nibuya et al. 1995, 1996; Zhao et al. 2008; Fujimaki et al. 2000; Thome et al. 2000; Castagné et al. 2009). The control groups re- ceived vehicle (saline) for 21 days, and four groups were treated repeatedly with the appropriate drug for 21, 14, 7, and 3 days prior to the end of the experi- ments. There were additional groups treated with a single dose of AD and sacrificed at the same time points (i.e., 21, 14, 7, and 3 days) and 1 day afterwards. The experimental paradigm is presented in Fig. 1a. Brains were removed 24 h after the last injection, rapidly frozen in a heptane/dry ice mixture, and kept at −80°C until preparation. 283 Tissue collection and RNA isolation Brains were always sectioned by the same investigator into 50-μm slices using a cryostat (Leica Microsystems GmbH). Neuroanatomical descriptions are based on the nomenclature provided by the rat brain atlas (Paxinos and Watson 1998). Four brain regions were excised with a laser microdissector (Leica Microsystems GmbH): the PCX, the CCX, the HIP, and the AMY (for the coronal diagrams and microdissected areas, see Fig. 1b). A MicroRnase Kit (Qiagen Inc.) was used to extract total RNA from the microdissected samples. The total RNA concentration was measured using a NanoDrop ND-1000 Spectrometer (ThermoScientific Inc.). The quality of the isolated RNA was checked using a microcapillary electrophoresis system (BioRad) according to the manufac- turer’s instructions, and samples that passed the quality thresh- old (RIN> 8.0) were used for further experiments. a 1 7 14 20 21 DAYS Fig. 1 (a) A schema of the experimental procedures used. The experiments lasted for a total of 21 days. The control groups received saline for 21 days. The repeatedly treated groups were treated either with DMI or CIT for 21, 14, 7, or 3 days. The single-dose treatment groups received drugs only once during the experiments at the same time points (21, 14, 7, and 3 days and one additional day before the end of the experiment), followed by a drug-free period of time and saline injections. (b) Drawings of the coronal diagrams adapted from the rat brain atlas (Paxinos and Watson 1998) and the microdissected brain regions in which gene expression was studied, i.e., the prefrontal cortex (PCX), the cingulate cortex (CCX), the dentate gyrus of the hippocampus (HIP), the basolateral nuclei of the amygdala (AMY) SALINE AD 14 days SALINE AD 7 days SALINE AD 3 days CONTROL - SALINE AD 21 days CCX SALINE SALINE SALINE SALINE SALINE AD 1x AD 1x AD 1x b PCX Bregma ~ 5.20 Bregma ~ 1.60 Bregma ~ - 2.56 AMY AD 1x DG AD 1x