282 sustained hyperactivity of the stress axis caused by the stress neurotransmitters, corticotrophin-releasing hormone (CRH) and urocortin, the effects of which are mediated through the CRHR1 and CRHR2 receptors (Leonard and Myint 2009). There is also evidence that inflammatory processes play an important role in depression, and most antidepressants have anti-inflammatory effects (Leonard and Myint 2009; Maes et al. 2009). Preclinical and clinical evidence suggest that neuro- peptides (such as galanin, neuropeptide Y, and proenkephalin) and their receptors may be directly involved in depression and related illnesses (Dziedzicka-Wasylewska and Papp 1996; Dziedzicka-Wasylewska et al. 2002; Kuteeva et al. 2008; Redrobe et al. 2002). Based on the above-mentioned data and literature survey (Boehm et al. 2006; Carr et al. 2010; Drigues et al. 2003; Frechilla et al. 1998; Orsetti et al. 2008; Urani et al. 2005), genes of interest that are believed to be crucial in ADs action were selected for our study. These genes encode receptors (e.g., Adra, Adrb, Cnr, Drd, GalR, Gria, and Opr), transcrip- tion factors (e.g., Creb, Crem, Egr, Fos, Jun, Jund, and Sp1), genes involved in inflammatory responses (e.g., Il10r, Il1r, Il6r, Stat, and Tnf), HPA activity (e.g., Crhbp, Crhr, and Ucn), neuronal apoptosis and synaptic plasticity (e.g., Bcl2, Bdnf, Cacna, Cdk, Ntf, and Vgf), and a number of other genes previously unlinked directly to ADs action but important because of their involvement in the transcriptional machinery of the cell (e.g., Aes, Arnt, Cebpb, Dbp, Esr2, Ets, Gata2, Hdac, Id1, Nr, Pax6, and Yy1). In contrast to the vast majority of preclinical studies, which have been conducted using ani- mals treated with ADs repeatedly (typically 7–14 days), we decided to conduct a temporal analysis of the expression of genes regulated by ADs in specific brain regions. The notion that the onset of ADs action generally takes weeks is widely accepted. However, questions concerning the sequence of molecular events that are necessary for the ther- apeutic effects to occur during the time following drug admin- istration remain unanswered. This delay in response suggests that antidepressant action is a complex multicellular process that requires the orchestration of many biochemical events, including changes in the transcription of genes that may not be directly involved in the neurotransmitter signaling cascade. Previous studies have shown that delayed onset of ADs action (e.g., changes in receptor density) is induced not only by different types of chronic treatments but also by single-dose antidepressant treatments followed by a certain period of drug-free time (Dziedzicka-Wasylewska and Rogoz 1997; Dziedzicka-Wasylewska et al. 2001; Kuśmider et al. 2006, 2007). Therefore, we additionally checked whether a single dose of a drug followed by a drug-free period induced changes similar to those observed after repeated AD treatments. Because the activation of the limbic system is a relatively consistent biological finding in patients with major depres- sion (aan het Rot et al. 2009; Hercher et al. 2009; Ressler Psychopharmacology (2012) 223:281–297 and Mayberg 2007; Tanti and Belzung 2010), we focused our study on the time-dependent effects of ADs on the expression of selected genes in the limbic regions of the brain, including the prefrontal cortex (PCX), the cingulate cortex (CCX), the dentate gyrus of the hippocampus (HIP), and the basolateral nucleus of the amygdala (AMY). Therefore, we applied automated laser microdissection, RNA isolation, and amplification (custom RT-PCR arrays) to determine the expression level of selected genes and to investigate the mode of action of the tricyclic antidepressant desipramine (DMI) and selective serotonin reuptake inhibitor citalopram (CIT) in the above-mentioned four regions of the rat brain. The laser microdissection technique allowed the precise excision of a defined population of cells, and the RT- PCR arrays allowed the quantification of minute amounts of transcripts. Additionally, we used an advanced bioinformatics approach, i.e., singular value decomposition (SVD) represen- tation of time-dependent multiple gene expression data, which allowed the comparison of global gene expression patterns across different brain regions and used ADs. The SVD is an analytic procedure which has been successfully adopted to analyze the time dynamics of multiple gene expression data- sets (Holter et al. 2000; 2001; Simek et al. 2004). Through the experimental design of our study, we expected to highlight some unresolved questions regarding the molec- ular mechanisms of antidepressant action. First, what is the time-course of antidepressant action, i.e., which molecular targets are affected by a specific AD and at what time during treatment does it occur? What are the structure-specific changes in gene expression that occur during ADs treatment? Are there common end points of antidepressant action, i.e., do ADs belonging to different pharmacological classes target a final common molecular pathway? Finally, does acute treat- ment followed by a drug-free period of time induce similar molecular changes to those caused by repeated treatment? Methods Animals Male Wistar rats weighting 250–310 g at the beginning of the experiments were used. Animals were housed six to eight per cage under a 12-h dark/light cycle, with food and water supplied ad libitum. All animal procedures used in this study were approved by the local Bioethics Commission at the Institute of Pharmacology, Polish Academy of Scien- ces (Krakow, Poland). Drug treatment DMI and CIT were administered in doses of 15 mg/kg in a volume of 2 ml i.p. daily, and all animals were