Psychopharmacology (2012) 223:281–297 c-fos mRNA in response to acute restraint stress in rat frontal cortex (Morinobu et al. 1995). A similar observation is made for Egr1 after ECS or repeated tranylcypromine or imipramine in PCX (Park et al. 2011). In our study, we also observed downregulation of Egr1 and c-fos mRNA levels in PCX at different time points during DMI treatment. Another correlation observed in our study concerned the expression profiles of c-fos and Nr4a1. Nr4a1 (NGFI-B, Nur77) is a member of a subgroup of nuclear steroid recep- tors that do not have any recognized ligands and function independently as orphan receptors with transcriptional reg- ulatory activity. Pharmacological studies indicate that NGFI-B mRNA and protein levels are elevated via the activation of adrenergic receptors (Humphries et al. 2004). Evaluation of mRNA level by ISH in groups of animals treated with CIT confirmed the upregulation of Egr1, Nr4a1, and c-fos mRNA level after 3 days of repeated treatment in the CCX. But the changes observed by ISH were not so pronounced as evaluated by an RT-PCR meth- od. The difference in mRNA fold change may reflect the technical differences and sensitivity of these two methods used to evaluate the gene expression. Additionally, both of these methods are semiquantitative, so the most important is the direction of observed changes, not the absolute value. In gene expression studies, RT-PCR is the most sensitive meth- od available, while ISH allows evaluating and visualizing the localization of transcripts in various other brain regions at the same time (Bartlett 2002), so they both can be con- sidered as complementary. As has been previously reported, the activation of IEGs, associated with synaptic activity, learning, and memory, is under the control of the SRF (Knoll and Nordheim 2009). Interestingly, our analysis of TFBSs revealed an over- representation of SRF binding sites in the promoters of genes that changed over time after both the DMI and CIT treatments in all brain regions. Also, binding sites for MEF2A, which belongs with SRF to the MADS-box tran- scription factors family, were over-represented. Although these transcription factors have different DNA binding prop- erties and are required for opposing cellular processes (Schlesinger et al. 2011), an interaction between them has been recently predicted by bioinformatics approaches and confirmed experimentally (Pustylnik 2011). One of the most interesting changes in gene expression found in this study concerned the Dbp gene, which showed a progressive decrease during repeated DMI treatment. The D site of the albumin promoter (albumin D-box)-binding protein, also known as DBP, belongs to the clock genes and is expressed according to circadian rhythms (LopezMolina et al. 1997). As sleep disturbances are common in major depressive disorders and sleep deprivation is one possible treatment for depression, Dbp may be involved in antide- pressant action. Dbp has been already identified as a 293 potential candidate gene in bipolar disorder and alcoholism by Niculescu and colleagues (Niculescu et al. 2000; Rodd et al. 2007; Le-Niculescu et al. 2008). According to our pres- ent study, Dbp may also be an interesting target for studies on the mechanisms underlying antidepressant action. Interestingly, the repeated and single-dose treatments resulted in opposite regulation of the Id1 gene, which was increased upon repeated DMI treatment. Id (inhibitor of differentiation) functions as a negative regulator of differen- tiation during development. ID1, together with ID2 and ID3, increases the self-renewal and proliferation potential of the cortical neural stem cells (Jung et al. 2010). In the present study, we have shown, for the first time, alterations in Id1 expression after antidepressant treatment. Neuroplasticity is believed to be involved in ADs action, so changes in Id1 gene expression in the brain cortex may be another potential target for further studies concerning ADs and depression. In addition to the evaluation of temporal changes in gene expression and comparison of the effects of two ADs, the aim of our study was to verify the hypothesis that single- dose treatment induces similar molecular changes to those seen under repeated treatment. This hypothesis fits into the idea of time-dependent sensitization (TDS) (Antelman and Gershon 1998; Antelman et al. 2000) which assumes that the therapeutic effects of a drug may not be due to the drug’s action per se but they rather develop as a function of time. A drug is viewed as a foreign substance and stressor that induces a time-dependent cascade of molecular events, irre- spective of whether it is given repeatedly or only once. There are many pharmacological examples of TDS (Antelman et al. 2000). In the case of ADs, the increased level of monoamines at the synapse may only be an early step in a potentially complex cascade of molecular events that results in an AD’s therapeutic effects (Pineyro and Blier 1999). The results of the present study cannot definitely confirm nor reject the hypothesis of TDS in ADs action. Interestingly, the expression of most affected genes was observed to change with time (time factor in a two-way ANOVA) but not with the treatment type, especially after CIT treatment. As described in the “Introduction” section, the genes chosen for investigation in the present study are either strongly believed, on the basis of available data, to be involved in ADs action or are novel targets for new ADs therapies. The changes in expression of many of these genes did not show any significant difference between treatment types (repeated or single-dose, followed by the same period of time). This may confirm the role of time in ADs treatment as more important than the role of the drug itself, although the two drugs used in our study activated different molecular processes and pathways, as revealed by our bioinformatics analysis. On the other hand, if we discard the TDS hypoth- esis, we have to question the role of many of the selected