290 Table 2 (continued) Region GO GO term AMY GO:0043231~intracellular membrane- bounded organelle GO:0005654~nucleoplasm GO:0060255~regulation of macromolecule metabolic Fold changes (p value) 1.16 (0.026) 1.60 (0.037) 1.20 (0.041) Psychopharmacology (2012) 223:281–297 TFBS over-representation Binding site TFBS class MEF2A (MA0052) CREB1 (MA0018) NFKB1 (MA0105) SRF (MA0083) ESR1 (MA0112) MADS bZIP REL MADS NUCLEAR RECEPTOR Fold change (p value) 11.7 (0.0000) 6.1 (0.0195) 5.7 (0.0281) 5.5 (0.0371) 5.2 (0.0474) The statistical significance of GO enrichment for the time-dependent changes in gene expression was computed using algorithms implemented in the DAVID 2008 database. The analyses were performed on lists of genes that correspond to changes in gene expression that reached the level of statistical significance after two-way ANOVA for the time factor. An empty chart report means that there were no annotations that passed the specified threshold (maximum EASE score, 0.05). For TFBS searches, the cREMaG software was used. The fold change of the detected number of identified TFBSs compared to the number expected by chance and the statistical significance of the over-representation of the TFBS-containing genes compared to the number expected by chance were computed using a z score test whereas the expression level was constant for the single-dose treatment. CIT induced entirely different changes in the ex- pression of Fos and Nr4a1, e.g., the significant increases in the mRNA levels for these genes in the CCX and almost no differences between time-courses in gene expression after repeated and acute treatment were observed. In the CCX, HIP, and AMY, there was a peak in gene expression on day 3. Not all of the changes in the expression profiles presented in Fig. 3 are described in detail, they were meant only to show that the changes in expression for a subset of individual genes (which reached the statistical threshold of significance) oc- curred at different time points. Some of the profiles were similar when comparing the repeated and single-dose treat- ments or brain regions, while others differed. Evaluation of Egr1, c-fos, Nr4a1, and Id1 mRNA levels by an in situ hybridization As a second, independent analysis of gene expression changes induced by AD treatment, we used in situ hybridization (ISH) to compare the regulation of genes with the most pronounced changes after CIT treatment. ISH also allowed us to evaluate potential changes in other brain areas. The mRNA encoding Egr1, c-fos, Nr4a1, and Id1 was measured in the CIT-treated animals. Since the fold change of expression after CIT treat- ment were the most significant in CCX after 3 days, this brain region was selected for quantification at that specific time point to validate changes obtained in RT-PCR experiments. A modest but consistent increase (~20 %) in mRNA encoding Egr1, Nr4a1, and c-fos was observed after 3 days of repeated treatment in CCX. Overall two-way ANOVA (factors: time/ treatment) revealed significant effect of time on Egr1 (F1,220 5.42, p0 0.0295) and c-fos (F1,140 11.51, p0 0.004) mRNA level. In case of Nr4a1 (F1,21 0 3.52, p 0 0.0746) and Id1 (F1,120 2.67, p0 0.1283), the effect of time on mRNA level did not reach the level of significance, although in both cases, the increase after 3 days of repeated treatment was observed (Fig. 4). Discussion Studies based on microarray analyses of gene expression following antidepressant treatment usually compare region- specific gene expression changes either after different ADs treatment (Altar et al. 2004; Boehm et al. 2006; Conti et al. 2007; Drigues et al. 2003; Jungke et al. 2011; Knuuttila et al. 2004; Palotas et al. 2004; Ploski et al. 2006; Sillaber et al. 2008; Takahashi et al. 2006; Wong et al. 2004) or in different animal models of depression (Andrus et al. 2010; Bergstrom et al. 2007; Orsetti et al. 2008; Surget et al. 2009). In our study, we broadened the perspective by evaluating the time-course of gene expression changes during antidepressant treatment. Additionally, in our study, we applied RT-PCR arrays which have significant advantages in specificity and sensitivity com- pared with whole-genome cDNA microarrays whose results may often be ambiguous due to technical reasons (Ostrowski and Wyrwicz 2009). The present study indicates that two ADs of different pharmacological profiles influenced the expression of dif- ferent genes, although some common changes induced by these drugs were also observed. For the DMI treatment,