Proteomic analysis of hippocampus and frontal cortex in a rat model of depression with gene-environment interaction and antidepressant treatment.

The wide-scale analysis of protein expression provides a powerful strategy for the exploration at a molecular level of complex pathophysiological mechanisms, such as the response to treatment with psychotropic agents. GENDEP is an Integrated Project that combines large-scale clinical pharmacogenomic studies on depressed patients with preclinical investigations on animal models of disease, focusing on treatment with antidepressants. In this work, the Flinders Sensitive Line (FSL), a genetically selected rat model of depression displaying good face, predictive and construct validity (Overstreet 2005) was investigated by a two dimensional proteomic approach. As a control, analyses were carried out on the corresponding Flinders Resistant Line (FRL), which does not show the depressive-like behaviour. To evaluate gene–environment interactions, the FSL and FRL animals were subjected to maternal separation (MS), since early life trauma is considered to be an important antecedent of major depression. Following weaning, FSL and FRL rats were treated with escitalopram (ES), a selective serotonin reuptake inhibitor. FSL and FRL rats were subjected to 180 min. maternal separation from postnatal day 2 to 14. Control groups were not maternally separated. Both stressed and control animals were split into groups receiving ES admixed to food pellets (25 mg/kg/day) or vehicle for 30 days. One week before the end of treatments, the animals were subjected to the Porsolt swim test in order to assess the antidepressant effect of ES. After sacrifice, extracts were prepared from frontal cortex and hippocampus and proteins were separated by two-dimensional electrophoresis. Spots were detected by staining with a fluorescent dye. Image analysis was carried out on maps with PDQuest software to compare protein levels in different gels. Protein level comparisons were carried out with both univariate (Student’s t test) and multivariate (Partial Least Squares) statistical analysis methods. Spots displaying statistically significant differences were cut and proteins were identified using peptide fingerprinting mass spectrometry. Comparisons were carried out between groups in different electrophoretic runs to detect changes induced by genetic background, stress exposure and pharmacological treatment in each brain region. Modified proteins were identified. By the comparison FRL vs FSL, information was gathered about genetic differences between the lines, which are probably involved in the depressive-like behaviour. In the comparison (FSL+noMS+vehicle) vs (FSL+no MS+ES) vs (FSL+MS+ES) the different effect of pharmacological treatment was revealed on control and maternally separated FSL. By the comparison (FSL+noMS+vehicle) vs (FSL+MS+vehicle) vs (FSL+MS+ES), information was collected about the effect of the pharmacological treatment on the modifications induced by maternal separation. The same designs were also analysed on the FRL. Proteins involved in the different pharmacological response in different genetic backgrounds were revealed in the comparison (FRL+MS+ES) vs (FSL+MS+ ES). Results on proteome analysis of ES treated rats allowed the identification of molecular correlates of vulnerability to the disease, the effect of early life stress and the response to pharmacological treatment.