Chronic psychosocial stress and citalopram modulate the expression of the glial proteins GFAP and NDRG2 in the hippocampus

Abstract

Rationale: It has been suggested that there are causal relationships between alterations in brain glia and major depression.

Objectives: To investigate whether a depressive-like state induces changes in brain astrocytes, we used chronic social stress in male rats, an established preclinical model of depression. Expression of two astrocytic proteins, the intermediate filament component glial fibrillary acidic protein (GFAP) and the cytoplasmic protein N-myc downregulated gene 2 (NDRG2), was analyzed in the hippocampus. For comparison, expression of the neuronal protein syntaxin-1A was also determined.

Methods: Adult male rats were subjected to daily social defeat for 5 weeks and were concomitantly treated with citalopram (30 mg/kg/day, via the drinking water) for 4 weeks.

Results: Western blot analysis showed that the chronic stress downregulated GFAP but upregulated NDRG2 protein. Citalopram did not prevent these stress effects, but the antidepressant per se downregulated GFAP and upregulated NDRG2 in nonstressed rats. In contrast, citalopram prevented the stress-induced upregulation of the neuronal protein syntaxin-1A.

Conclusions: These data suggest that chronic stress and citalopram differentially affect expression of astrocytic genes while the antidepressant drug does not prevent the stress effects. The inverse regulation of the cytoskeletal protein GFAP and the cytoplasmic protein NDRG2 indicates that the cells undergo profound metabolic changes during stress and citalopram treatment. Furthermore, the present findings indicate that a 4-week treatment with citalopram does not restore normal glial function in the hippocampus, although the behavior of the animals was normalized within this treatment period, as reported previously.

Fig. 1

Design of the chronic stress and chronic citalopram experiment. Four groups of male Wistar rats were analyzed, control, stress, control CIT, and stress CIT. After 1 week of habituation (no stress), stress animals were subjected to daily social defeat for 5 weeks. Animals in the control CIT group received the drug during 4 weeks, daily via the drinking water. Animals in the stress CIT group also received the drug during 4 weeks while the daily stress exposure was continued

Fig. 2

Effects of chronic psychosocial stress on body weight gain and adrenal weight. a Chronic social defeat reduced body weight gain in stressed rats compared to control. Weekly data are expressed as percentages of body weight at the beginning of the experiment (mean ± SEM); **P < 0.01, ***P < 0.001 significant difference to control as determined by two-way repeated measures ANOVA with Bonferroni’s post hoc test. b Stress also caused a significant increase in adrenal gland weight; **P < 0.01, significant difference between all controls (including control CIT) and all stressed animals (including stress CIT) determined with the t test. C control, S stress, SC stress CIT, CC control CIT

Fig. 3

NDRG2 colocalizes with GFAP but not with the neuronal marker protein NSE. a NDRG2 immunofluorescence (green) in an astrocyte in the stratum radiatum of the hippocampus. Please note that NDRG2 is found throughout the cytoplasm of the astrocyte. b GFAP immunofluorescence (red) in the same astrocyte as in A. c Merged picture showing NDRG2-GFAP colocalization (yellow) in the cytoplasm of the astrocyte (arrow). d Double immunofluorescence for NDRG2 (red) and NSE (green) reveals no colocalization in any neuron

Fig. 4

Effects of chronic psychosocial stress and concomitant citalopram treatment on GFAP and NDRG2 expression in the hippocampus (Western blot analysis; β-actin as reference protein). Representative blotting membranes are shown beneath the bar graphs. Results are presented as percentages of the mean control value (mean ± SEM). Statistical differences resulting from one-way ANOVA with Tukey’s multiple comparison test: *P < 0.05, **P < 0.01 vs. control. Statistical difference to control as determined with the t test: ^# P < 0.05; ^### P < 0.001. C control, S stress, SC stress citalopram, CC control citalopram

Fig. 5

Representative images of NDRG2-immunoreactive astrocytes in hippocampal regions stratum radiatum and stratum oriens, in a control (a, c) and a stressed rat (b, d)

Fig. 6

Quantification of NDRG2-positive cells in hippocampal region CA1. a Schematic illustrating that labeled cells (asterisks) were counted in areas of approximately 90,000 μm² in the stratum oriens and 185,000 μm² in the stratum radiatum. b No significant differences with respect to the densities of NDRG2-positive cells in stress and control were observed. Results are presented as numbers of immunopositive cells/μm², multiplied by 1,000 (mean ± SEM)

Fig. 7

Effects of chronic psychosocial stress and concomitant citalopram treatment on syntaxin-1A expression in the hippocampus (Western blot analysis; β-actin as reference protein). Representative blotting membranes are shown beneath the bar graph. Results are presented as percentages of the mean control value (mean ± SEM). Statistical differences resulting from one-way with Tukey’s multiple comparison test: *P < 0.05 vs. control; ⁺ P < 0.05, ⁺⁺⁺ P < 0.001, vs. stress. C control, S stress, SC stress CIT, CC control CIT