Imipramine and Venlafaxine Differentially Affect Primary Glial Cultures of Prenatally Stressed Rats

Abstract

Here, we examine the effects of prenatal administration of two antidepressants-imipramine (IMI) and venlafaxine (VEN)-on morphology and activity of a primary glial culture. Microglia are targeted by antidepressants used for antenatal depression and are important regulators of central nervous system development. In this study, female Wistar rats were assigned to one of four groups: a control group that received water ad libitum (1), and groups that received additionally once daily either water (2), IMI (10 mg/kg) (3), or VEN (20 mg/kg) (4) by oral gavage from gestation day 7 to 22. Oral gavage administration induced prenatal stress. Cell cultures were obtained from the brains of 1-day-old pups. Prenatal stress caused a disturbance of sensorimotor function in pups. Prenatal stress also produced alterations in the glial cultures, specifically, an increased percentage of microglia in the mixed glial cultures and an increased percentage of dead cells. Moreover, increased levels of IL1-β, TNF-α, NO, and an increased expression of CX3CR1 mRNA were found in microglia. However, the ratio of Bax/Bcl2 mRNA was reduced. Prenatal stress increased the vulnerability of microglia to lipopolysaccharide (LPS). The mixed glial culture derived from pups exposed to IMI showed greater morphological changes and the highest percentage of microglia. Microglia were characterized by the largest increase in the production of pro-inflammatory cytokines and NO, and the greatest reduction in the expression of CX3CR1 mRNA. Exposure to IMI reduced the effects of LPS on IL-1β production and Bax/Bcl2 mRNA, and exacerbated the effects of LPS on CX3CR1 mRNA expression. Prenatal administration of VEN induced protective effects on microglia, as measured by all studied parameters. Taken together, our data suggest that, by disturbing microglia function, exposure to even mild forms of chronic prenatal stress may predispose individuals to psychiatric or neurodevelopmental disorders. These data also indicate that chronic mild stress sensitizes microglia to immune challenges, which may lead to enhanced neuronal damage in the embryonic brain. The observed detrimental effects of IMI on microglial activity under conditions of prenatal stress may help to explain the teratogenic effects of IMI reported in the literature.

Keywords: imipramine; prenatal; primary glial cell cultures; rat; stress; venlafaxine.

Figure 1

Effects of prenatal stress and administration of imipramine (IMI) or venlafaxine (VEN) during pregnancy on morphology of mixed glial and a microglia culture. IMI and VEN were administered via oral gavage. Representative images of mixed glial culture on day 8 in the bright field (using cell analyzer JuLI, magnification 20×) are shown for (A) control group, (D) prenatal stress group, (G) stress+IMI group, and (J) stress+VEN group. Representative phase contrast images were taken on microglia separated from the mixed glial culture on day 13. Cellular nuclei were stained using Hoechst 33342 (blue), using a fluorescent microscope (Nikon TS-100F, magnification 40×): (B) control group, (E) prenatal stress group, (H) stress+IMI group, and (K) stress+VEN group. Microglial cells were stained with fluorescein conjugated agglutinin-1 (R. communis), which is a marker of surface glycoproteins on microglia (green), using a fluorescent microscope (Nikon TS-100F, magnification 40×): (C) control group, (F) prenatal stress group, (I) stress+IMI group, and (L) stress+VEN group. For interpretation of the references to color in this figure legend, please see the web version of this article.

Figure 2

The effects of exposure to imipramine (IMI) or venlafaxine (VEN) combined with prenatal stress on IL-1β and IL-1β mRNA (A), TNF-α and TNF-α mRNA (B), and NO and iNOS mRNA (C) in primary microglial cell culture. IMI (10 mg/kg) or VEN (20 mg/kg) was administered once daily by oral gavage from gestational day (GD) 7 to GD 22. Cultures were prepared from the brains of pups delivered by dams from one of four groups: (1) drinking water during pregnancy (control group); (2) receiving water (prenatal stress group), (3) IMI (stress+IMI group), or (4) VEN (stress+VEN group) additionally once daily by oral gavage. Data are presented as mean ± SEM from three independent experiments (n = 9). Data were analyzed using a one-way ANOVA followed by Bonferroni post hoc test or by Kruskal–Wallis test followed by Dunn's test (NO level in culture supernatants); ^*p < 0.05, ****p < 0.0001 vs. control group; ⁺p < 0.05, ⁺⁺p < 0.01, ⁺⁺⁺⁺p < 0.0001 vs. prenatally stressed group.

Figure 3

The effects of exposure to imipramine (IMI) or venlafaxine (VEN) combined with prenatal stress on LPS-stimulated IL-1β and IL-1β mRNA (A), TNF-α and TNF-α mRNA (B), NO and iNOS mRNA (C), in primary microglial cell cultures. Experimental details are the same as in Figure 2. Briefly, cultures were exposed to LPS (1 µg/ml) to induce TNF-α release (for 6 h), IL-1β and NO release (for 24 h), or to stimulate TNF-α mRNA expression (for 4 h), or IL-1β or iNOS mRNA (for 12 h). Data are presented as mean ± SEM from three independent experiments (n = 9). Data were analyzed using a Kruskal–Wallis test followed by Dunn's test or with one-way ANOVA followed by a Bonferroni post hoc test (iNOS mRNA expression); ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 vs control+LPS group; ^&p < 0.05, ^&&p < 0.01, ^&&&p < 0.001, ^&&&&p < 0.0001 vs. prenatally stressed +LPS group.

Figure 4

The effects of exposure to imipramine (IMI) or venlafaxine (VEN) combined with prenatal stress on the ratio of Bax/Bcl2 mRNA (A), and the ratio of Bax/Bcl2 mRNA following LPS induction (B). Details of the experimental procedure are shown in Figure 2. Microglial cultures were exposed to LPS (1 µg/ml) for 12 h. Data are presented as mean ± SEM from three independent experiments (n = 9). Results were analyzed using a Kruskal–Wallis test followed by Dunn's test; **p < 0.01 vs. control group; ⁺⁺⁺⁺p < 0.0001 vs. prenatally stressed group; ^##p < 0.01 vs. control+LPS group; ^&&&&p < 0.0001 vs. prenatally stressed +LPS group.

Figure 5

The effects of exposure to imipramine (IMI) or venlafaxine (VEN) combined with prenatal stress on the CX3CR1 mRNA (A), and CX3CR1 mRNA following LPS induction (B). The primary microglial cell cultures were exposed to LPS (1 µg/ml) for 12 h. Experimental details are provided in Figure 2. Data are presented as mean ± SEM from three independent experiments (n = 9). Data were analyzed using a Kruskal–Wallis test followed by Dunn's test. The effects of LPS on CX3CR1 mRNA expression were examined using a one way-ANOVA followed by Bonferroni post hoc test; * p < 0.05, ^****p < 0.0001 vs. control group; ⁺⁺⁺⁺p < 0.0001 vs. prenatally stressed group; ^####p < 0.0001 vs. control+LPS group; ^&&&&p < 0.0001 vs. prenatally stressed+LPS group.