Therapeutic treatment with fluoxetine using the chronic unpredictable stress model induces changes in neurotransmitters and circulating miRNAs in extracellular vesicles

Abstract

The most widely prescribed antidepressant, fluoxetine (FLX), is known for its antioxidant and anti-inflammatory effects when administered post-stress. Few studies have evaluated the effects of FLX treatment when chronic stress has induced deleterious effects in patients. Our objective was to evaluate FLX treatment (20 mg/kg/day, i.v.) once these effects are manifested, and the drug's relation to extracellular circulating microRNAs associated with inflammation, a hedonic response (sucrose intake), the forced swim test (FST), and corticosterone levels (CORT) and monoamine concentrations in limbic areas. A group of Wistar rats was divided into groups: Control; FLX; CUMS (for six weeks of exposure to chronic, unpredictable mild stress); and CUMS + FLX, a mixed group. After CUMS, the rats performed the FST, and serum levels of CORT and six microRNAs (miR-16, -21, -144, -155, -146a, -223) were analyzed, as were levels of dopamine, noradrenaline, and serotonin in the prefrontal cortex, hippocampus, and hypothalamus. CUMS reduced body weight, sucrose intake, and hippocampal noradrenaline levels, but increased CORT, immobility behavior on the FST, dopamine concentrations in the prefrontal cortex, and all miRNAs except miR-146a expression. Administering FLX during CUMS reduced CORT levels and immobility behavior on the FST and increased the expression of miR-16, -21, -146a, -223, and dopamine. FLX protects against the deleterious effects of stress by reducing CORT and has an antidepressant effect on the FST, with minimally-modified neurotransmitter levels. FLX increased the expression of miRNAs as part of the antidepressant effect. It also regulates both neuroinflammation and serotoninergic neurotransmission through miRNAs, such as the miR-16.

Keywords: Chronic unpredictable mild stress; Corticosterone; Fluoxetine; Neurotransmitters; Serotonin; miRNAs.

Fig. 1

Experimental design. The rats were assigned to the different experimental groups (CON, FLX, CUMS and CUMS + FLX). Both VCUG and VCUG + FLX groups were subjected to stress for 42 days (6 weeks). One day after the end of CUMS or FLX administration (day 43). The rats in each experimental group underwent FST for 15 min. After a period of 24 h (day 44), the test was repeated for 5 min. Finally, 72 h after completing CUMS (day 45), the rats were sacrificed by decapitation to obtain blood serum and dissect three brain structures: the hypothalamus, the hippocampus and the prefrontal cortex, to analyze the concentrations of DA, NA and 5-HT. The expression of miR-16, -21, -144, -155, -146a and -223 and the levels of CORT were analyzed in plasma and serum, respectively.

Fig. 2

Effects of CUMS and FLX on weekly body weight gain. Body weight gain in the CUMS + FLX, CUMS, and FLX groups was significantly les than in the CTRL group from the 4th week of the study. Data expressed as Means ± Standard Error (n = 6 per group), analyzed by a two-way ANOVA with a post hoc Tukey test. *p < 0.05 compared to CTRL; +p < 0.05 vs. FLX, ^ap<0.05 vs. CUMS.

Fig. 3

Effects of CUMS and FLX on the sucrose preference test showing (A) the percentage of the sucrose solution consumed in 1 h; and (B) consumption of water and sucrose. The preference rate for sucrose and total ingestion of liquid decreased significantly in the CUMS and FLX rats compared to the CTRL and FLX groups. During the final 2 weeks of stress, no difference was observed between water and sucrose consumption in the CUMS and CUMS + FLX groups. Data expressed as Means ± Standard Error (n = 6 per group), analyzed by a two-way ANOVA with a post hoc Tukey test. *p < 0.05 compared to CTRL; +p < 0.05 vs. FLX, ^ap<0.05 vs. CUMS.

Fig. 4

Effect of CUMS and FLX on the FST (A) and serum CORT levels (B). CUMS showed an increase in immobility behavior with a decrease in swimming behavior. Treatment with FLX reduced immobility behavior and increased swimming behavior. CORT increased due to the effect of CUMS, and decreased with FLX treatment. Data expressed as Means ± Standard Error (n = 6 per group). ANOVA followed by a Tukey test. *p < 0.05 compared to CTRL; +p < 0.05 vs. FLX, ^ap<0.05 vs. CUMS.

Fig. 5

Effect of CUMS and FLX on DA (A), NA (B), and 5-HT (C) levels. CUMS and FLX induced neurochemical changes in all the brain structures analyzed. An increased in the levels of NA and DA in the PFC in the CUMS and CUMS + FLX groups compared to the CTRL and FLX group. Data expressed as means ± standard error (n = 6 per group). Statistical analysis was performed by an ANOVA followed by a Tukey test. *p < 0.05 compared to CTRL; +p < 0.05 vs. FLX, ^a p < 0.05 vs. CUMS. NA = noradrenalin; DA = dopamine; 5-HT = serotonin. HYP=Hypothalamus, HIPP= Hipocampus, PFC= Prefrontal Cortex.

Fig. 6

Effect of CUMS and FLX on levels of miRNA expression. FLX induced changes in the expression of miRNAs in CUMS rats. Data expressed as Means ± Standard Error (n = 6 per group). Statistical analysis was performed by an ANOVA followed by a Tukey test. *p < 0.05 compared to CTRL; +p < 0.05 vs. FLX, ^ap<0.05 vs. CUMS.