Adolescent fluoxetine treatment mediates a persistent anxiety-like outcome in female C57BL/6 mice that is ameliorated by fluoxetine re-exposure in adulthood

Abstract

The objective of this study was to evaluate whether juvenile fluoxetine (FLX) exposure induces long-term changes in baseline responses to anxiety-inducing environments, and if so, whether its re-exposure in adulthood would ameliorate this anxiety-like phenotype. An additional goal was to assess the impact of adolescent FLX pretreatment, and its re-exposure in adulthood, on serotonin transporters (5-HTT) and brain-derived-neurotrophic-factor (BDNF)-related signaling markers (TrkB-ERK1/2-CREB-proBDNF-mBDNF) within the hippocampus and prefrontal cortex. To do this, female C57BL/6 mice were exposed to FLX in drinking water during postnatal-days (PD) 35-49. After a 21-day washout-period (PD70), mice were either euthanized (tissue collection) or evaluated on anxiety-related tests (open field, light/dark box, elevated plus-maze). Juvenile FLX history resulted in a persistent avoidance-like profile, along with decreases in BDNF-signaling markers, but not 5-HTTs or TrkB receptors, within both brain regions. Interestingly, FLX re-exposure in adulthood reversed the enduring FLX-induced anxiety-related responses across all behavioral tasks, while restoring ERK2-CREB-proBDNF markers to control levels and increasing mBDNF within the prefrontal cortex, but not the hippocampus. Collectively, these results indicate that adolescent FLX history mediates neurobehavioral adaptations that endure into adulthood, which are indicative of a generalized anxiety-like phenotype, and that this persistent effect is ameliorated by later-life FLX re-exposure, in a prefrontal cortex-specific manner.

Figure 1

Timeline of juvenile fluoxetine (FLX) treatment and experimental procedures. (A) Postnatal day (PD)-28 female C57BL/6 mice arrived to our animal colony. One week later (PD35), they were randomly assigned to receive FLX in their drinking water (250 mg/l) for 15 consecutive days (PD35-49), or water alone (vehicle, VEH). After a 21-day washout period (PD70), separate groups of mice were tested on the open field test (OFT), light/dark box (LDB), elevated plus maze (EPM), or euthanized for tissue extraction (Long-term groups). (B) Separate groups of PD28 mice arrived to our animal colony. One-week later (PD35) they were exposed to VEH or FLX in their drinking water (PD35-49). After a 21-day washout period (PD70), FLX or VEH was re-administered for two additional weeks (PD70-84). Twenty-four h after FLX or VEH re-exposure (PD85), mice were either tested on the OFT, LDB, EPM, or euthanized for tissue extraction (Re-exposure groups). (C) Blood was collected from a separate group of animals on PD49 to evaluate serum FLX concentrations after exposure to FLX in their drinking water for 15 days.

Figure 2

Long-term effects of juvenile fluoxetine (FLX) treatment, and its re-exposure in adulthood, on the open field test. (A) FLX pretreatment during adolescence (PD35-49) resulted in a decrease in the time spent in the center area of the open field arena at PD70 (Long-Term). (B) FLX pretreatment did not influence total distance traveled during the 5-min session of the open field test. (C) Re-exposure to FLX in adulthood (PD70-84) ameliorated the long-lasting anxiety-like effect of adolescent FLX pre-exposure, as no differences in total time spent in the center between the groups were observed. (D) At PD85, no differences in distance traveled were apparent between the experimental groups. Data are presented as mean ± SEM. *p < 0.05.

Figure 3

Long-term effects of juvenile fluoxetine (FLX) treatment, and its re-exposure in adulthood, on the light/dark box. (A) Juvenile FLX pretreatment (PD35-49) led to a significantly decreased time spent in the lighted-side of the light/dark box at PD70 (Long-Term), when compared to water (VEH)-pretreated controls. (B) When compared to VEH-pretreated controls, juvenile FLX pretreatment did not influence the time it took the animals to first enter the lighted-side of the apparatus. (C) FLX re-exposure in adulthood (PD70-84) ameliorated the long-lasting anxiety-like effect of juvenile FLX pretreatment, as no differences in total time spent in the center between the groups were observed. (D) At PD85, no differences in the time it took for animals to first enter the lighted-side of the apparatus were apparent between the experimental groups. Data are presented as mean ± SEM. *p < 0.05.

Figure 4

Long-term effects of juvenile fluoxetine (FLX) treatment, and its re-exposure in adulthood, on the elevated plus maze. (A) Adolescent FLX pretreatment (PD35-49) resulted in a significant increase in time spent in the closed arms of the maze at PD70 (Long-Term), when compared to water (VEH)-treated controls. (B) No differences in distance traveled during the 5-min test were observed between the groups. (C) Re-exposure of FLX in adulthood (PD70-84) ameliorated the long-lasting anxiety-like effect of juvenile FLX pre-exposure since no differences in total time spent in the closed arms were noted between the groups (PD85). (D) No differences in distance traveled were observed between the experimental groups. Data are presented as mean ± SEM. *p < 0.05.

Figure 5

Long-term effects of juvenile FLX treatment, and its re-exposure in adulthood, on ERK1/2, CREB, BDNF TrkB, and 5-HTT proteins in the hippocampus of female C57BL/6 mice. (A) Adolescent FLX pretreatment (PD35-49) significantly decreased phosphorylated (p) ERK1, (p)ERK2, and (p)CREB without altering total (t) proBDNF or mBDNF at PD70 (Long-term), when compared to water (VEH)-treated controls. (B) No differences in (t)ERK1/2, (t)CREB, (t)TrkB, or (t)5-HTT were noted between the groups 21-days after adolescent FLX pretreatment. (C) Re-exposure to FLX in adulthood (PD70-84), in female mice with adolescent FLX history, decreased phosphorylated (p) levels of ERK1, ERK2, and CREB, with no differences in total proBDNF or mBDNF, 24 h after the end of treatment (PD85). (D) No differences in total ERK1/2, CREB, TrkB, or 5-HTT were observed on PD85, as a function of juvenile FLX exposure (PD35-49) and re-exposure in adulthood (PD70-84). Western blots of hippocampal tissue, with α-Tubulin as loading control, were performed under identical protocols. Representative images were cropped from different blots (Supplemental Figure S1). Data are presented as mean ± SEM. *p < 0.05.

Figure 6

Long-term effects of juvenile FLX treatment, and its re-exposure in adulthood, on ERK1/2, CREB, BDNF TrkB, and 5-HTT proteins in the prefrontal cortex of female C57BL/6 mice. (A) Adolescent FLX pretreatment (PD35-49) resulted in significant decreases in phosphorylated (p) ERK2, (p)CREB, (t)proBDNF, and (t)mBDNF at PD70 (Long-term), when compared to water (VEH)-treated controls (B) No significant differences in total protein levels of ERK1/2, CREB, TrkB were apparent 21-days after juvenile FLX treatment. (C) Re-exposure to FLX in adulthood (PD70-84) normalized (p)ERK, (p)CREB, and (t)proBDNF, and significantly increased (t)mBDNF, 24 h after the end of antidepressant re-exposure (PD85). (D) No differences in (t)ERK1/2, (t)CREB, (t)TrkB, or (t)5-HTT were observed on PD85, as a function of juvenile FLX exposure (PD35-49) and re-exposure in adulthood (PD70-84). Western blots of prefrontal cortex tissue, with α-Tubulin as loading control, were performed under identical protocols. Representative images were cropped from different blots (Supplemental Figure S2). Data are presented as mean ± SEM. *p < 0.05.

Figure 7

Plasma fluoxetine levels. Plasma fluoxetine levels are shown in ng/ml for adolescent (postnatal day 49) female C57BL/6 mice receiving 250 mg/l of fluoxetine in their drinking water (for 15 days). The gray area displays plasma fluoxetine levels in humans taking 20–80 mg fluoxetine per day^,.