Melatonin Prevents Depression but Not Anxiety-like Behavior Produced by the Chemotherapeutic Agent Temozolomide: Implication of Doublecortin Cells and Hilar Oligodendrocytes

Abstract

Melatonin is a hormone synthesized by the pineal gland with neuroprotective and neurodevelopmental effects. Also, melatonin acts as an antidepressant by modulating the generation of new neurons in the dentate gyrus of the hippocampus. The positive effects of melatonin on behavior and neural development may suggest it is used for reverting stress but also for the alterations produced by chemotherapeutic drugs influencing behavior and brain plasticity. In this sense, temozolomide, an alkylating/anti-proliferating agent used in treating brain cancer, is associated with decreased cognitive functions and depression. We hypothesized that melatonin might prevent the effects of temozolomide on depression- and anxiety-like behavior by modulating some aspects of the neurogenic process in adult Balb/C mice. Mice were treated with temozolomide (25 mg/kg) for three days of two weeks, followed by melatonin (8 mg/kg) for fourteen days. Temozolomide produced short- and long-term decrements in cell proliferation (Ki67-positive cells: 54.89% and 53.38%, respectively) and intermediate stages of the neurogenic process (doublecortin-positive cells: 68.23% and 50.08%, respectively). However, melatonin prevented the long-term effects of temozolomide with the increased number of doublecortin-positive cells (47.21%) and the immunoreactivity of 2' 3'-Cyclic-nucleotide-3 phosphodiesterase (CNPase: 82.66%), an enzyme expressed by mature oligodendrocytes, in the hilar portion of the dentate gyrus. The effects of melatonin in the temozolomide group occurred with decreased immobility in the forced swim test (45.55%) but not anxiety-like behavior. Thus, our results suggest that melatonin prevents the harmful effects of temozolomide by modulating doublecortin cells, hilar oligodendrocytes, and depression-like behavior tested in the forced swim test. Our study could point out melatonin's beneficial effects for counteracting temozolomide's side effects.

Keywords: adult neurogenesis; anxiety; depression; hippocampus; melatonin; oligodendrocytes; temozolomide.

Figure 1

Temozolomide induced anxiety- and depression-like effects, but melatonin only prevented depression-like effects. (A) Experimental design. Female Balb/C mice received two cycles of temozolomide (TMZ: 25 mg/Kg) or saline solution (control, CTR: 0.9% NaCl) for three days (1–3 and 8–10 days) for two weeks. After the last administration of TMZ or saline solution, mice received melatonin (MEL: 8 mg/Kg) or the vehicle of MEL (VEH, ethanol: saline solution) for 14 days. Thus, the behavioral tests were performed from day 26 to 30. (B,C) Open field test. For five minutes, individual rodents were placed in a plexiglass box with clean sawdust on the floor. The time spent in the field’s periphery (B) or the center (C) was quantified. (D–H) Elevated plus maze. Mice were gently put at the center of the elevated plus maze face to open arms, and for five minutes we filmed the animal behavior to analyze the number of events in the closed (D) or open (F) arms and the time spent in the closed (E) or open (G) arms. (H) The anxiety index was calculated as indicated in the Materials and Methods section. (I) Forced swim test (FST). The immobility time in the forced swim test (FST) is shown. n = 6–8. Results were analyzed with a one-way ANOVA, followed by the Bonferroni post hoc test. But, when the normality test failed a Kruskal–Wallis one-way analysis of variance on ranks followed by a Student–Newman–Keuls post hoc test was applied (panel (G), open arms time). Differences were considered statistically significant at p ≤ 0.05. Significant differences are indicated with * used for those groups which are different from the control group (* vs. CTR group); # for those groups which are different from the melatonin group (# vs. MEL group); and $ for those groups which are different from the temozolomide group ($ vs. TMZ group).

Figure 2

Temozolomide’s short- and long-term effects on cell proliferation and intermediate stages of hippocampal neurogenesis. (A) Experimental design. Female Balb/C mice received TMZ or saline solution on days 1–3 and 8–10. Mice were divided into control (CTR short-term and CTR long-term: N = 6) and TMZ (TMZ short-term, TMZ long-term: N = 6). For short-term evaluation, mice were euthanized at day 11, but for the long-term assessment mice were euthanized at day 25, and representative micrographs of Ki67 (B) or DCX (D) cells are shown. Scale bars 200 µm, respectively. Cumulative quantifications of Ki67- (C) or DCX-labeled cells (E) are shown. Results were analyzed with a two-way ANOVA, followed by the Bonferroni post hoc test. Factors were treatment and time of evaluation. Differences were considered statistically significant at p ≤ 0.05. n = 3. Significant differences are indicated with * for those groups different from the control group (* vs. CTR group) short- or long-term.

Figure 3

Melatonin prevents the effects of temozolomide on intermediate stages of hippocampal neurogenesis. Female Balb/C mice received temozolomide (TMZ) (25 mg/kg) or saline solution (0.9% NaCl) on days 1–3 and 8–10. Thus, mice were separated into four groups: control (CTR), (2) TMZ, (3) melatonin (MEL), and (4) TMZ + MEL; CTR and TMZ were injected intraperitoneally with saline solution from day 11 to 24. Mice of the MEL and TMZ + MEL groups were injected intraperitoneally with melatonin at the beginning of the dark phase of the light–dark cycle (8 mg/kg). Representative micrographs of Ki67 (A) or DCX (C) cells are shown. Scale bars 200 µm, respectively. The cumulative quantifications of Ki67 (B) or DCX (D) cells is shown. Results were analyzed with a one-way ANOVA, followed by the Bonferroni post hoc test. Differences were considered statistically significant at p ≤ 0.05. Differences were considered statistically significant at p ≤ 0.05. n = 4–7. Significant differences are indicated with * for those groups which are different from the control group (* vs. CTR group), # for those groups which are different from the melatonin group (# vs. MEL group).

Figure 4

Melatonin increases the volume of the infrapyramidal mossy fiber tract. Female Balb/C mice received temozolomide (TMZ) (25 mg/kg) or saline solution (0.9% NaCl) on days 1–3 and 8–10. Thus, mice were separated into four groups: control (CTR), (2) TMZ, (3) melatonin (MEL), and (4) TMZ + MEL; CTR and TMZ were injected intraperitoneally with saline solution from day 11 to 24. Mice of the MEL and TMZ + MEL groups were injected intraperitoneally with MEL at the beginning of the dark phase of the light–dark cycle (8 mg/kg). Representative micrographs of calbindin (A) immunoreactivity are shown. Scale bar 400 µm, respectively. The volume of the infra- and suprapyramidal mossy fiber tract is shown in (B,C) as cubic micrometers. Results were analyzed with a one-way ANOVA, followed by the Bonferroni post hoc test. Differences were considered statistically significant at p ≤ 0.05. n = 3. Significant differences are indicated with * for those groups which are different from the control group (* vs. CTR group), # for those groups which are different from the melatonin group (# vs. MEL group).

Figure 5

Melatonin prevented the decreased immunoreactivity of CNPase caused by temozolomide in the hilus of the dentate gyrus. Female Balb/C mice received temozolomide (TMZ) (25 mg/kg) or saline solution (0.9% NaCl) on days 1–3 and 8–10. Thus, mice were separated into four groups: control (CTR), (2) TMZ, (3) melatonin (MEL), and (4) TMZ + MEL; CTR and TMZ were injected intraperitoneally with saline solution from day 11 to 24. Mice of the MEL and TMZ + MEL groups were injected intraperitoneally with MEL at the beginning of the dark phase of the light–dark cycle (8 mg/kg). Representative micrographs of CNPase immunoreactivity are shown (A). Scale bar 200 µm, respectively. Immunoreactivity quantification of CNPase in the granular cell layer (GCL), the molecular layer, or in the hilus is shown (B–D) as optical density (O.D.). Results were analyzed with a one-way ANOVA, followed by the Bonferroni post hoc test. Differences were considered statistically significant at p ≤ 0.05. n = 3–4. Significant differences are indicated with * for all those groups different from the control group (* vs. CTR group), and $ indicates differences for all those groups different from the TMZ group ($ vs. TMZ group).

Figure 6

Principal component analysis. Principal component 1 (Dim1) versus 2 (Dim2) with individual dispersion are shown. Analysis included behavioral and physiological values using five behavioral and histological measurements across 12 samples and four treatments of mice divided into four groups: control (CTR), (2) TMZ, (3) melatonin (MEL), and (4) TMZ + MEL. The points correspond to each mouse’s PC1 and PC2 scores, and a 95% confidence interval defines the ellipses. Centroid ellipses are separated, indicating notable differences between treatments.

Figure 7

Model of the melatonin effects on structural dentate gyrus plasticity. The model suggests that melatonin (MEL) modulates several neurogenic events in the dentate gyrus (DG). Depending on the strain of mice, melatonin influences cell proliferation (Ki67), the intermediate stages based on the expression of doublecortin (DCX), and the increased volume of the infrapyramidal mossy fibers (Calbindin (IFPMFs)), which include the axons of new neurons projecting to the CA3. In mice treated with temozolomide (TMZ), melatonin prevents decreased Ki67 and DCX cells and the immunoreactivity for CNPase, a protein expressed in oligodendrocytes. The effects of melatonin may imply membrane receptor activation, and the regulation of cytoskeleton-associated proteins and signaling pathways. Illustration shows: RGC, radial glial cells; NPC, neural progenitor cells; IN, immature neuron; MN, mature neuron; IFPMFs, infrapyramidal mossy fibers; Mel, melatonin; TMZ: temozolomide.