Electroconvulsive seizures regulate various stages of hippocampal cell genesis and mBDNF at different times after treatment in adolescent and adult rats of both sexes

Abstract

Electroconvulsive therapy, a fast-acting option for treatment-resistant depression, is modeled at the preclinical level through the induction of electroconvulsive seizures (ECS) in rodents. Recent studies from our group proved sex- and age-differences in the antidepressant-like response elicited by ECS in rats; while an antidepressant-like response was observed in male adolescent and adult rats (although with greater efficacy in adulthood), the same parameters rendered inefficacious in females of any age. To better understand the potential sex differences taking place at the molecular level that might be mediating these behavioral disparities, we evaluated the impact of a repeated treatment with ECS (95 mA for 0.6 s, 100 Hz, 0.6 ms) in adolescent and adult rats of both sexes. Several hippocampal markers of neuroplasticity, commonly regulated by most antidepressants, such as those of neurogenesis (cell proliferation, neurogenic differentiation, long-term cell survival) or mBDNF and associated signaling (e.g., mTOR and ERK1/2) were evaluated at different time-points after treatment (1-, 8-, 15- and up to 30-days post-treatment). The main results demonstrated that ECS improved the survival rate of new cells born in the dentate gryus before treatment. Moreover, ECS increased cell proliferation and neurogenic differentiation at different times post-treatment, paired with persistent increases in mBDNF, observed long after treatment. In general, effects were different for each sex and varied with the age of the animal (adolescent vs. adulthood). The present study is the first-one to demonstrate that such persistent molecular changes induced by ECS in hippocampus, some of them observed up to 30-days post-treatment, also occurred in female rats and adolescence. Although these molecular changes could not justify the lack of ECS efficacy described by these same parameters of ECS in female rats (vs. male rats), they proposed certain beneficial effects common to both sexes, and age periods studied, opening the avenue for further studies. Based on these neurochemical effects, ECS should have displayed similar efficacies for both biological sexes. Therefore, the reason behind these disparities should be further explored to better translate efficacious treatments specific and/or personalized for each sex to the clinic.

Keywords: BDNF; BrdU; ERK1/2; Ki-67; NeuroD; mTOR; neurogenesis.

Figure 1

(A) Diagram of hippocampal markers selected for evaluation: 1. Neurogenesis markers (i.e., Ki-67, NeuroD, BrdU); 2. Other neuroplasticity makers (i.e., BDNF, p-mTOR/mTOR, p-ERK1/2/ERK1/2). (B) Experimental timeline in adolescent rats. (C) Experimental timeline in adult rats. BDNF, brain-derived neurotrophic factor; BrdU, 5-bromo-2′-deoxyuridine; ECS, electroconvulsive seizures; ERK, extracellular signal-regulated kinase; d, day; mTOR, mammalian target of rapamycin; NSC, neural stem cells; PND, post-natal day; TrkB, tropomyosin receptor kinase B.

Figure 2

Modulation of hippocampal cell survival (BrdU +cells) by repeated ECS treatment in male (A) and female (B) adolescent rats as measured 1-day and 8-days after treatment, or in male (C) and female (D) adult rats as measured 1-, 8-, 15- and up to 30-days post-treatment. Data represent mean ± SEM of BrdU total estimated +cells in the dentate gyrus as measured by immunohistochemistry analysis. Individual values are shown for each rat (symbols). Pair comparisons for each sex and age of study were done by Student’s t-tests: *p < 0.05 and **p < 0.01 vs. SHAM rats. Representative images of BrdU + cells (dark brown labeling in a lighter granular layer background), taken with a light microscope and quantified with a 63x objective lens, are shown next to the bar graphs.

Figure 3

Modulation of hippocampal (A) cell proliferation (Ki-67 + cells) and (B) neurogenic differentiation (NeuroD +cells) by repeated ECS treatment in female adult rats as measured 1-, 8-, 15- and up to 30-days post-treatment. Data represent mean ± SEM of the number of total estimated +cells in the dentate gyrus as measured by immunohistochemistry analysis. Individual values are shown for each rat (symbols). Pair comparisons for each sex and age of study were done by Student’s t-tests: *p < 0.05, **p < 0.01 and ***p < 0.001 vs. SHAM rats. Representative images of (A) Ki-67 + cells (dark brown labeling in a blue granular layer background) or (B) NeuroD +cells (dark blue labeling in a lighter blue granular layer background), taken with a light microscope and quantified with a 63x objective lens, are shown below the bar graphs.

Figure 4

Modulation of hippocampal mBDNF protein content by repeated ECS treatment in male (A) and female (B) adolescent rats as measured 1-day and 8-days after treatment, or in male (C) and female (D) adult rats as measured 1-, 8-, 15- and up to 30-days post-treatment. Data represent mean ± SEM of mBDNF protein content expressed as % change vs. SHAM-treated control rats at each particular time and as ascertained by western blot analysis. Individual values are shown for each rat (symbols). Pair comparisons for each sex and age of study were done by Student’s t-tests: *p < 0.05, **p < 0.01 and ***p < 0.001 vs. SHAM rats. Representative immunoblots depicting mBDNF and β-actin (as a loading control) are shown below each bar graph.

Figure 5

Evaluation of the protein content of certain neuroplasticity markers in hippocampus following repeated ECS treatment by western blot analysis. (A) p-ERK1/2/ERK1/2 and (B) p-mTOR/mTOR in male and female adolescent rats as measured 1-day and 8-days after treatment. (C) p-ERK1/2/ERK1/2 and (D) p-mTOR/mTOR in male and female adult rats as measured 1-, 8-, 15- and up to 30-days post-treatment. Data represents mean ± SEM of the ratio between the phosphorylated vs. total form of the protein evaluated and expressed as % change vs. SHAM-treated control rats at each particular time. Individual rates are shown for each rat (symbols). Pair comparisons for each sex and age of study were done by Student’s t-tests: **p < 0.01 vs. SHAM rats. Representative immunoblots depicting p-ERK1/2, ERK1/2, p-mTOR and mTOR are shown below each bar graph.