Electroconvulsive seizure and VEGF increase the proliferation of neural stem-like cells in rat hippocampus

Abstract

All classes of antidepressants increase hippocampal cell proliferation and neurogenesis, which contributes, in part, to the behavioral actions of these treatments. Among antidepressant treatments, electroconvulsive seizure (ECS) is the most robust stimulator of hippocampal cell proliferation and the most efficacious treatment for depression, but the cellular mechanisms underlying the actions of ECS are unknown. To address this question, we investigated the effect of ECS on proliferation of neural stem-like and/or progenitor cells in the subgranular zone of rat dentate gyrus. We define the neural differentiation cascade from stem-like cells to early neural progenitors (also referred to as quiescent and amplifying neural progenitors, respectively) by coexpression of selective cellular and mitotic activity markers. We find that at an early mitotic phase ECS increases the proliferation of quiescent progenitors and then at a later phase increases the proliferation of amplifying progenitors. We further demonstrate that vascular endothelial growth factor (VEGF) signaling is necessary for ECS induction of quiescent neural progenitor cell proliferation and is sufficient to produce this effect. These findings demonstrate that ECS and subsequent induction of VEGF stimulates the proliferation of neural stem-like cells and neural progenitor cells, thereby accounting for the superior neurogenic actions of ECS compared with chemical antidepressants.

Fig. 1.

Analysis of dividing cells using markers for QNPs and ANPs in rat SGZ. Staining for Sox-2 (blue) is used as a marker for both QNPs and ANPs. Staining for GFAP (green) is used as a marker for QNPs. Staining for BrdU (red) is used to measure mitotic activity. (A) Confocal micrographs of BrdU-labeled Sox-2⁺ and GFAP⁺ QNPs (BrdU⁺QNP) in rat SGZ 2 h after BrdU injection (yellow arrowheads). (Left) Low magnification photomicrograph of hilus and SGZ, which borders the hilus and the granule cell layer (GCL). Among QNPs within SGZ, only a few cells were BrdU⁺ (indicated by arrowhead). Astrocytes also show Sox-2⁺ and GFAP⁺ immunostaining, but most astrocytes in hilus exhibit stellate morphology (white arrows). (Right) Magnification of each square. (B–D) High magnifications of BrdU-labeled Sox-2⁺ but GFAP-negative ANPs (BrdU⁺ANP; white arrowheads) and BrdU⁺QNP (yellow arrowheads). The orthogonal projections are shown to confirm triple or double labeling throughout the cells. (Scale bars: A, 20 μm; B–D, 5 μm.)

Fig. 2.

ECS increases the proliferation of QNPs 2 h after BrdU labeling. (A) Experimental schema. Rats received injections of BrdU 48 h after sham or a single ECS and were killed 2 h later. (B) Representative coronal sections through the GCL with BrdU-labeled cells are shown from sham and single ECS. (Scale bars: 250 μm.) (C–E) Quantification of BrdU⁺ and Sox-2⁺ cells (C); BrdU⁺, Sox-2⁺, and GFAP⁺ triple-labeled (BrdU⁺QNP) cells (D); and BrdU⁺, Sox-2⁺, but GFAP-negative (BrdU⁺ANP) cells (E) in SGZ of hippocampus after a single ECS compared with sham-handled controls. Data are shown as the means ± SEM, n = 6 for each group; *, P < 0.05; n.s., P > 0.05.

Fig. 3.

ECS increases the proliferation of both QNPs and ANPs 24 h after BrdU labeling. (A) Experimental schema. Arrowheads show ECS treatment. ECS-S, single ECS; ECS-D7, multiple ECS, (seven times, once a day). Control group was sham-handled same as ECS-D7 group but without ECS. Rats received injections of BrdU after single or multiple ECS treatments, and all animals were killed 24 h later. (B) Representative coronal sections through the GCL with BrdU-labeled cells, from sham, ECS-S and ECS-D7. (Scale bars: 250 μm.) (C–E) Quantification of BrdU⁺ and Sox-2⁺cells (C); BrdU⁺, Sox-2⁺, and GFAP⁺ triple-labeled (BrdU⁺QNP) cells (D); and BrdU⁺, Sox-2⁺, but GFAP-negative (BrdU⁺ANP) cells (E) in SGZ of hippocampus after a single or multiple ECS compared with sham-handled controls. Data are shown as the means ± SEM., n = 6 per group; *, P < 0.05; one-way ANOVA, followed by the post hoc Bonferroni test.

Fig. 4.

Role of VEGF-Flk-1 signaling in the regulation of QNP and ANP proliferation in rat DG. (A–C) Experimental schema. (A) Recombinant rat VEGF₁₆₄ was infused i.c.v. for 3 days via osmotic minipump, with or without irradiation (IRR) exposure. (B) Flk-1 inhibitor SU5416 was delivered (i.c.v.) 30 min before ECS, followed by three daily infusions. (C) VEGF₁₆₄ was continuously infused (i.c.v.) for 24 h via osmotic minipump. All animals were injected with BrdU at the indicated time point and killed 2 h after injection. (D–F) VEGF increases the proliferation of both QNPs and ANPs with or without irradiation exposure. Note that irradiation exposure preferentially reduced proliferation of the ANP population. (G–I) VEGF-Flk-1 signaling mediates ECS-induced QNP and ANP proliferation. (J–L) VEGF administration for 24 h preferentially increases the proliferation of QNPs. VEH, vehicle. Data are shown as the means ± SEM, n = 4–6 per group; *, P < 0.05 compared with vehicle-infused controls. One-way ANOVA, followed by Bonferroni (D–F and J), t test (F–H) or Kruskal–Wallis followed by Dunn's multiple comparison post hoc test (K) were used. #, P < 0.05; compared with nonirradiation controls; two-way ANOVA, followed by Bonferroni post hoc test (B–D).

Fig. 5.

Expression of VEGF and Flk-1 in rat hippocampus. (A) (Left and Center) VEGF (green) and GFAP (red) immunoreactivity (IR) in rat hilus and GCL. VEGF-IR in hilus is mainly colocalized with GFAP. (Scale bars: 50 μm.) (Right) VEGF expression in the GCL shows a punctuate appearance. (Scale bar: 25 μm.) (B and C) Representative autoradiogram of Flk-1 mRNA expression (B). (Scale bar: 1 mm.) and Flk-1-IR (C). [Scale bars: 20 μm (Right) and 10 μm (Left).] (D–G) Confocal images of Flk-1-IR (green) and (D) the endothelial marker RECA (red), (E) the neural stem-like and progenitor cell marker Sox-2, (F) the neural stem-like or astrocytic marker GFAP (red), or (G) the cell proliferation marker Ki67 (red), and merged images (Right). Arrows in E show colocalization of Flk-1-IR and Sox-2-IR in SGZ. Arrow in F shows an Flk-1-expressing cell in the SGZ, which has a process through the DG that is visualized with GFAP. Arrows in G show colocalization of Flk-1-IR and Ki67-IR in the SGZ. [Scale bars: 20 μm except magnified panels (Right, 10 μm).]

Fig. 6.

Model of ECS induction of QNPs. Single or repeated ECS increases VEGF expression and signaling, thereby leading to induction of QNP cell proliferation. The induction of ANP cell proliferation may occur via asymmetric division of QNPs, or via direct effects of VEGF-Flk-1 signaling on ANP cells. This contrasts with the induction of ANP but not QNP cell proliferation in response to chronic fluoxetine administration.