VEGF is an essential mediator of the neurogenic and behavioral actions of antidepressants

Abstract

The neural mechanisms underlying the cellular and behavioral responses to antidepressants are not yet known. Up-regulation of growth factors and adult neurogenesis suggest a role for one or more of these factors in the action of antidepressants. One candidate of interest is vascular endothelial growth factor (VEGF), which was initially characterized for its role in angiogenesis, but also exerts direct mitogenic effects on neural progenitors in vitro. Results of this study demonstrate that VEGF is induced by multiple classes of antidepressants at time points consistent with the induction of cell proliferation and therapeutic action of these treatments. We find that VEGF signaling through the Flk-1 receptor is required for antidepressant-induced cell proliferation. We also show that VEGF-Flk-1 signaling is required and sufficient for behavioral responses in two chronic and two subchronic antidepressant models. Taken together, these studies identify VEGF and VEGF-Flk-1 signaling as mediators of antidepressant actions and potential targets for therapeutic intervention.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Hippocampal VEGF expression is increased by fluoxetine, desipramine, and ECS. VEGF mRNA expression was determined by ISH analysis. (A and B) Representative autoradiograms illustrate induction of VEGF mRNA in the dentate gyrus granule cell layer (DG), CA3 and CA1 pyramidal cell layers of the hippocampus 6 h after sham (A) or ECS treatment (B). The graphs show optical density values as a percent of sham-handled controls. (C) The time course of VEGF mRNA induction was quantified in rats receiving a single ECS [DG, F(3, 12) = 7.612, P < 0.01; CA3, F(3, 12) = 0.608, n.s.; CA1, F(3, 12) = 1.970, P > 0.05]. (D) Quantification of VEGF protein levels in whole hippocampus homogenates, determined by ELISA after a single ECS [F(2, 14) = 6.965, P < 0.01]. (E) VEGF mRNA expression was quantified following 5 or 14 days of fluoxetine treatment [FLX-14d: DG, F(1, 6) = 6.211, P < 0.05; CA3, F(1, 6) = 15.272, P < 0.01; CA1, F(1, 6) = 32.514, P < 0.01; FLX-5d: DG, F(1, 8) = 0.001, n.s.; CA3, F(1, 8) = 0.093, n.s.; CA1, F(1, 8) = 2.206, n.s.]. (G) VEGF mRNA induction by 14 days of desipramine [DMI-14d: DG, F(1, 10) = 4.261, P = 0.06; CA3, F(1, 10) = 0.532, n.s.; CA1, F(1, 10) = 2.901, P = 0.1]. VEGF protein levels after fluoxetine (F) or desipramine (H) treatment [FLX-14d, F(1, 8) = 5.247, P ≤ 0.05; FLX-5d, F(1, 8) = 0.231, n.s.; DMI-14d, F(1, 10) = 16.042, P < 0.01; n = 4–7 per group in each experiment]. ∗, P ≤ 0.05; ∗∗, P ≤ 0.01; +, P ≤ 0.1; n.s., P > 0.1.

Fig. 2.

Antidepressant-induced cell proliferation in the SGZ is mediated by VEGF-Flk-1 signaling. Immunohistochemical visualization of BrdU was followed by confocal analysis and z-sectioning to identify newborn endothelial cells. (A) Representative coronal section through the dentate gyrus with immunoperoxidase labeled BrdU (black arrowheads). (Scale bar, 50 μm.) (B) Representative colabeled cell in the SGZ labeled expressing both BrdU (red) and the endothelial cell marker RECA (green) at 63× magnification. (Scale bar, 10 μm.) (C) Quantification of BrdU-positive cells in the SGZ of the hippocampus after chronic fluoxetine injections (14 days) compared with saline controls [F(1, 8) = 34.129, P < 0.01]. (D) BrdU-positive cells 24 or 72 h after a single ECS compared with sham-handled controls [F(2, 9) = 24.575, P < 0.01]. Quantification of BrdU/RECA-positive cells following fluoxetine treatment (E) [F(1, 8) = 18.289, P < 0.01] or ECS (F) [F(2, 9) = 6.358, P < 0.05; n = 4–5 per group in each experiment]. ∗, P ≤ 0.05; ∗∗, P ≤ 0.01; +, P ≤ 0.1; n.s., P > 0.1.

Fig. 3.

VEGF-Flk-1 signaling is required for the induction of cell proliferation by antidepressants. (A) The graph represents the number of BrdU-positive cells in the SGZ of rats receiving sham or ECS and i.c.v. infusions of vehicle or SU5416 [main effect ECS: F(1, 22) = 13.376; P < 0.01; main effect SU5416: F(1, 22) = 12.629; P < 0.01; ECS × SU5416: F(1, 22) = 6.644; P < 0.05; n = 6–8 per group]. The same experiment was performed by using SU1498 [main effect ECS: F (1, 23) = 21.249; P < 0.01; SU1498 F (1, 23) = 0.316, n.s.; ECS × SU1498: F(1, 23) = 3.123; P = 0.09; n = 5–8 per group] and with a kinase inhibitor that does not block Flk-1 signaling, K252a [main effect ECS: F(1, 10) = 28.038, P < 0.01; K252a: F(1, 10) = 1.998; n.s.; K252a × ECS: F(1, 10) = 0.196; n.s.; n = 3–4 per group]. (B) Representative image of a cell in the SGZ coexpressing BrdU (red) and early neuronal marker TUJ-1 (green) (white arrow) and BrdU-positive, TUJ-1-negative cells (arrowhead). (Scale bar, 10 μm.) (C) Percentage of 40 BrdU-positive cells that coexpress TUJ-1 (n.s.; n = 5 per group). (D) Graph illustrating the number of BrdU-positive cells counted per SGZ in animals treated for 14 days with fluoxetine or desipramine and SU5416 or vehicle infusions on days 8, 10, 12, and 14 [main effect AD: F(2, 30) = 2.94, P = 0.06; main effect SU5416: F(1, 30) = 16.277, P < 0.01; AD × SU5416: F(2, 30) = 1.521, n.s.; n = 5–11 per group]. ∗, P ≤ 0.05; ∗∗, P ≤ 0.01; +, P ≤ 0.1; n.s., P > 0.1.

Fig. 4.

VEGF mediates behavioral responses to chronic antidepressant treatment. For NSF, the latency to feed (maximum of 6 min) is quantified in (A) for animals receiving SU5416 or vehicle infusions and injections of saline or desipramine [main effect DMI: F(1, 27) = 20.033, P < 0.01; main effect SU5416: F(1, 27) = 3.9725, P = 0.05), and in (B) for animals infused with recombinant VEGF [F(1, 10) = 4.639, P = 0.05]. Treatments had no effect on home-cage feeding for a 6-min period (C and D) (n = 5–6 per group). For the chronic stress paradigm, a sucrose preference test was performed after 4 weeks of CUS exposure and antidepressant treatment. (E) Graph represents the average sucrose consumption over three 1-h tests [main effect DMI: F(1, 28) = 2.243, P = 0.1; SU5416: F(1, 28) = 7.871, P < 0.01; DMI × SU5416: F(1, 28) = 3.849, P = 0.05]. Water consumption was not significantly affected by treatments (F) (n = 5–11 per group). ∗, P ≤ 0.05; ∗∗, P ≤ 0.01; +, P ≤ 0.1; n.s., P > 0.1.

Fig. 5.

VEGF-Flk-1 receptor signaling is required and sufficient for subchronic behavioral responses to desipramine. (A–B) Following LH training, rats received desipramine/saline and infusions of SU5416/vehicle and were tested in LH and FST. Quantification of escape failures and escape latency in (A). [Failures: main effect DMI: F(1, 28) = 13.790, P < 0.01; main effect SU5416: F(1, 28) = 5.062, P < 0.05; DMI × SU5416: F(1, 28) = 1.623, n.s.; Latency: main effect DMI: F(1, 28) = 9.725, P < 0.01; main effect SU5416: F(1, 28) = 3.681, P = 0.06; DMI × SU5416: F(1, 28) = 0.702, n.s.] The FST consisted of one 15-min session, and behaviors were scored by using a sampling technique described in Materials and Methods. (B) Quantification of immobility and climbing behaviors (see Results for swimming) [Immobility: main effect DMI: F(1, 18) = 7.355, P < 0.05; main effect SU5416: F(1, 18) = 0.926, n.s.; DMI × SU5416: F(1, 18) = 11.791, P < 0.01; Climbing: main effect DMI: F(1, 18) = 29.443, P < 0.01; main effect SU5416: F(1, 18) = 4.860, P < 0.05; DMI × SU5416: F(1, 18) = 4.1974, P = 0.05]. Learned helplessness training was followed by continuous i.c.v. infusions of recombinant VEGF164 (10 ng/h). (C) Quantification of escape failures and escape latency from LH test [Failures: F(1, 17) = 4.021, P ≤ 0.05; Latency: F(1, 17) = 3.202, P = 0.09]. (D) Quantification of FST data [Immobility: F(1, 10) = 10.737, P < 0.01; Swim: F(1, 10) = 7.173, P < 0.05; Climb: F(1, 10) = 6.047, P < 0.05; n = 5–6 per group]. ∗, P ≤ 0.05; ∗∗, P ≤ 0.01; +, P ≤ 0.1; n.s., P > 0.1.