Induction of neuronal vascular endothelial growth factor expression by cAMP in the dentate gyrus of the hippocampus is required for antidepressant-like behaviors

Abstract

The cAMP cascade and vascular endothelial growth factor (VEGF) are critical modulators of depression. Here we have tested whether the antidepressive effect of the cAMP cascade is mediated by VEGF in the adult hippocampus. We used a conditional genetic system in which the Aplysia octopamine receptor (Ap oa(1)), a G(s)-coupled receptor, is transgenically expressed in the forebrain neurons of mice. Chronic activation of the heterologous Ap oa(1) by its natural ligand evoked antidepressant-like behaviors, accompanied by enhanced phosphorylation of cAMP response element-binding protein and transcription of VEGF in hippocampal dentate gyrus (DG) neurons. Selective knockdown of VEGF in these cells during the period of cAMP elevation inhibited the antidepressant-like behaviors. These findings reveal a molecular interaction between the cAMP cascade and VEGF expression, and the pronounced behavioral consequences of this interaction shed light on the mechanism underlying neuronal VEGF functions in antidepression.

Figure 1.

Antidepressant-like behaviors induced by chronic octopamine treatment in Ap oa₁ mice. a, Experimental design. Mice treated with chronic octopamine for 2 weeks or fluoxetine for 3 weeks were divided into two groups for behavioral measurements. b, FST. Immobility time was measured 24 h after the last injection of drug (n = 13–16 animals per group). Significant effects of octopamine are seen for genotype (Ap oa₁ vs WT littermates, **p < 0.01, two-way ANOVA) and for treatment in Ap oa₁ mice (vehicle vs octopamine, **p < 0.01, two-way ANOVA). Significant effects of fluoxetine in WT littermates are seen (**p < 0.01, two-way ANOVA). c, NSFT. Latency to feed was measured 2 weeks after the last injection of chronic octopamine or fluoxetine (n = 8–10 animals per group). Chronic octopamine reduced latency to feed in Ap oa₁ mice compared with WT mice that received fluoxetine or Ap oa₁ mice that received vehicle (**p < 0.01, two-way ANOVA). Fluoxetine did not reduce latency in WT littermates (octopamine vs fluoxetine, p > 0.05, two-way ANOVA). d, SCT. The test was performed 2 weeks after octopamine or fluoxetine treatment (n = 13–16 animals per group). Chronic octopamine in Ap oa₁ mice increased sucrose consumption compared with WT mice treated with fluoxetine (*p < 0.05, two-way ANOVA) or Ap oa₁ mice treated with vehicle (**p < 0.01, two-way ANOVA). There was no difference in water consumption between groups (p > 0.05). e, FST. Immobility time was measured 2 weeks after the last injection of drug (n = 13–16 animals per group). Significant effects of octopamine are seen for genotype (Ap oa₁ vs WT littermates, **p < 0.01, two-way ANOVA) and for treatment in Ap oa₁ mice (vehicle vs octopamine, **p < 0.01, two-way ANOVA). Significant effects of fluoxetine in WT littermates were not seen (p > 0.05, two-way ANOVA). f, OFT (n = 16–18 animals per group). The total distance moved in the box was similar in all groups (p > 0.05 vs WT mice given octopamine, two-way ANOVA). g, OFT (n = 16–18 animals per group). The number of center square crossings (left), and the distance moved in the center zone (right) expressed as a percentage of the total distance moved in the box were similar in all groups (p > 0.05 vs WT mice given octopamine, two-way ANOVA). h, EPMT (n = 16–18 animals per group). The number of visits to the open arms and time spent in the open arms, expressed as a percentage of the total time spent in the open and closed arms, did not differ among groups (p > 0.05 vs WT mice given octopamine, two-way ANOVA). i, Locomotor activity in the EPMT. Total arm entries and total distance moved were not different (p > 0.05 vs WT mice given octopamine, two-way ANOVA).

Figure 2.

Increased octopamine-induced neurogenesis in Ap oa₁ transgenic mice. a, Experimental design for proliferation studies. b, Octopamine increased proliferation in the SGZ of the hippocampus more than vehicle in Ap oa₁ mice. BrdU (red) and DAPI (blue) immunoreactivities. c, Survival paradigm. d, Octopamine increased survival of BrdU⁺ cells in Ap oa₁ mice. BrdU (red) and DAPI (blue) immunoreactivities. e, Labeling of the immature neuronal marker DCX (green) in the DG when mice are subjected to the survival paradigm of c. f, High-power confocal image of a cell (arrowhead in e) coexpressing BrdU (red) and DCX (green). The bottom and right panels show images merged across the x- and z-axis, respectively. g, Quantification of BrdU⁺ cells in the SGZ after proliferation (***p < 0.001, Student's t test; n = 10–13 animals per genotype) and survival paradigm (***p < 0.001, Student's t test; n = 7–10 animals per genotype). Quantification of DCX⁺ cells in the DG of e. Octopamine increased the number of DCX⁺ cells compared with that of the vehicle in Ap oa₁ mice (***p < 0.001, Student's t test; n = 7 animals per genotype).

Figure 3.

Enhanced CREB phosphorylation and VEGF expression in the hippocampus of Ap oa₁ mice. a, pCREB levels in the hippocampus 2 h after the last injection of octopamine (1 mg/kg) treatment for 14 d in WT and Ap oa₁ mice. Top: pCREB immunostaining in the whole hippocampus. Bottom: Magnified images of the boxed areas of the DG. b, Left: Densitometric analysis of DG granule cells in a. Significant effects are seen by genotype (Ap oa₁ vs WT mice, ***p < 0.001, Student's t test; n = 5 animals per genotype). Data are shown relative to the level in WT mice given octopamine. Right: Quantitative immunoblot analysis of pCREB in whole hippocampi. Top: Representative immunoblots. Bottom: Densitometric analyses of total immunoreactivity. Data are normalized to total CREB in each group, and are shown relative to the level in WT mice given octopamine (**p < 0.01, Student's t test; n = 4 animals per genotype). c, VEGF levels in the hippocampi of Ap oa₁ mice and WT littermates receiving chronic octopamine treatment. Top: Whole hippocampi. Bottom: Magnified images of the boxed areas of the DG. d, Left: Densitometric analyses of DG granule cells in c. Significant effects on genotype are seen after chronic octopamine treatment (***p < 0.001, Student's t test; n = 5 animals per genotype). Data are normalized to WT mice. Right: VEGF mRNA and protein expression by quantitative RT-PCR and immunoblot analysis of whole hippocampal homogenates. Significant effects on genotype are seen after chronic octopamine (Ap oa₁ vs WT, ***p < 0.001, Student's t test; n = 4 animals per genotype). Data are normalized to GAPDH (in mRNA) or β-actin (in protein) in each group and are shown relative to the level in WT mice. e, f, Colocalization of VEGF (green) with the neuronal marker NeuN (red) or the astrocytic marker GFAP (red) in GCL. VEGF expression was enhanced in neuronal, but not astroglial, cells of the Ap oa₁ mice. The top and right panels show images merged across the z- and x-axis, respectively. g, The vascular marker RECA-1 (red). The number of RECA⁺ vessels is similar to that in the DG, including GCL, molecular layer and hilus from Ap oa₁ and WT mice (n = 5 animals per genotype).

Figure 4.

VEGF expression by Ap oa₁ activation. a, pCREB was induced by octopamine and Ap oa₁ in the HEK293 in vitro model system. Left: Representative immunoblots of pCREB, total CREB and β-actin: pCREB was induced only in the presence of both Ap oa₁ and octopamine treatment. Right: Densitometric analyses of three independent experiments. Forskolin alone (***p < 0.001 vs control, Student's t test; n = 4) or transfection of Ap oa₁ and octopamine treatment (***p < 0.001, Student's t test) increased the level of pCREB. Cotransfection with Ap oa₁ and CREB plasmids gave the strongest CREB activation on octopamine treatment (***p < 0.001, Student's t test). pCREB levels were normalized to the amount of β-actin, and are shown relative to expression in nontransfected control cells. b, Human VEGF165 mRNA induction. Ap oa₁ was transfected into HEK293 cells, and human VEGF165 mRNA was measured by quantitative RT-PCR. Forskolin (10 μm) added to nontransfected cells for 6 h demonstrates the cAMP inducibility of VEGF (***p < 0.001 vs control, Student's t test; n = 4). Octopamine (1 μm; 6 h treatment) increased VEGF mRNA (***p < 0.001 vs control, Student's t test). c, VEGF promoter assay. Top: VEGF promoter-reporter constructs used in the reporter assays. Bottom: CRE2 for the induction of VEGF promoter by octopamine. HEK293 cells were transiently transfected with VEGF promoter plasmids, Ap oa₁ and CREB expression plasmids and treated with octopamine. pVEGF(W) showed an increase in VEGF promoter activity in the presence of octopamine (**p < 0.01 vs octopamine nontreated, Student's t test; n = 7), and so was pCRE2 (^###p < 0.001 vs control vector; ^###p < 0.001 vs octopamine nontreated, Student's t test). pCRE1 did not increase promoter activity in response to octopamine treatment (p > 0.05 vs no octopamine). d, EMSA. The presence of rhCREB in the VEGF CRE2 binding complex was demonstrated by gel-supershift analysis (arrow). Incubation with cold CRE2 or mutant CRE2 decreased the CRE2 binding. Preincubation of rhCREB with anti-CREB resulted in the formation of a supershifted CRE2 band (arrowhead). Representative image from five independent experiments.

Figure 5.

DG specific VEGF expression required for the ability of chronic octopamine to induce antidepressant-like behaviors in Ap oa₁ transgenic mice. a, Knockdown efficiency of lenti-shVEGF by RNA interference (RNAi). Lenti-shVEGF was transfected into mouse primary hippocampal neuronal cells, and mouse VEGF and β-actin mRNA levels were measured by RT-PCR. Forskolin-induced VEGF mRNA expression was reduced by lenti-shVEGF1 and 3 in vitro. b, Experimental design. c, Confirmation of the location of viral injection into the DG with lenti-EGFP. EGFP expression was revealed by IHC. d, DG-specific knockdown of VEGF and pCREB protein levels. Top: VEGF immunostaining in the whole hippocampus. Middle: Magnified images of DG in the boxed areas. Bottom: pCREB in DG. e, Left: Densitometric analyses of VEGF in the DG in d. Lenti-shVEGF reduced the VEGF protein level in the DG of Ap oa₁ transgenic mice (***p < 0.001 vs lenti-EGFP; ^###p < 0.001 vs WT mice treated with lenti-EGFP or lenti-shVEGF, two-way ANOVA, Tukey's post hoc; n = 10 animals per group). shVEGF had no significant effects on basal VEGF protein levels (p > 0.05, lenti-EGFP vs lenti-shVEGF in WT mice, two-way ANOVA, n = 8 animals per group). Right: Quantitative RT-PCR. Lenti-shVEGF specifically reduced VEGF mRNA expression in whole hippocampi from Ap oa₁ mice on octopamine treatment (**p < 0.01 vs lenti-EGFP, Student's t test; n = 5 animals per group). f, VEGF⁺ and NeuN⁺ doubly labeled cells at GCL in lenti-EGFP or lenti-shVEGF injected Ap oa₁ mice. VEGF expression was reduced in NeuN⁺ cells by lenti-shVEGF. g, FST. Lenti-shVEGF increased immobility time in Ap oa₁ transgenic mice (***p < 0.001 vs lenti-EGFP; ^###p < 0.001 vs WT mice treated with lenti-EGFP or lenti-shVEGF, two-way ANOVA, Tukey's post hoc; n = 8 animals per group). Lenti-shVEGF had no significant effects on immobility time in WT mice (p > 0.05, lenti-EGFP vs lenti-shVEGF, two-way ANOVA; n = 10 animals per group). h, DCX⁺ cells. Top: DCX immunoreactivity. Bottom: Ap oa₁ mice treated with lenti-EGFP increased in DCX⁺ cells more than WT mice did (^###p < 0.001, two-way ANOVA, Tukey's post hoc; n = 4 animals per group). Ap oa₁ mice injected with lenti-shVEGF had significantly fewer DCX⁺ cells than lenti-EGFP-injected Ap oa₁ mice (**p < 0.01, two-way ANOVA, Tukey's post hoc). i, Experimental design for CUS. j, SCT. Left: Injecting mice with Adeno-VEGF before CUS enhanced consumption of sucrose more than injecting mice with control adenovirus before CUS (***p < 0.001, two-way ANOVA, Tukey's post hoc; n = 9 animals per group). (Right) There was no difference in water consumption between groups (p > 0.05). k, FST. Injecting mice with Adeno-VEGF before CUS reduced immobility time over injecting control mice with control virus and exposing them to CUS (**p < 0.01, two-way ANOVA, Tukey's post hoc; n = 9 animals per group).

Figure 6.

Blockade of fluoxetine-induced antidepressive behavioral effects by RNAi knockdown of VEGF in DG. a, Experimental design. Fluoxetine (10 mg/kg) was administered daily for 21 d. b, Fluoxetine-induced pCREB and neurogenesis. Top: Fluoxetine-induced pCREB is not reduced by lenti-shVEGF. Bottom: Chronic fluoxetine-treated mice exposed to lenti-shVEGF form fewer DCX⁺ cells than those exposed to lenti-EGFP. c, The number of DCX⁺ cells in the DG in b. (**p < 0.01, two-way ANOVA, Tukey's post hoc; n = 7 animals per group). d, FST. Fluoxetine decreases immobility time compared with vehicle treatment (*p < 0.05). In mice given fluoxetine and lenti-shVEGF increases immobility time to the level of the vehicle-treated controls (**p < 0.01 vs lenti-EGFP; two-way ANOVA, Tukey's post hoc; n = 10 animals per group).