Figure 1.
Age-dependent effect of systemic imipramine on TrkB phosphorylation and signaling in the mouse brain.
(a) Phosphorylation of TrkB phospholipase-Cγ1 (PLCγ1) binding site (Y816) after acute imipramine treatment (30 mg/kg, 30 min, i.p.) in prefrontal cortex (PFC) and hippocampus (HC). Phospho-TrkB values are normalized against total TrkB levels. (b) Phosphorylation of CREB (Ser133) after acute imipramine treatment (30 mg/kg, 30 min, i.p.) in prefrontal cortex (PFC) and hippocampus (HC). Phospho-CREB values are normalized against total CREB levels. (c) The effect of acute imipramine treatment (30 mg/kg, 30 min, i.p.) on the association of phosphorylated PLCγ1 (Tyr783) with catalytic TrkB receptors in P8 mouse pup hippocampus. (d) The effect of acute imipramine treatment (30 mg/kg, 30 min, i.p.) on the association of phosphorylated PLCγ1 (Tyr783) with catalytic TrkB receptors in P25 mouse pup hippocampus. Results are expressed as percentage of respective control. A t-test was performed between each control and treated group of animals at the different ages; *P<0.05, ** P<0.01, *** P<0.001. n = 6–7 per group.
Figure 2.
Developmental regulation of BDNF-induced TrkB receptor phosphorylation.
(a) Representative blots of experiments showing age-dependent modification of BDNF-induced (50 ng/ml, 5 min, at 37°C) TrkB tyrosine phosphorylation (Y816) response in mouse cortical (PFC) and hippocampal (HC) microslices ex vivo. (b) Representative blots of experiments showing that BDNF (50 ng/ml, 15 min, 37°C) readily induces TrkB phosphorylation at sites Y515 and Y705/6 in P8 hippocampal microslices whereas neither site is effectively phosphorylated by BDNF in P24 hippocampal microslices. (c) NGF (50 ng/ml, 15 min, at 37°C) readily induces TrkA tyrosine phosphorylation (Y674/5) in P24 hippocampal microslices whereas BDNF (50 ng/ml, 15 min, at 37°C) has no effect on TrkB phosphorylation (Y705/6) under the same conditions. For statistical analysis, a one-way ANOVA followed with Bonferroni post hoc test was performed; ***P<0.001. n = 4 per group. Phospho-TrkB values are normalized against total TrkB levels.
Figure 3.
Immature, but not adult, BDNF deficient mice show reduced basal hippocampal TrkB phosphorylation.
(a) Basal hippocampal TrkB phosphorylation (Y816, Y705/6) is significantly reduced in P11 old Bdnf+/− (+/−) and Bdnf−/− (−/−) mouse pups compared to age-matched wild-type (WT) mice. (b) Basal hippocampal TrkB phosphorylation (Y816) is not altered in mice with conditional deletion of BDNF in the forebrain (cBdnf−/− or (c) −/−). (c) Basal hippocampal TrkB phosphorylation (Y705/6) is not altered in adult Bdnf+/− (+/−) mouse. For statistical analysis, a t-test was performed; **P<0.01. n = 3–6 per group. Phospho-TrkB values are normalized against total TrkB levels.
Figure 4.
TrkB responsiveness to ex vivo BDNF and systemic imipramine is not altered in trkB.T1−/− mice.
BDNF-induced (ex vivo, 50 ng/ml, 5 min) TrkB phosphorylation (Y816) in hippocampal microslices prepared from P10 (a) or P20 (b) wild-type and trkB.T1−/− KO pups. (c) Imipramine-induced (30 mg/g, i.p., 30 min) TrkB phosphorylation (Y816) in adult male wild-type and trkB.T1−/− mouse hippocampus. Two-Way ANOVA followed with Bonferroni post hoc test was performed for statistical analysis; ** P<0.01, *** P<0.001 compared to respective control, #P<0.05 compared to wt/control. The CTRL bar represents control treatments at each age. Phospho-TrkB values are normalized against total TrkB levels. n = 3–5 per group.
Figure 5.
TrkB responsiveness to ex vivo BDNF is not altered by antidepressant drugs or cAMP signalling.
(a) P60 mice were pretreated acutely with imipramine (Imi; 30 mg/kg i.p., 60 min) or saline (Sal), hippocampi were collected, sliced and incubated ex vivo with or without BDNF (50 ng/ml) for 5 minutes at 37°C. TrkB phosphorylation (Y816) was analyzed with western blotting. (b) P24 hippocampal microslices were pre-incubated at 37°C for 15 minutes with vehicle or sp-cAMP (10 µM) and then exposed to BDNF (50 ng/ml) or vehicle at 37°C for another 15 minutes. TrkB phosphorylation (Y705/6) was analyzed with western blotting. (c) Adult animals were chronically treated for 21 days with vehicle (Veh) or fluoxetine (0.08 mg/ml) in drinking water, hippocampi were collected, sliced and incubated ex vivo with or without BDNF (50 ng/ml) for 5 minutes at 37°C. TrkB phosphorylation (Y816) was analyzed with western blotting. Phospho-TrkB values are normalized against total TrkB levels. Two-Way ANOVA followed with Bonferroni post hoc test was performed for statistical analysis; **P<0.01, *** P<0.001. n = 3–6 per group.
Figure 6.
BDNF, but not antidepressant drugs, induce TrkB phosphorylation in a cell-free kinase assay.
(a) Incubation of adult cortical or hippocampal brain lysates with BDNF (50 ng/ml) induces the tyrosine phosphorylation of Shc (Y515) and phospholipase-Cγ1 (Y816) binding sites of TrkB. * indicate unidentified phospho-proteins detected by the antibodies. (b) BDNF (50 ng/ml), but not imipramine or fluoxetine, induces TrkB phosphorylation in P20 mouse hippocampal lysates. Overall tyrosine phosphorylation of TrkB was analyzed using phospho-TrkB ELISA. For statistical analysis, a one-way ANOVA followed with Bonferroni post hoc test was performed; ***P<0.001 compared to respective control, ###P<0.001 compared to ATP/Control. n = 4 per group.
Figure 7.
Postnatal systemic clomipramine treatments lead to long-lasting and distinct behaviours depending onexposure period.
(a) Schematic figure showing treatment groups. Animals were treated with a daily dose of clomipramine (20 mg/kg, i.p.) during early (P4-9; E-PS) or late (P16-21; L-PS) postnatal period. At 3 months of age the animals were subjected to behavioural analyses (b) Age-dependent phosphorylation of TrkB receptor (Y816) in mouse brain after acute treatment with clomipramine (20 mg/g, i.p., 60 min). (c) Clomipramine treatment during late postnatal period (activates TrkB) produces anxiolytic-like behaviour in the light-dark test. (d) Clomipramine treatment during the early postnatal period (does not activate TrkB) produces anxiolytic-like behaviour in the novelty-suppressed feeding task. A t-test (b) or Two-Way ANOVA followed with Bonferroni post hoc test (c–d) was performed for statistical analysis; *P<0.05, ** P<0.01, *** P<0.001 compared to the respective age saline (Sal) treated group. The CTRL bar is representative of each respective control at each age. n = 6/group (biochemical analysis) or n = 10–15 per group (behavioural analysis).