Figure 1.
Imipramine specifically activates TrkB receptors in the mouse brain.
A) The ability of imipramine (30 mg/kg, i.p., 30 min; n = 6/group) to induce rapid TrkB phosphorylation in the hippocampus and medial prefrontal cortex of TrkBF616A mutant mice is abolished with 1NaPP1 pretreatment (25 µM for 1-week in drinking water+83 ng/g co-injection with imipramine). B) A representative blot showing TrkB kinase dependent action of imipramine-induced phosphorylation of TrkB (Y816) and the ∼105 kDa protein. C) A representative blot showing imipramine-induced TrkB phosphorylation in flag-precipitated pool of protein from the brains of mice over-expressing flag-tagged catalytic TrkB receptors. Data is presented as percentage of control/saline ± standard error of mean (SEM). *<0.05, **<0.01; two-way ANOVA with Newmann-Keuls post hoc test.
Figure 2.
Antidepressant drugs activate the immaturely glycosylated form of TrkB.
A) Acute imipramine treatment induces the phosphorylation (Y816) of full-length and low-molecular weight (LMW; ∼105 kDa) TrkB receptors in mouse brain. n = 6/group. B) Antidepressant-induced ∼105 kDa protein is sensitive to Endo-H digestion. A representative blot of triplicate data. C) Total TrkB, phosphorylated TrkB (Y816) and phosphorylated ∼105 kDa protein levels are increased in the brains of mice over-expressing catalytic TrkB receptors. n = 5/group. Data is presented as percentage of control ± standard error of mean (SEM). *<0.05, ***0.005; unpaired two-tailed t-test.
Figure 3.
Role of BDNF in antidepressant-induced rapid TrkB activation in brain.
Imipramine (30 mg/kg, 30 min, i.p.) readily increases the phosphorylation of TrkB receptors (Y816) in forebrain specific BDNF−/− knock-out mice (BDNF2L/2LCk-cre) n = 4/group. Data is presented as percentage of control ± standard error of mean (SEM). *<0.05; unpaired two-tailed t-test.
Figure 4.
Antidepressant drugs amitriptyline and imipramine do not regulate TrkB phosphorylation in primary neurons.
A) Whereas BDNF (20 ng/ml; 15 min) robustly increases the phosphorylation of TrkB (Y816) in E18 rat cortical and hippocampal neurons (14 DIV), amitriptyline (left & middle; 0.5 µM, 5 µM; 15 min) and imipramine (0.5 µM, 5 µM; 50 µM; 15 min) produces no change on TrkB phosphorylation. Representative blot of triplicate data. B) Imipramine pre-treatment (4, 12, 40 µM; 15 min) did not facilitate BDNF-induced (5 ng/ml; 15 min) TrkB phosphorylation in E18 rat cortical neurons as measured with phospho-TrkB ELISA. n = 4/group. C) Imipramine pre-treatment (4, 12, 40 µM; 15 min) did not regulate TrkB phosphorylation in its own or in combination with depolarization stimuli (50 mM KCl; 15 min) as measured with phospho-TrkB ELISA. n = 4/group. Data is presented as percentage of control ± standard error of mean (SEM). *<0.05; one-way ANOVA with Newmann-Keuls post hoc test.
Figure 5.
Monoamines and monoamine reuptake in TrkB activation in vitro and in vivo.
A) Serotonin selective reuptake inhibitor fluoxetine produced essentially similar changes on hippocampal TrkB phosphorylation in the brains of wild-type and serotonin transporter KO mice, sert−/−. n = 3–5/group. B) Whereas the ability of serotonergic antidepressant citalopram (20 mg/kg, i.p., 60 min) and norepinephrinergic antidepressant reboxetine (20 mg/kg, i.p., 30 min) to induce full-length TrkB receptor phosphorylation (Y816) in mice depleted of serotonin (with pCPA) or norepinephrine (with DSP-4) are reduced, ∼105 kDa protein is heavily phosphorylated by both drugs in the hippocampi of these mice. Representative blots. n = 6–7/group. C) Norepinephrine (NE; 10 nM-10 µM; 15 min) and serotonin (5-HT; 10 nM-10 µM; 15 min) produced no changes on TrkB phosphorylation in BDNF-responsive E18 rat cortical neurons (15DIV) as measured with phospho-TrkB ELISA. n = 4/group. Data is presented as percentage of control ± standard error of mean (SEM). *<0.05; two-way ANOVA with Newmann-Keuls post hoc test.