mTOR-dependent synapse formation underlies the rapid antidepressant effects of NMDA antagonists

Abstract

The rapid antidepressant response after ketamine administration in treatment-resistant depressed patients suggests a possible new approach for treating mood disorders compared to the weeks or months required for standard medications. However, the mechanisms underlying this action of ketamine [a glutamate N-methyl-D-aspartic acid (NMDA) receptor antagonist] have not been identified. We observed that ketamine rapidly activated the mammalian target of rapamycin (mTOR) pathway, leading to increased synaptic signaling proteins and increased number and function of new spine synapses in the prefrontal cortex of rats. Moreover, blockade of mTOR signaling completely blocked ketamine induction of synaptogenesis and behavioral responses in models of depression. Our results demonstrate that these effects of ketamine are opposite to the synaptic deficits that result from exposure to stress and could contribute to the fast antidepressant actions of ketamine.

Fig. 1

Ketamine transiently and dose-dependently activates mTOR signaling in rat prefrontal cortex (PFC). (A) Time course of ketamine (10 mg/kg, i.p.) induced mTOR signaling determined by Western blot analysis of phospho-mTOR (pmTOR), phospho-4E-BP1 (p4E-BP1), and phospho-p70S6K (pp70S6K) in synaptoneurosomes of PFC. Levels of total mTOR, GAPDH and p70S6K were also determined. (B) Dose-dependent activation, determined 1 hr after ketamine administration, of pmTOR, p4E-BP1 and pp70S6K. (C) Pre-treatment (10 min) with NBQX (10 mg/kg, i.p.) blocked ketamine (10 mg/kg, i.p.) activation of pmTOR, p4E-BP1, and pp70S6K, as well as upstream signaling kinases phospho-ERK (pERK) and phospho-Akt (pAkt) (analyzed 1 hr after ketamine). Levels of pERK1 and pERK2 were similarly regulated and were combined for quantitative analysis. (D) Pre-treatment (30 min) with inhibitors of ERK (U0126, 20 nmol, ICV) or PI-3k/Akt (LY294002, 20 nmol, ICV) abolished ketamine (10 mg/kg, i.p.) activation of mTOR signaling proteins (analyzed 1 hr after ketamine administration). Values represent mean ± SEM [n = 4 animals; * P < 0.05; ** P < 0.01, Analysis of Variance (ANOVA)].

Fig. 2

Ketamine rapidly increases synaptic proteins and spine number. (A) Time course for ketamine (10 mg/kg, ip) induction of synaptic proteins ARC, synapsin I, PSD95, and GluR1 in synaptoneurosomes of prefrontal cortex (PFC), and (B) blockade by pre-treatment (30 min) with a selective mTOR inhibitor rapamycin (0.2 nmol, ICV) (values represent mean ± SEM, n = 4 – 6 animals; *P < 0.05; **P < 0.01, ANOVA.) (C) Ketamine increased spine density in medial PFC, analyzed by two-photon microscopy 24 hr after treatment. Representative images are shown of high magnification Z-stack projections of apical tuft segments of Neurobiotin-labeled layer V pyramidal cells (Scale: 5 μm). (D) Results are the mean ± SEM (8 cells from 4 rats in each group; *P < 0.05; **P < 0.01, t-test). (E) Ketamine enhanced mPFC layer V pyramidal cell EPSC responses. Sample whole cell voltage-clamp recordings of 5-HT and hypocretin-induced EPSCs in slices (24 hr post-ketamine). (F) Cumulative probability distributions showing significant increases in amplitude (p < 0.0001, KS-z vale = 6.5 for 5-HT and 6.7 for Hcrt) and (H) frequency of 5-HT- and hypocretin-induced EPSCs (n = 12 neurons/group; *P < 0.05, t-test). Ketamine-induction of spine density and function were blocked by rapamycin infusions (C–G)

Fig. 3

Rapid behavioral actions of ketamine require mTOR signaling. Rapamycin was infused (0.2 nmol, ICV) 30 min prior to ketamine (10 mg/kg, ip), and responses in the FST (A), NSFT (B), or LH (C) paradigms was determined. Infusion of rapamycin (0.01 nmol) into the medial prefrontal cortex (PFC) blocked the antidepressant actions of ketamine (10 mg/kg, ip) in the FST (D) and NSFT (E). (F) Location of rapamycin infusions in the PFC (infusion sites are the same on the right and left sides due to the use of bilateral cannulae). Pre-treatment with inhibitors of ERK (U0126, 20 nmol, ICV) or PI3 kinase/Akt (LY294002, 20 nmol, ICV) blocked the behavioral effects of ketamine in FST (G) and NSFT (H). (I) Low (10 mg/kg) but not a high anesthetic dose (80 mg/kg) of ketamine produced an antidepressant action in the FST. Values represent mean ± SEM (n = 6–8 animals; *P < 0.05; **P < 0.01, ANOVA).

Fig. 4

Selective NR2B antagonist Ro 25-6981 activates mTOR signaling and produces rapamycin-sensitive behavioral effects. (A, B) Effects of Ro 25-6981 (10 mg/kg, ip) on pmTOR, p4E-BP1, pp70S6K, pERK, pAkt, Arc, synapsin I, PSD95, and GluR1 in prefrontal cortex. (C–D) Pre-treatment with rapamycin (0.2 nmol, ICV) abolished the actions of Ro 25-6981 in FST (C) and NSFT (D). Values represent mean ± SEM (n = 6–8 animals; *P < 0.05; **P < 0.01, ANOVA).