N-Methyl-D-aspartate receptor antagonist d-methadone produces rapid, mTORC1-dependent antidepressant effects

Abstract

Currently available antidepressants have a delayed onset and limited efficacy, highlighting the need for new, rapid and more efficacious agents. Ketamine, an NMDA receptor antagonist, has emerged as a new rapid-acting antidepressant, effective even in treatment resistant patients. However, ketamine induces undesired psychotomimetic and dissociative side effects that limit its clinical use. The d-stereoisomer of methadone (dextromethadone; REL-1017) is a noncompetitive NMDA receptor antagonist with an apparently favorable safety and tolerability profile. The current study examined the rapid and sustained antidepressant actions of d-methadone in several behavioral paradigms, as well as on mTORC1 signaling and synaptic changes in the medial prefrontal cortex (mPFC). A single dose of d-methadone promoted rapid and sustained antidepressant responses in the novelty-suppressed feeding test (NSFT), a measure of anxiety, and in the female urine sniffing test (FUST), a measure of motivation and reward. D-methadone also produced a rapid reversal of the sucrose preference deficit, a measure of anhedonia, in rats exposed to chronic unpredictable stress. D-methadone increased phospho-p70S6 kinase, a downstream target of mTORC1 in the mPFC, and intra-mPFC infusion of the selective mTORC1 inhibitor rapamycin blocked the antidepressant actions of d-methadone in the FUST and NSFT. D-methadone administration also increased levels of the synaptic proteins, PSD95, GluA1, and Synapsin 1 and enhanced synaptic function in the mPFC. Studies in primary cortical cultures show that d-methadone also increases BDNF release, as well as phospho-p70S6 kinase. These findings indicate that d-methadone induces rapid antidepressant actions through mTORC1-mediated synaptic plasticity in the mPFC similar to ketamine.

Fig. 1

D-methadone induces rapid and sustained antidepressant effects. a Experimental time course for d-methadone/ketamine injections and the behavioral tests. b A single injection of d-methadone (20 mg/kg, s.c.) or ketamine (10 mg/kg, i.p.) increased time in the female urine sniffing test (FUST) 24 h after administration (two-way ANOVA followed by Duncan test, effect of drug treatment: F_1,88 = 8.89, p < 0.05; effect of water/urine: F_1,88 = 64.19, p < 0.05; interaction: F_1,88 = 10.50, p < 0.05). c There was no effect of either d-methadone or ketamine on beam breaks to measure locomotor activity (LMA, one-way ANOVA, F_3,27 = 0.13, p > 0.05). d Administration of d-methadone or ketamine also decreased the latency to feed in the novelty-suppressed feeding test (NSFT, 72 h after dosing; one-way ANOVA followed by Duncan test, F_3,26 = 5.29, p < 0.05). e There was no effect of either drug on home cage food consumption (F_3,26 = 0.56, p > 0.05). Each bar represents the mean ± standard error of the mean (S.E.M.). *p < 0.05 compared with the vehicle groups. n = 7–12/group

Fig. 2

D-methadone rapidly reverses the pro-depressive behaviors induced by chronic unpredictable stress (CUS). a Experimental time course for CUS, drug injection and behavioral tests. b CUS decreased sucrose preference (one-way ANOVA followed by Duncan test, F_3,45 = 2.99, p < 0.05), c decreased female urine sniffing time (F_3,45 = 5.19, p < 0.05), and d increased the latency to feed in the novelty-suppressed feeding test (F_3,45 = 6.67, p < 0.05). These effects were reversed by a single injection of d-methadone (s.c., 20 mg/kg) or ketamine (i.p., 10 mg/kg). The behavioral tests were conducted 24, 48, and 72 h after dosing as indicated. There was no effect of CUS or drug dosing on (c) time sniffing water (F_3,45 = 0.15, p > 0.05) or e home cage food consumption (F_3,45 = 0.19, p > 0.05). Each bar represents the mean ± standard error of the mean (S.E.M.). *p < 0.05 compared with the non-stressed vehicle group; ^#p < 0.05 compared to the stressed vehicle group. n = 9–15/group

Fig. 3

The rapid antidepressant effects of d-methadone are mediated by mTORC1 signaling in the mPFC. a Experimental time course for d-methadone injection and brain tissues collection. b D-methadone (s.c., 20 mg/kg) increased the expression of p-p70S6K (Student t-test, t₂₀ = 2.08, p < 0.05), PSD95 (t₂₀ = 2.14, p < 0.05), Synapsin 1 (t₂₀ = 2.17, p < 0.05) and GluA1 (t₁₉ = 2.82, p < 0.05) 24 h after drug administration. No effect was found for p-mTOR (t₂₀ = 1.29, p > 0.05) and p-4EBP1 (t₂₀ = 0.89, p > 0.05). c Experimental time course for surgery, drug injection and behavioral tests. Intra-mPFC infusions of rapamycin (10 nmol, 0.2 µL/side) abolished the antidepressant effects of d-methadone (s.c., 20 mg/kg) in the d FUST (three-way ANOVA followed by Duncan test, effect of treatment 1, vehicle/rapamycin: F_1,88 = 4.54, p < 0.05; effect of treatment 2, vehicle/d-methadone: F_1,88 = 5.33, p < 0.05; effect of water/urine: F_1,88 = 161.11, p < 0.05; interaction treatment 1*treatment 2*water/urine: F_1,88 = 4.50, p < 0.05), and e NSFT (two-way ANOVA followed by Duncan test, effect of treatment 1, vehicle/rapamycin: F_1,44 = 1.14, p > 0.05; effect of treatment 2, vehicle/d-methadone: F_1,44 = 0.53, p > 0.05; interaction treatment 1*treatment 2: F_1,44 = 5.98, p < 0.05). There was no effect on d time sniffing water or f home cage food consumption (one-way ANOVA, F_3,44 = 0.49, p > 0.05). Each bar represents the mean ± standard error of the mean (S.E.M.). *p < 0.05 compared with the vehicle group; ^#p < 0.05 compared with the d-methadone group. n = 10–13/group

Fig. 4

D-methadone enhances 5-HT- and NMDA-induced EPSCs and increases spines head diameter in layer V mPFC pyramidal neurons. Rats were treated with saline or d-methadone (s.c., 20 mg/kg) and 24 h later slices of mPFC were prepared for patch clamp recording. Pyramidal neurons were filled with neurobiotin during patch clamp recording. a Representative traces of postsynaptic currents (PSCs) in layer V pyramidal cells from saline and d-methadone-treated rats. b D-methadone increased pyramidal cell excitatory postsynaptic current (EPSC) frequency induced by 5-HT (Student’s t-test, t₄₉ = 2.10; p < 0.05) or NMDA (t₃₃ = 2.03, p < 0.05). There was no effect on hypocretin-induced EPSCs frequency (Student’s t test, t₄₃ = 2.02, p > 0.05). There was no difference in NMDA-, 5-HT- and hypocretin-induced inhibitory postsynaptic current (IPSC) frequency (Student’s t test, NMDA: t₃₈ = 2.02; 5-HT: t₃₁ = 2.04; Hcrt: t₁₀ = 2.23, p > 0.05; n = 19–23 cells for vehicle group; n = 22–30 cells for d-methadone group). c Representative confocal image of neurobiotin-filled layer V neurons, and enlarged images of the apical dendrite branch segments from vehicle control and d-methadone treated rats (24 h after dosing). d D-methadone (s.c., 20 mg/kg) increased spine head diameter shown in the cumulative fraction curve (Kolmogorov–Smirnov test, D = 0.08, p < 0.05; control, n = 3784 spines from seven cells; d-methadone, n = 5194 spines from 10 cells). e D-methadone did not change dendritic spine density (Student’s t-test, p > 0.05. All: t₁₅ = 0.11; stubby: t₁₅ = 0.99; thin: t₆ = 1.21; mushroom: t₁₅ = 0.94; stubby + mushroom: t₁₆ = 1.38; control, n = 7 cells from four rats; d-methadone, n = 10 cells from six rats). Each bar represents the mean ± standard error of the mean (S.E.M.). *p < 0.05 compared with the vehicle group

Fig. 5

Incubation with d-methadone activates Erk1/2 and mTORC1-p70S6K pathways and increases BDNF release in primary cortical cultures. a Experimental time line for d-methadone incubation and cell or media collection. D-methadone treatment (60 min) induced a concentration-dependent increase in b p-Erk1/2 levels (one-way ANOVA followed by Duncan test, F_5,49 = 3.10, p < 0.05) and c p-p70S6K levels at doses of 100 and 500 nM (F_3,36 = 3.15, p < 0.05. n = 8–10 wells/group). Representative images of western blots are shown above each bar graph for vehicle and d-methadone treatments. d D-methadone incubation (60 min) increased BDNF release into the media at the higher dose (F_2,19 = 5.08, p < 0.05. n = 6–8 samples/group). Each bar represents the mean ± standard error of the mean (S.E.M.). *p < 0.05 compared with the control (vehicle) group