Activity-dependent brain-derived neurotrophic factor signaling is required for the antidepressant actions of (2 R,6 R)-hydroxynorketamine

Abstract

Ketamine, a noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist, produces rapid and long-lasting antidepressant effects in major depressive disorder (MDD) patients. (2R,6R)-Hydroxynorketamine [(2R,6R)-HNK], a metabolite of ketamine, is reported to produce rapid antidepressant effects in rodent models without the side effects of ketamine. Importantly, (2R,6R)-HNK does not block NMDA receptors like ketamine, and the molecular signaling mechanisms for (2R,6R)-HNK remain unknown. Here, we examined the involvement of BDNF/TrkB/mechanistic target of rapamycin complex 1 (mTORC1) signaling in the antidepressant actions of (2R,6R)-HNK. Intramedial prefrontal cortex (intra-mPFC) infusion or systemic (2R,6R)-HNK administration induces rapid and long-lasting antidepressant effects in behavioral tests, identifying the mPFC as a key region for the actions of (2R,6R)-HNK. The antidepressant actions of (2R,6R)-HNK are blocked in mice with a knockin of the BDNF Val66Met allele (which blocks the processing and activity-dependent release of BDNF) or by intra-mPFC microinjection of an anti-BDNF neutralizing antibody. Blockade of L-type voltage-dependent Ca2+ channels (VDCCs), required for activity-dependent BDNF release, also blocks the actions of (2R,6R)-HNK. Intra-mPFC infusion of pharmacological inhibitors of TrkB or mTORC1 signaling, which are downstream of BDNF, also block the actions of (2R,6R)-HNK. Moreover, (2R,6R)-HNK increases synaptic function in the mPFC. These findings indicate that activity-dependent BDNF release and downstream TrkB and mTORC1 signaling, which increase synaptic function in the mPFC, are required for the rapid and long-lasting antidepressant effects of (2R,6R)-HNK, supporting the potential use of this metabolite for the treatment of MDD.

Keywords: (2R,6R)-hydroxynorketamine; BDNF; depression; ketamine; mTORC1.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Systemic or mPFC infusion of (2R,6R)-HNK induces antidepressant effects. (A) Mice were administered vehicle, (2R,6R)-HNK (3 to 30 mg/kg, i.p.) or ketamine (10 mg/kg, i.p.), followed by behavioral tests. (B and C) (2R,6R)-HNK and ketamine significantly decreased both immobility time 24 h after dosing in the FST and latency to feed 3 d after treatment in the NSFT. (D) Four days after treatment, (2R,6R)-HNK had no effect in the FUST, even though ketamine significantly increased female urine sniffing at this time point. (E–G) Studies of FUST 1 d after dosing show that (2R,6R)-HNK (30 mg/kg) or ketamine (10 mg/kg) administration induced antidepressant effects; there were also significant effects 5 d after treatment in the FST. (H) Mice received bilateral infusion of (2R,6R)-HNK (10 ng per side) in the mPFC, followed by behavioral tests. (I and J) Infusions of (2R,6R)-HNK into the mPFC induced significant antidepressant responses in the FST and NSFT. (K) Location of cannula placements in the mPFC. Bars represent mean ± SEM, n = 4 to 12 per group. ###P < 0.001, ##P < 0.01 compared with vehicle group; Dunnett’s multiple comparison test after significant results of one-way ANOVA. ***P < 0.001, **P < 0.01 compared with vehicle group; Student’s t test.

Fig. 2.

Antidepressant effects of (2R,6R)-HNK require BDNF release. (A) Val/Val (WT), Val/Met, and Met/Met mice were administered vehicle or (2R,6R)-HNK (30 mg/kg, i.p.), followed by behavioral testing. (B–D) (2R,6R)-HNK produced significant antidepressant effects in the FST and NSFT in Val/Val mice, but these effects were blocked in Val/Met and Met/Met mice. There were no effects on locomotor activity and no effects of genotype on immobility time or latency to feed. (E) Mice received bilateral infusions of BDNF nAb (0.2 μg per side) into the mPFC before administration of (2R,6R)-HNK, followed by behavioral tests. (F) Location of cannula placements in the mPFC. (G and I) Infusions of BDNF nAb blocked the antidepressant actions of (2R,6R)-HNK, including the reduction of immobility time in the FST and latency in the NSFT. (H) There were no effects of (2R,6R)-HNK or BDNF nAb infusion on locomotor activity and no effects of BDNF nAb alone on immobility time or latency to feed. Bars represent mean ± SEM, n = 5 to 8 per group. ***P < 0.001, *P < 0.05 compared with vehicle-treated WT group or vehicle-treated vehicle group; ###P < 0.001, ##P < 0.01 compared with (2R,6R)-HNK–treated WT group or (2R,6R)-HNK–treated vehicle group; Tukey’s multiple comparison test after significant results of two-way ANOVA.

Fig. 3.

(2R,6R)-HNK stimulates BDNF release in primary neuronal cultures. (A) Schematic showing timeline for primary cortical culture experiments. After drug incubations, cells were collected, and the phosphorylation levels of ERK and total ERK were determined by Western blot analysis (B) and media was collected and BDNF release was determined by ELISA (C and D). (B) Primary cortical cultures were treated with (2R,6R)-HNK (100 pM to 500 nM) for 60 min. (Left) Representative Western blot images are shown. (Right) Levels of phospho-ERK are presented as a ratio by dividing phopho-ERK by total ERK. Treatment with (2R,6R)-HNK (1, 10, and 50 nM; 60 min) significantly increased phospho-ERK levels. (C) (2R,6R)-HNK at both 10 and 50 nM (60 min) significantly increased BDNF release into the media. (D) Influence of preincubation (20 min) with NBQX (50 μM) or verapamil (10 µM) on (2R,6R)-HNK (10 nM; 60 min) stimulation of BDNF release in primary cultured neurons. (2R,6R)-HNK–stimulated BDNF release was completely blocked by pretreatment with verapamil. BDNF levels were normalized to levels of protein and are presented as a ratio compared with vehicle to allow for values to be compared across experiments. The mean absolute value for BDNF level in the vehicle group is 15.2 ± 4.7 pg/mL. Bars represent mean ± SEM, n = 4 to 8 per group. **P < 0.01, *P < 0.05 compared with vehicle group or vehicle-treated vehicle group; #P < 0.05 compared with vehicle-treated (2R,6R)-HNK group; Fisher’s least significant difference multiple comparison test after significant results of one-way ANOVA.

Fig. 4.

Inhibition of mTORC1 signaling blocks the antidepressant actions of (2R,6R)-HNK. (A) Mice received administration of (2R,6R)-HNK (30 mg/kg, i.p.) or ketamine (10 mg/kg, i.p.), and PFC dissections were collected 30 min later. (B) (2R,6R)-HNK and ketamine significantly increased phospho-mTOR. Levels of phospho-mTOR were determined by Western blot analysis (Right) and are presented as a ratio divided by total mTOR (Left). Bars represent mean ± SEM, n = 5 to 6 per group. **P < 0.01 compared with vehicle group, Fisher’s least significant difference multiple comparison test after significant results of one-way ANOVA. (C) Mice received bilateral infusion of rapamycin (0.02 nmol per side) in the mPFC before administration of (2R,6R)-HNK (30 mg/kg, i.p.), followed by behavioral tests starting 24 h later. (D) Location of cannula placements in the mPFC. (E–G) Infusions of rapamycin blocked the reduction of immobility time and latency to feed induced by (2R,6R)-HNK in the FST and NSFT; there were no effects on locomotor activity, and rapamycin alone had no effect on immobility time or latency to feed. Bars represent mean ± SEM, n = 6 to 8 per group. ***P < 0.001 compared with vehicle-treated vehicle group; ###P < 0.001 compared with vehicle-treated (2R,6R)-HNK group; Tukey’s multiple comparison test after significant results of two-way ANOVA.

Fig. 5.

(2R,6R)-HNK increases synaptic function in the mPFC. Mice were administered vehicle or (2R,6R)-HNK (30 mg/kg, i.p.) 24 h before slice preparation and electrophysiological recordings. Layer V pyramidal neurons were patched and 5-HT–induced or hypocretin (Hcrt)-induced EPSCs were determined. (A) Representative traces of EPSC recordings from saline vehicle and (2R,6R)-HNK–treated mice. (B) (2R,6R)-HNK administration significantly increased the frequency of hypocretin-induced EPSCs in layer V pyramidal neurons; there was also a tendency for increased 5-HT–induced EPSC frequency. EPSC frequencies are mean ± SEM, percent of control, n = 20 cells from six mice for vehicle, n = 23 cells from six mice for (2R,6R)-HNK; *P < 0.05 compared with vehicle group; Student’s t test. (C) Cumulative probability distributions showing that (2R,6R)-HNK significantly increased the amplitude of 5-HT–induced EPSCs as well as hypocretin-induced EPSCs. Cumulative probability distributions showing significantly increased amplitudes (Kolmogorov–Smirnov two-sample test). Veh, vehicle.