NMDA receptor blockade at rest triggers rapid behavioural antidepressant responses

Abstract

Clinical studies consistently demonstrate that a single sub-psychomimetic dose of ketamine, an ionotropic glutamatergic NMDAR (N-methyl-D-aspartate receptor) antagonist, produces fast-acting antidepressant responses in patients suffering from major depressive disorder, although the underlying mechanism is unclear. Depressed patients report the alleviation of major depressive disorder symptoms within two hours of a single, low-dose intravenous infusion of ketamine, with effects lasting up to two weeks, unlike traditional antidepressants (serotonin re-uptake inhibitors), which take weeks to reach efficacy. This delay is a major drawback to current therapies for major depressive disorder and faster-acting antidepressants are needed, particularly for suicide-risk patients. The ability of ketamine to produce rapidly acting, long-lasting antidepressant responses in depressed patients provides a unique opportunity to investigate underlying cellular mechanisms. Here we show that ketamine and other NMDAR antagonists produce fast-acting behavioural antidepressant-like effects in mouse models, and that these effects depend on the rapid synthesis of brain-derived neurotrophic factor. We find that the ketamine-mediated blockade of NMDAR at rest deactivates eukaryotic elongation factor 2 (eEF2) kinase (also called CaMKIII), resulting in reduced eEF2 phosphorylation and de-suppression of translation of brain-derived neurotrophic factor. Furthermore, we find that inhibitors of eEF2 kinase induce fast-acting behavioural antidepressant-like effects. Our findings indicate that the regulation of protein synthesis by spontaneous neurotransmission may serve as a viable therapeutic target for the development of fast-acting antidepressants.

Figure 1. Time-course of NMDAR antagonist-mediated antidepressant-like behavioural effects

Mean immobility±SEM of C57BL/6 mice in FST following acute treatment of ketamine, CPP or MK801. Independent groups of mice were used at each time point and drug treatment to avoid behavioural habituation. ANOVA analysis F_3,27 =30.31, P<.0001 for treatment groups, F_3,27 =19.06, P<.0001 for duration of response, and F_9,81=9.32, P<.0001 for treatment-duration interaction; therefore, we examined treatment effects by time-point. a, Ketamine (3.0 mg/kg) significantly reduced immobility, suggestive of an AD-like response, at 30-min., 3-hrs, 24-hrs, and 1-week compared to vehicle treatment (*P<0.05). b, CPP (0.5 mg/kg) significantly reduced immobility at 30-min., 3-hrs, and 24-hrs (*P<0.05) compared to vehicle treatment. c, MK-801 (0.1 mg/kg) produced significant decreases in immobility at 30-min and 3-hrs compared to vehicle treatment (*P<0.05), (n=10/group/time-point). Here and in all figures error bars represent Standard Error of the Mean (SEM).

Figure 2. BDNF translation in antidepressant effects of NMDAR antagonists

a, Immobility in FST following acute ketamine (3.0 mg/kg). At 30-min, ANOVA F_1,35=17.13, P=0.0002 for drug, F_1,35=7.57, P=0.0093 for genotype-drug interaction, multiple comparisons with t-test (*P<0.05). At 24-hr, in a separate cohort, ANOVA F_1,29=3.77, P=0.0619 for treatment, multiple comparisons (*P<0.05) (n=7-12/group). b, Densitometric analysis of BDNF (normalized-GAPDH) in hippocampus following ketamine (3.0 mg/kg) or MK801 (0.1 mg/kg). 30-min., ANOVA F_2,12=6.77, P=0 .0108 for treatment, Bonferroni post-hoc test *P<0.05. 24-hrs, no significance (n=5-6/group). c, Anisomycin and actinomycinD protocol. d, Immobility at 30-min., ANOVA F_1,34=11.83, P=0.0016 for treatment and F_{1, 34} = 10.91, P =0.0023 for treatment-inhibitor interaction, multiple comparisons (*P<0.05)(n=8-10/group). e, Immobility at 24 hrs, ANOVA F_{1, 31}=9.34, P =0.0046 for treatment, multiple comparisons (*P<0.05)(n=8-10/group). f, Immobility of WTs given vehicle or NMDA (75 mg/kg), tested 30-minutes later in FST. g, Immobility of WTs given NBQX (10 mg/kg) or PTX (1.0 mg/kg), tested 30-minutes later in FST. h, Immobility of WTs given vehicle, ketamine (3.0 mg/kg), ketamine+NBQX (10 mg/kg), tested 30-minutes later in FST. ANOVA F_2,26=8.226, P<0.0019, Bonferroni post-hoc analysis shows ketamine effect reversed by NBQX, *P<0.05. i, Densitometric analysis of p-mTOR (normalized-mTOR) in hippocampus 30-minutes after vehicle or ketamine.

Figure 3. Ketamine blocks NMDAR spontaneous activity, reduces the level of eEF2 phosphorylation, and strengthens synaptic responses

a, Representative western blots from hippocampal primary cultures. b, (left) Densitometric analysis of peEF2 (normalized-total eEF2). Data expressed as mean percentage±SEM. TTX alone does not alter peEF2 while AP5 or ketamine, with or without TTX, significantly decreases peEF2 as assessed by t-test analysis (*P<0.05). (right) Application of 1, 5 and 50 μM of ketamine produces dose-dependent decreases in peEF2 assessed by t-test analysis (*P<0.05). c, Representative traces of NMDAR spontaneous activity after application of 1, 5 and 50 μM. d, Quantification of charge transfer (10 sec) reveals significant effects (*P<0.05) for all ketamine concentrations compared to control (n=6-16) assessed by t-test analysis (*P<0.05). e, Field potential slopes are plotted as a function of time. Representative field potential traces, (average 2-min) are shown during baseline (1) and at 45-min (2). The asterisk refers to significantly different field potentials values (*P<0.05). For statistical analysis we used two-way repeated ANOVA with Bonferroni post-hoc analysis. The drug-time interaction was significant (F_143,1430=6.723 P<0.001).

Figure 4. Rapid antidepressant-like behaviour mediated by decreased p-eEF2 and increased BDNF translation

a, Images of CA1 pyramidal and stratum radiatum layers after acute vehicle, ketamine, or MK-801; scale bar=100 μm (red: peEF2, blue: DAPI). b, Stratum radiatum magnification; scale bar=20 μm. c, ImageJ analysis of average fluorescence intensity. ANOVA cell layer F_2,23=13.13, P=0.0002 for treatment, dendrites F_2,23=14.06, P=0.0001 for treatment (n=4/group). d, Densitometric analysis of peEF2 (normalized-total eEF2) in hippocampus after NMDAR antagonist. ANOVA F_2,23=3.183, P=0.03 for treatment (n=8/group). e-h, Densitometric analysis. e, g, Significant increases in hippocampal BDNF protein (normalized-GAPDH) with rottlerin (5.0 mg/kg) versus vehicle (t-test *P<0.05), and NH125 (5.0 mg/kg) versus vehicle (*P<0.05). f, h, Significant decreases in peEF2 (normalized-total eEF2) versus vehicle (*P<0.05) and NH125 versus vehicle (t-test *P<0.05). i, Immobility in FST of WTs given acute rottlerin (5.0 mg/kg) or NH125 (5.0 mg/kg). ANOVA F_{3, 44}= 8.13, P=0.0002 for treatment, Bonferroni post-hoc analysis shows significance with rottlerin or NH125 versus vehicle (*P<0.05), but not ERK inhibitor SL327 (10mg/kg). j, Immobility of BDNF KO or littermate CTLs given acute rottlerin (5.0 mg/kg), tested 30-minutes later in FST. ANOVA F_{1, 19}=5.77, P=0.0267 for treatment, Bonferroni post-hoc analysis for rottlerin versus vehicle CTLs (*P<0.05) (n=5-7/group).