Chronic Administration of the N-Methyl-D-Aspartate Receptor Antagonist Ketamine Improves Rett Syndrome Phenotype

Abstract

Background: Rett syndrome (RTT) is a neurological disorder caused by mutation of the X-linked MECP2 gene, which results in the progressive disruption of excitatory and inhibitory neuronal circuits. To date, there is no effective treatment available for the disorder. Studies conducted in RTT patients and murine models have shown altered expression of N-methyl-D-aspartate receptors (NMDARs). Genetic deletion of the NMDAR subunit, GluN2A, in mice lacking Mecp2 is sufficient to prevent RTT phenotypes, including regression of vision.

Methods: We performed a systematic, randomized preclinical trial of chronic administration of low-dose (8 mg/kg, intraperitoneal) ketamine, an NMDAR antagonist, starting either early in development or at the onset of RTT phenotype in Mecp2-null mice.

Results: Daily exposure to ketamine ameliorated RTT symptoms and extended the life span of treated Mecp2-null mice without adverse side effects. Furthermore, significant improvement was observed in cortical processing and connectivity, which were fully restored to a wild-type level, particularly when treatment was started at the onset of regression.

Conclusions: Our findings provide strong evidence that targeting NMDA receptors can be a safe and effective treatment for RTT.

Keywords: Behavior; Breathing; Cortical activity; Parvalbumin connectivity; Preclinical trial; Survival.

Conflict of interest statement

Competing interests: All authors report no biomedical financial interests or potential conflicts of interest.

Copyright © 2016. Published by Elsevier Inc.

Figures

Figure. 1.

Ketamine 8 mg/kg is rapidly absorbed in the brain and does not induce side effects. (A) Pharmacokinetic analysis of total ketamine concentration in plasma (left) and brain (right) in WT adult mice after a single intraperitoneal injection (n = 3 mice per time point). (B) A single dose of ketamine 8 mg/kg did not affect spontaneous locomotor activity (Two-way ANOVA, p ≥ 0.05. Empty square: WT-saline, n = 6 mice; gray square: WT-ketamine, n = 15 mice) (C) High dose of ketamine (56 mg/kg) significantly reduced PPI response (Kruskal-Wallis, ** p ≤ 0.01, Dunn’s post-test. WT–saline, n = 10; WT-ketamine 8 mg/kg, n = 10; WT-ketamine 56 mg/kg, n = 8 mice).

Figure. 2.

Prolonged ketamine treatment improves key RTT-like phenotypes. (A) Survival curves in WT (empty circle) and in Mecp2 KO treated with vehicle (black-filled circle) or ketamine from P15 (magenta) or from P30 (green) revealed prolonged ketamine treatment improved the lifespan of Mecp2 KO mice. (B) No improvement of the body weight of P55 treated Mecp2 KO mice was observed (Kruskal-Wallis, ** p ≤ 0.01; *** p ≤ 0.001, Dunn’s post-test). Data are expressed as mean ± SEM. (C) The phenotypic severity score spans three groups: absent (white), mild (light polka dots), severe RTT phenotype (dark polka dots). Notably, almost 80% of the adult KO-k15 mice had a mild score compared to their age matched KO-v. KO-k30 mice did not show an improvement in the RTT score severity. (D) The clasping phenotypic score was improved mainly in the KO-k15 mice (WT-v, n = 13; KO-v, n = 18; KO-k15, n = 14; KO-k30, n = 17 mice). Data are expressed as mean ± SEM.

Figure. 3.

Ketamine delays the worsening of the respiratory function. (A) Representative plethysmography traces illustrating breathing pattern. (B) Quantification of the number of apneas per minute. Ketamine treatment from P30 prevented the developmental increase of apneic episodes (Wilcoxon signed-rank test, * p < 0.05, ** p ≤ 0.01. WT-v, n = 8; KO-v, n = 11; KO-k15, n = 7; KO-k30, n = 8 mice).

Figure. 4.

Ketamine delays regression of the visual acuity. (A) Example of stimulation used to measure visual acuity by optomotor task (OPT). (B) Average OPT visual in WT-v (n = 14), KO-v (n = 20), KO-k15 (n =11) and KO-k30 (n = 21) mice. Visual acuity was significantly reduced in KO-v (Two-way ANOVA, ** p ≤ 0.01,*** p ≤ 0.001, Bonferroni post-test), whereas ketamine treatment delayed the visual regression. At P55, KO-k15 and KO-k30 acuity was significantly higher than KO-v (Two-way ANOVA, # p ≤ 0.05, Bonferroni post-test), but still statistically lower than WT-v (Two-way ANOVA, * p ≤ 0.05,** p ≤ 0.01, Bonferroni post-test).

Figure. 5.

Ketamine increases neuronal activities and response reliability in visual cortex. (A) Representative spike trains and corresponding peristimulus time histogram (PSTH) in response to two oriented gratings or a uniform gray stimulus (8 presentations each). (B, C) Averages of maximal evoked and spontaneous activities. Ketamine treatment significantly increased both types of activity in both treatment paradigms (Kruskal-Wallis, * p ≤ 0.05, ** p ≤ 0.01,*** p ≤ 0.001, Dunn’s post-test). (D, E) Coefficient of variation of the response to the preferred gratings. Only KO-k30 had a restored coefficient of variation value (Kolmogorov-Smirnov test, ** p ≤ 0.01; *** p ≤ 0.001. WT-v15, n = 74; KO-v15, n = 66; KO-k15, n = 44; WT-v30, n = 57; KO-v30, n = 60; KO-k30, n = 138 cells).

Figure. 6.

Prolonged ketamine treatment restores parvalbumin-circuit inputs onto pyramidal cells. (A) Representative confocal high magnification images showing parvalbumin (PV, green) and GAD65 (GAD, red) in WT-v, KO-v, KO-k15 and KO-k30. Scale bar 10 mm. (B) PV-cell innervations of pyramidal cell somata were statistically increased in KO-v compared to WT-v (Kruskal-Wallis, ** p ≤ 0.01, Dunn’s post-test). Both ketamine treatments reduced PV-innervations towards WT levels. (C) PV-PV connections were not affected by the loss of Mecp2 or by the ketamine treatments. (WT-v15, n = 3; KO-v15, n = 3; KO-k15, n = 4; WT-v30, n = 3; KO-v30, n = 4; KO-k30, n = 4 mice). Data are expressed as mean ± SEM.

Figure. 7.

Ketamine preferentially modulates parvalbumin cortical circuits leading to a rebalancing of cortical activity in Mecp2 KO mice. Excitatory/inhibitory (E/I) imbalance underlies impairment in cortical processing. Increased parvalbumin (PV) puncta density onto pyramidal cells leads to a silent cortex. The NMDAR antagonist ketamine primarily acts on NMDARs localized on PV-cells. It reduces their spiking activity, results in the disinhibition of pyramidal cells, and thereby renormalizes the E/I balance.