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

Open AccessPublished:August 24, 2015DOI:https://doi.org/10.1016/j.biopsych.2015.08.018

      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

      Rett syndrome (RTT) is a severe progressive neurological disorder that mainly affects girls and is characterized by early neurological regression followed by loss of acquired cognitive, social, and motor skills, together with development of autistic behavior (
      • Hagberg B.
      Clinical manifestations and stages of Rett syndrome.
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      The story of Rett syndrome: From clinic to neurobiology.
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      Rett syndrome: Revised diagnostic criteria and nomenclature.
      ). The disease typically manifests around 6 to 18 months and represents the second most common cause of severe intellectual disability in the female gender (
      • Amir R.E.
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      • Wan M.
      • Tran C.Q.
      • Francke U.
      • Zoghbi H.Y.
      Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2.
      ) with a frequency of approximately 1:10,000 (
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      • Bower C.
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      Rett syndrome in Australia: A review of the epidemiology.
      ). Classic RTT is due to de novo mutations in an X-linked gene that encodes for methyl-CpG-binding protein 2 (MECP2) (
      • Amir R.E.
      • Van den Veyver I.B.
      • Wan M.
      • Tran C.Q.
      • Francke U.
      • Zoghbi H.Y.
      Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2.
      ). MeCP2 is a multifunctional chromatin protein that regulates gene expression by either repressing or activating transcription or by functioning at a post-transcriptional level (
      • Lyst M.J.
      • Bird A.
      Rett syndrome: A complex disorder with simple roots.
      ).
      Mecp2-null mice recapitulate key neurological phenotypes observed in RTT patients beginning at 5 to 6 weeks of age, whereas female heterozygous mice show delayed onset of overt signs (4 to 12 months) (
      • Chen R.Z.
      • Akbarian S.
      • Tudor M.
      • Jaenisch R.
      Deficiency of methyl-CpG binding protein-2 in CNS neurons results in a Rett-like phenotype in mice.
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      • Hendrich B.
      • Holmes M.
      • Martin J.E.
      • Bird A.
      A mouse Mecp2-null mutation causes neurological symptoms that mimic Rett syndrome.
      ). Similar to the neuroanatomical findings in humans, murine Rett models also display microcephaly without gross neuropathological changes or neurodegeneration (
      • Chen R.Z.
      • Akbarian S.
      • Tudor M.
      • Jaenisch R.
      Deficiency of methyl-CpG binding protein-2 in CNS neurons results in a Rett-like phenotype in mice.
      ,
      • Guy J.
      • Hendrich B.
      • Holmes M.
      • Martin J.E.
      • Bird A.
      A mouse Mecp2-null mutation causes neurological symptoms that mimic Rett syndrome.
      ). A decrease in synaptic plasticity, or the ability of neurons to change their synaptic strength in response to activity, is also observed in many neuronal types (
      • Asaka Y.
      • Jugloff D.G.M.
      • Zhang L.
      • Eubanks J.H.
      • Fitzsimonds R.M.
      Hippocampal synaptic plasticity is impaired in the Mecp2-null mouse model of Rett syndrome.
      ,
      • Guy J.
      • Gan J.
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      • Bird A.
      Reversal of neurological defects in a mouse model of Rett syndrome.
      ). The loss of MECP2 mainly induces a progressive imbalance of excitatory-inhibitory synaptic activity across brain circuits (
      • Shepherd G.M.G.
      • Katz D.M.
      Synaptic microcircuit dysfunction in genetic models of neurodevelopmental disorders: Focus on Mecp2 and Met.
      ,
      • Kron M.
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      • Christian D.
      • Ogier M.
      • Katz D.M.
      Brain activity mapping in Mecp2 mutant mice reveals functional deficits in forebrain circuits, including key nodes in the default mode network, that are reversed with ketamine treatment.
      ,
      • Durand S.
      • Patrizi A.
      • Quast K.B.
      • Hachigian L.
      • Pavlyuk R.
      • Saxena A.
      • et al.
      NMDA receptor regulation prevents regression of visual cortical function in the absence of Mecp2.
      ), either in favor of excitation or inhibition depending on the brain region and the developmental time point. Notably, hyperexcitability is a widespread feature of brainstem nuclei (
      • Kron M.
      • Howell C.J.
      • Adams I.T.
      • Ransbottom M.
      • Christian D.
      • Ogier M.
      • Katz D.M.
      Brain activity mapping in Mecp2 mutant mice reveals functional deficits in forebrain circuits, including key nodes in the default mode network, that are reversed with ketamine treatment.
      ,
      • Kline D.D.
      • Ogier M.
      • Kunze D.L.
      • Katz D.M.
      Exogenous brain-derived neurotrophic factor rescues synaptic dysfunction in Mecp2-null mice.
      ,
      • Medrihan L.
      • Tantalaki E.
      • Aramuni G.
      • Sargsyan V.
      • Dudanova I.
      • Missler M.
      • Zhang W.
      Early defects of GABAergic synapses in the brainstem of a MeCP2 mouse model of Rett syndrome.
      ,
      • Taneja P.
      • Ogier M.
      • Brooks-Harris G.
      • Schmid D.A.
      • Katz D.M.
      • Nelson S.B.
      Pathophysiology of locus ceruleus neurons in a mouse model of Rett syndrome.
      ) and hippocampus (
      • Zhang L.
      • He J.
      • Jugloff D.G.M.
      • Eubanks J.H.
      The MeCP2-null mouse hippocampus displays altered basal inhibitory rhythms and is prone to hyperexcitability.
      ,
      • Calfa G.
      • Hablitz J.J.
      • Pozzo-Miller L.
      Network hyperexcitability in hippocampal slices from Mecp2 mutant mice revealed by voltage-sensitive dye imaging.
      ), while hypoexcitability characterizes forebrain regions (
      • Kron M.
      • Howell C.J.
      • Adams I.T.
      • Ransbottom M.
      • Christian D.
      • Ogier M.
      • Katz D.M.
      Brain activity mapping in Mecp2 mutant mice reveals functional deficits in forebrain circuits, including key nodes in the default mode network, that are reversed with ketamine treatment.
      ,
      • Durand S.
      • Patrizi A.
      • Quast K.B.
      • Hachigian L.
      • Pavlyuk R.
      • Saxena A.
      • et al.
      NMDA receptor regulation prevents regression of visual cortical function in the absence of Mecp2.
      ,
      • Dani V.S.
      • Chang Q.
      • Maffei A.
      • Turrigiano G.G.
      • Jaenisch R.
      • Nelson S.B.
      Reduced cortical activity due to a shift in the balance between excitation and inhibition in a mouse model of Rett syndrome.
      ,
      • Wood L.
      • Gray N.W.
      • Zhou Z.
      • Greenberg M.E.
      • Shepherd G.M.G.
      Synaptic circuit abnormalities of motor-frontal layer 2/3 pyramidal neurons in an RNA interference model of methyl-CpG-binding protein 2 deficiency.
      ,
      • Wood L.
      • Shepherd G.M.G.
      Synaptic circuit abnormalities of motor-frontal layer 2/3 pyramidal neurons in a mutant mouse model of Rett syndrome.
      ). This overall network imbalance culminates with the onset of RTT regression.
      Accumulating evidence suggests that N-methyl-D-aspartate receptor (NMDAR) dysfunction significantly contributes to the regression process. Indeed, postmortem studies in RTT patients have shown irregular expression of NMDARs and an elevated glutamate/glutamine peak across brain regions (
      • Blue M.E.
      • Naidu S.
      • Johnston M.V.
      Development of amino acid receptors in frontal cortex from girls with Rett syndrome.
      ,
      • Blue M.E.
      • Naidu S.
      • Johnston M.V.
      Altered development of glutamate and GABA receptors in the basal ganglia of girls with Rett syndrome.
      ). Similar alterations in NMDAR expression and subunit composition are also present in Mecp2-null mice (
      • Asaka Y.
      • Jugloff D.G.M.
      • Zhang L.
      • Eubanks J.H.
      • Fitzsimonds R.M.
      Hippocampal synaptic plasticity is impaired in the Mecp2-null mouse model of Rett syndrome.
      ,
      • Durand S.
      • Patrizi A.
      • Quast K.B.
      • Hachigian L.
      • Pavlyuk R.
      • Saxena A.
      • et al.
      NMDA receptor regulation prevents regression of visual cortical function in the absence of Mecp2.
      ,
      • Blue M.E.
      • Kaufmann W.E.
      • Bressler J.
      • Eyring C.
      • O’driscoll C.
      • Naidu S.
      • Johnston M.V.
      Temporal and regional alterations in NMDA receptor expression in Mecp2-null mice.
      ,
      • Mierau S.
      • Patrizi A.
      • Hensch T.
      • Fagiolini M.
      Cell-specific regulation of NMDA receptor maturation by MeCP2 in cortical circuits.
      ). Renormalizing NMDAR composition in Mecp2-null mice by genetically deleting the NMDAR subunit, GluN2A, strikingly prevents regression of visual cortical function and preserves neuronal cortical activity (
      • Durand S.
      • Patrizi A.
      • Quast K.B.
      • Hachigian L.
      • Pavlyuk R.
      • Saxena A.
      • et al.
      NMDA receptor regulation prevents regression of visual cortical function in the absence of Mecp2.
      ). Moreover, acute administration of a subanesthetic dosage of ketamine, an ionotropic glutamatergic NMDAR antagonist, is sufficient to reverse forebrain hypofunction and rescue sensorimotor gating behavioral abnormalities in Mecp2-null mice (
      • Kron M.
      • Howell C.J.
      • Adams I.T.
      • Ransbottom M.
      • Christian D.
      • Ogier M.
      • Katz D.M.
      Brain activity mapping in Mecp2 mutant mice reveals functional deficits in forebrain circuits, including key nodes in the default mode network, that are reversed with ketamine treatment.
      ). Ketamine induces cortical disinhibition in human and in behaving animal studies (
      • Breier A.
      • Malhotra A.K.
      • Pinals D.A.
      • Weisenfeld N.I.
      • Pickar D.
      Association of ketamine-induced psychosis with focal activation of the prefrontal cortex in healthy volunteers.
      ,
      • Jackson M.E.
      • Homayoun H.
      • Moghaddam B.
      NMDA receptor hypofunction produces concomitant firing rate potentiation and burst activity reduction in the prefrontal cortex.
      ) by preferentially modulating NMDAR activity onto parvalbumin (PV) fast-spiking inhibitory neurons (
      • Kinney J.W.
      • Davis C.N.
      • Tabarean I.
      • Conti B.
      • Bartfai T.
      • Behrens M.M.
      A specific role for NR2A-containing NMDA receptors in the maintenance of parvalbumin and GAD67 immunoreactivity in cultured interneurons.
      ,
      • Behrens M.M.
      • Ali S.S.
      • Dao D.N.
      • Lucero J.
      • Shekhtman G.
      • Quick K.L.
      • Dugan L.L.
      Ketamine-induced loss of phenotype of fast-spiking interneurons is mediated by NADPH-oxidase.
      ). Notably, the loss of MECP2 induces an early maturation of PV-positive cells, which leads to increased inhibitory innervations onto pyramidal cells and reduced neuronal activity in the visual cortex (
      • Durand S.
      • Patrizi A.
      • Quast K.B.
      • Hachigian L.
      • Pavlyuk R.
      • Saxena A.
      • et al.
      NMDA receptor regulation prevents regression of visual cortical function in the absence of Mecp2.
      ).
      In the present study, we tested the hypothesis that a chronic pharmacologic manipulation of the NMDAR can rescue or prevent onset of RTT phenotypes. Here, we evaluated the effects of low-dose, daily ketamine treatment in Mecp2-null mice. In particular, we assessed the safety and efficacy of two treatment paradigms starting at different key time points of development: 1) from eye opening at postnatal day (P) 15, when the first alteration in the PV cortical circuits is observed but before the onset of RTT phenotypes; and 2) from P30, at the onset of RTT symptoms and when the visual regression begins (
      • Durand S.
      • Patrizi A.
      • Quast K.B.
      • Hachigian L.
      • Pavlyuk R.
      • Saxena A.
      • et al.
      NMDA receptor regulation prevents regression of visual cortical function in the absence of Mecp2.
      ). Our results indicate that both prolonged ketamine treatment paradigms were well tolerated and significantly extended the life span of Mecp2-null mice. At the cortical level, PV-circuits were no longer hyperconnected and neuronal excitatory circuits exhibited wild-type (WT) level of neuronal activity.

      Methods and Materials

      Study Design

      The primary outcome measures of the study included 1) ketamine tolerance in healthy animals, 2) behavioral analysis of ketamine-treated Mecp2-null mice and 3) analysis of visual cortical circuits.
      The treatment and genotype were randomized and blinded from team members (Supplemental Figure S1). End points were predefined as the presence or absence of statistically significant differences of the above-listed parameters. No outliers were eliminated.

      Animals

      All procedures were approved Boston Children’s Hospital Institutional Animal Care and Use Committee and conducted in Mecp2-null mouse line (B6.129P2(C)-Mecp2tm1.1Bird/J) crossed with C57BL/6J mice (
      • Guy J.
      • Hendrich B.
      • Holmes M.
      • Martin J.E.
      • Bird A.
      A mouse Mecp2-null mutation causes neurological symptoms that mimic Rett syndrome.
      ). In vivo recordings were also performed on GLUN2A knockout (KO) mice (from M. Mishina, Ph.D., University of Tokyo) (
      • Fagiolini M.
      • Katagiri H.
      • Miyamoto H.
      • Mori H.
      • Grant S.G.N.
      • Mishina M.
      • Hensch T.K.
      Separable features of visual cortical plasticity revealed by N-methyl-D-aspartate receptor 2A signaling.
      ).

      Drug Treatment

      Ketamine HCl (Hospira, Lack Forest, Illinois) was dissolved in saline (.9% sodium chloride [NaCl]), which also served as the vehicle control. Ketamine (8 mg/kg) was administered daily via intraperitoneal (IP) injection at the same time each day. Animals across multiple litters were randomly assigned to a treatment group. Each litter contributed at least a Mecp2 KO and a WT mouse to the study.
      Mecp2 WT and KO mice were divided into two groups: 1) P15 to P55 paradigm (40 days) and 2) P30 to P55 paradigm (25 days).

      Pharmacokinetic Analysis

      P15, P30, and adult C57BL/6J mice received a single intraperitoneal dose of ketamine (8 mg/kg; n = 3/dose/time point). Blood and brains were collected for analysis at specific time points (Supplement).

      Neurobehavioral Characterization

      Weight and general condition of the animals were evaluated daily. All tests were performed at the same time of day and in the same dedicated observation room within the Neurodevelopmental Behavioral Core at Boston Children’s Hospital (Supplement).

      Spontaneous Locomotor Activity

      The distance traveled (in centimeters) and the mean velocity (in cm/s) were recorded in 5-minute periods with ActiTrack software (Panlab/Harvard Apparatus, Cornellà, Spain).

      Phenotypic Scoring

      Animals were scored using the RTT phenotypic severity scoring system described previously (
      • Guy J.
      • Gan J.
      • Selfridge J.
      • Cobb S.
      • Bird A.
      Reversal of neurological defects in a mouse model of Rett syndrome.
      ).

      Rotarod

      Animals were placed on a rotating rod apparatus (Economex Enclosure, Columbus Instruments, Columbus, Ohio), at a constant speed of 4 rpm for 10 seconds for acclimatization. The test session ended when the animal fell off the rod.

      Prepulse Inhibition of the Startle Reflex

      Prepulse inhibition of the startle index (PPI) was defined as the percentage reduction in mean startle response magnitude for each mouse at each prepulse and control trial.
      %PPI=100×[(pulsealone)(prepulse+pulse)]/pulsealone


      Optomotor Task

      Visual threshold acuity was evaluated using the optomotor task (
      • Prusky G.T.
      • Alam N.M.
      • Beekman S.
      • Douglas R.M.
      Rapid quantification of adult and developing mouse spatial vision using a virtual optomotor system.
      ) (Cerebral Mechanics, Lethbridge, Alberta, Canada). Vehicle- and ketamine-treated mice were tested at P30, P40, and P55.

      Whole-Body Plethysmography

      Breathing was recorded from unrestrained awake mice at P30 and between P48 and P55 using a constant flow whole-body plethysmograph (200 mL chamber) (EMKA Technologies, Paris, France) (
      • Zanella S.
      • Mebarek S.
      • Lajard A.M.
      • Picard N.
      • Dutschmann M.
      • Hilaire G.
      Oral treatment with desipramine improves breathing and life span in Rett syndrome mouse model.
      ). Mice were kept for 1 hour in the chamber. Only periods of quiet breathing during the last 20 minutes were analyzed to measure the number of apneas per minute. Apneas were defined when the breath holding was longer than two normal respiratory cycles.

      In Vivo Single Unit Recordings

      In vivo recordings were performed at P55 to P60 under Nembutal (50 mg/kg, IP)/chlorprothixene (.025 mg/kg, intramuscular) anesthesia using standard techniques (
      • Durand S.
      • Patrizi A.
      • Quast K.B.
      • Hachigian L.
      • Pavlyuk R.
      • Saxena A.
      • et al.
      NMDA receptor regulation prevents regression of visual cortical function in the absence of Mecp2.
      ). Cortical activity in the binocular zone of primary visual cortex was recorded using multichannel probes (A1x16-3mm50-177; Neuronexus Technologies, Ann Arbor, Michigan) (Supplement).

      Immunohistochemistry

      Primary antibodies and dilutions are detailed in the Supplement. Quantitative analyses of the binocular zone of visual cortex across all layers were performed blind to genotype and treatment. Mean pixel intensity of the PV signal in each field (1024 × 1024) was measured using MacBiophotonics ImageJ software (Bethesda, Maryland). The number of perisomatic synapses (at 100×) was determined on triple-stained images (PV, glutamic acid decarboxylase 65, DAPI) using the particle analysis function (ImageJ). NeuN-positive cell density was quantified per area by using ImageJ software and per volume with Volocity (version 5.5; PerkinElmer, Cambridge, Massachusetts).

      Western Blot

      Mecp2 WT and KO mice were acutely injected with ketamine 8 mg/kg either at P15 or P30. Visual cortices were dissected an hour later (Supplement).

      Statistical Analysis

      All data are presented as mean ± standard error. Behavioral differences between treatment groups were carried out using Kruskal-Wallis test, Kaplan-Meier, chi-square, and two-way analysis of variance as appropriate. In vivo recordings and immunohistochemistry quantification were compared using Kruskal-Wallis and Kolmogorov-Smirnov tests as appropriate. Statistical significance was defined as p ≤ .05. All statistics were performed using GraphPad Prism (version 5.0) software (GraphPad, La Jolla, California).

      Results

      Low-Dose Ketamine Does Not Induce Negative Behavioral Outcomes

      Despite the fact ketamine is widely used as an anesthetic and analgesic in pediatric clinical practice, it is well known that it may cause adverse effects when administered at high doses (
      • Morgan C.J.A.
      • Curran H.V.
      Ketamine use: A review.
      ). Therefore, we decided to perform a series of studies to evaluate brain penetrance and exclude any detrimental effect of the low dosage of ketamine (8 mg/kg).
      We first conducted a pharmacokinetic analysis to quantify ketamine penetrance in the brain following a single intraperitoneal injection of 8 mg/kg in adult WT mice (Figure 1A). As previously reported, ketamine showed preferential distribution with a brain to plasma ratio of approximately 2 to 1 (Figure 1A; Table 1) (
      • Shaffer C.L.
      • Osgood S.M.
      • Smith D.L.
      • Liu J.
      • Trapa P.E.
      Enhancing ketamine translational pharmacology via receptor occupancy normalization.
      ) and was quickly eliminated (plasma elimination half-life = 1.1 ± .8 hours).
      Figure thumbnail gr1
      Figure 1Ketamine 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 wild-type (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 analysis of variance, p ≥ .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 prepulse inhibition response (Kruskal-Wallis, **p ≤ .01, Dunn’s post-test. WT-saline, n = 10; WT-ketamine 8 mg/kg, n = 10; WT-ketamine 56 mg/kg, n = 8 mice).
      Table 1Mouse Pharmacokinetic Parameters After 8 mg/kg Intraperitoneal Administration of Ketamine
      Cmax (ng/mL)V (L)AUCinf (ng/h/L)t1/2,α (h)t1/2,β (h)CLd (L/h)CL (L/h)B/P Ratio
      P15463 ± 87115 ± 38122 ± 26.05 ± .03.28 ± .17157 ± 7871 ± 1214.7 ± 1
      P301760 ± 23181 ± 12296 ± 48.09 ± .03.25 ± .13284 ± 8630 ± 7.28.7 ± 3.7
      Adults1209 ± 152.569 ± 4366 ± 59.07 ± .011.11 ± .8326 ± 5148 ± 32.7 ± .5
      Data are expressed as mean ± SEM.
      AUCinf, area under the concentration time curve (AUC) with the last concentration extrapolated based on the elimination rate constant; B/P, brain to plasma ratio; CL, systemic clearance; CLd, distribution clearance; Cmax, maximum concentration; P, postnatal day; t1/2,α distribution of half-life; t1/2,β, elimination half-life; V, volume of distribution.
      Major side effects of ketamine administered at high, subanesthetic doses include agitation, confusion, and psychosis (
      • Rowland L.M.
      Subanesthetic ketamine: How it alters physiology and behavior in humans.
      ). Similarly in rodents, acute administration of a high dose of ketamine (more than 20 mg/kg) produces behavioral abnormalities such as disruption of sensorimotor gating, impaired cognitive function, and hyperlocomotor activity (
      • Irifune M.
      • Shimizu T.
      • Nomoto M.
      Ketamine-induced hyperlocomotion associated with alteration of presynaptic components of dopamine neurons in the nucleus accumbens of mice.
      ,
      • Verma A.
      • Moghaddam B.
      NMDA receptor antagonists impair prefrontal cortex function as assessed via spatial delayed alternation performance in rats: Modulation by dopamine.
      ,
      • Cilia J.
      • Hatcher P.
      • Reavill C.
      • Jones D.N.C.
      (+/-) Ketamine-induced prepulse inhibition deficits of an acoustic startle response in rats are not reversed by antipsychotics.
      ,
      • Imre G.
      • Fokkema D.S.
      • Boer J.A. Den
      • Ter Horst G.J.
      Dose-response characteristics of ketamine effect on locomotion, cognitive function and central neuronal activity.
      ). Importantly, lower dosages 2.5 - 10 mg/kg elicit few if any of these adverse effects (
      • Cilia J.
      • Hatcher P.
      • Reavill C.
      • Jones D.N.C.
      (+/-) Ketamine-induced prepulse inhibition deficits of an acoustic startle response in rats are not reversed by antipsychotics.
      ,
      • Imre G.
      • Fokkema D.S.
      • Boer J.A. Den
      • Ter Horst G.J.
      Dose-response characteristics of ketamine effect on locomotion, cognitive function and central neuronal activity.
      ).
      To confirm that 8 mg/kg of ketamine does not trigger major behavioral side effects in WT mice, we measured spontaneous locomotor activity and PPI of the acoustic startle reflex (
      • Braff D.L.
      • Geyer M.A.
      • Light G.A.
      • Sprock J.
      • Perry W.
      • Cadenhead K.S.
      • Swerdlow N.R.
      Impact of prepulse characteristics on the detection of sensorimotor gating deficits in schizophrenia.
      ). We used an open field to quantify the distance traveled (in centimeters) and the mean velocity (in cm/s) for 30 minutes immediately after a single injection of ketamine and found no significant difference compared with control mice injected with vehicle (Figure 1B). We then analyzed acoustic startle and PPI responses after an acute administration of ketamine or vehicle. Responses in both tests were unaffected by ketamine when administered at 8 mg/kg (Figure 1C). On the contrary, at a higher dose (56 mg/kg), ketamine induced a slight decrease in the startle response and a significant reduction of PPI (Figure 1C).
      Next, we evaluated chronic ketamine (k) treatments in WT mice that started either at P15 or P30 until P55 (WT-k15 or WT-k30, respectively). Both treatments failed to elicit any disruption of the basic acoustic startle and PPI responses (Supplemental Figure S2). In addition, prolonged ketamine administration in WT mice did not evoke weight loss (Supplemental Figure S3A) (
      • Venâncio C.
      • Magalhães A.
      • Antunes L.
      • Summavielle T.
      Impaired spatial memory after ketamine administration in chronic low doses.
      ) or neuronal cell death (Supplemental Figure S3B, C) (
      • Wang C.
      • Zheng D.
      • Xu J.
      • Lam W.
      • Yew D.T.
      Brain damages in ketamine addicts as revealed by magnetic resonance imaging.
      ).
      Finally, we tested whether acute administration of low-dosage ketamine would enhance neuronal activity of excitatory cortical circuits as previously reported (
      • Jackson M.E.
      • Homayoun H.
      • Moghaddam B.
      NMDA receptor hypofunction produces concomitant firing rate potentiation and burst activity reduction in the prefrontal cortex.
      ,
      • Kinney J.W.
      • Davis C.N.
      • Tabarean I.
      • Conti B.
      • Bartfai T.
      • Behrens M.M.
      A specific role for NR2A-containing NMDA receptors in the maintenance of parvalbumin and GAD67 immunoreactivity in cultured interneurons.
      ). We found that 8 mg/kg of ketamine was sufficient to increase spontaneous and evoked neurotransmission in pyramidal neurons as revealed by single unit recordings from primary visual cortex (Supplemental Figure S4A). Interestingly, we did not see such potentiation in GluN2A KO mice (Supplemental Figure S4B), supporting the notion that ketamine mainly acts through NMDARs containing GluN2A subunit.
      Overall, our data demonstrate that ketamine dosed at 8 mg/kg rapidly accumulates in the brain, does not induce negative side effects after acute or chronic administration, and significantly increases cortical activity by targeting NMDARs.

      Randomized Preclinical Study with Chronic Ketamine Administration

      Mecp2-null (KO) mice were injected daily IP with either vehicle (v) (KO-v) or ketamine (KO-k) starting at P15 (KO-15) or P30 (KO-30). The treatment was continued until P55 (Supplemental Figure S1A). Control groups were Mecp2 WT littermates treated with vehicle (WT-v15 and WT-v30). A total of 67 KO and 37 WT mice were used in the study.
      Littermates from multiple litters were randomly allocated to the different treatment groups. Drugs were administered at the same time each day. Every 15 days, starting at P30, a tailored battery of validated behavioral tests was carried out 23 hours after the last injection (Supplemental Figure S1 and Supplemental Methods and Materials). Each behavioral test was performed at the same time of day by the same person. Individuals caring for the animals, administering the drug, performing the experiments, and quantifying the outcomes were blinded to the genotype and treatments (Supplemental Figure S1B).

      Effect of Prolonged Ketamine Treatment on RTT Symptoms

      To determine whether ketamine could prevent or delay onset of RTT phenotype, we injected Mecp2-null mice with either vehicle (KO-v15; KO-v30) or ketamine (KO-k15; KO-k30) and evaluated multiple outcomes up to P55. Both groups of KO-v mice showed markedly reduced survival compared with WT-v mice, as expected for this RTT mouse model (
      • Guy J.
      • Hendrich B.
      • Holmes M.
      • Martin J.E.
      • Bird A.
      A mouse Mecp2-null mutation causes neurological symptoms that mimic Rett syndrome.
      ) (Supplemental Figure S5A; median survival P53). On the contrary, both groups of KO-k mice showed significantly extended survival compared with KO-v mice (median survival P83 for both KO-k15 and KO-k30). Interestingly, at P80, 47% of the KO-k15 and 36% of the KO-k30 mice were alive, while all of the KO-v mice were already deceased (Figure 2A, insert). Ketamine-treated Mecp2 KO mice still exhibited reduced body weight compared with WT littermates (Figure 2B).
      Figure thumbnail gr2
      Figure 2Prolonged ketamine treatment improves key Rett syndrome (RTT)-like phenotypes. (A) Survival curves in wild-type (WT) (empty circle) and in Mecp2 knockout (KO) treated with vehicle (v) (black-filled circle) or ketamine (k) from postnatal day (P)15 (magenta) or from P30 (green) revealed prolonged ketamine treatment improved the life span of Mecp2 KO mice. (B) No improvement of the body weight of P55 treated Mecp2 KO mice was observed (Kruskal-Wallis, **p ≤ .01; ***p ≤ .001, Dunn’s posttest). 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 mice treated with ketamine at P15 (KO-k15) had a mild score compared with their age-matched KO mice treated with vehicle (KO-v). KO mice treated with ketamine at P30 (KO-k30) did not show an improvement in the RTT score severity. (D) The clasping phenotypic score was improved mainly in the KO-k15 mice (WT mice treated with vehicle [WT-v], n = 13; KO-v, n = 18; KO-k15, n = 14; KO-k30, n = 17 mice). Data are expressed as mean ± SEM. ns, not significant.
      The prolonged life span was instead accompanied by overall improved health, particularly in KO-k15 mice, as measured using an established observational scoring system (Figure 2C; Supplemental Figure S5B) (
      • Guy J.
      • Gan J.
      • Selfridge J.
      • Cobb S.
      • Bird A.
      Reversal of neurological defects in a mouse model of Rett syndrome.
      ,
      • Weng S.M.
      • McLeod F.
      • Bailey M.E.S.
      • Cobb S.R.
      Synaptic plasticity deficits in an experimental model of rett syndrome: Long-term potentiation saturation and its pharmacological reversal.
      ). At P55, all WT-v mice had a score lower than 2, while 43% of the KO-v mice exhibited severe RTT-like symptoms (score > 6). Interestingly, ketamine treatment from P15 shifted the RTT score toward a mild RTT phenotype with only 25% of animals displaying the highest level of phenotypic severity (Figure 2C). Among the several RTT phenotypes scored, we found that the hindlimb-clasping phenotype, an indication of neurological and motor dysfunction (
      • Auerbach W.
      • Hurlbert M.S.
      • Hilditch-Maguire P.
      • Wadghiri Y.Z.
      • Wheeler V.C.
      • Cohen S.I.
      • et al.
      The HD mutation causes progressive lethal neurological disease in mice expressing reduced levels of huntingtin.
      ), was significantly reduced in KO-k15 (Figure 2D).
      We then evaluated the effect of ketamine treatments on motor performance and apnea incidence, two prominent features of RTT (
      • Katz D.M.
      • Dutschmann M.
      • Ramirez J.M.
      • Hilaire G.
      Breathing disorders in Rett syndrome: Progressive neurochemical dysfunction in the respiratory network after birth.
      ,
      • Neul J.L.
      • Kaufmann W.E.
      • Glaze D.G.
      • Christodoulou J.
      • Clarke A.J.
      • Bahi-Buisson N.
      • et al.
      Rett syndrome: Revised diagnostic criteria and nomenclature.
      ). Mice were tested on a nonaccelerating rotarod in four consecutive trials (Supplemental Figure S6A, B). As expected, motor performance of WT-v mice improved with repetition and age, whereas KO-v mice did not improve and instead became significantly worse with age. Both ketamine treatments partially improved motor coordination. Indeed, KO-k15 mice stayed longer on the rod compared with KO-v mice at both ages. KO-k30 mice started at the same level as the KO-v mice at P30 but did not regress with age (Supplemental Figure S6A). Learning performance, evaluated by testing the mice over successive trials, showed that KO-v mice did not show any improvement between the first and the last trials. KO-k15 mice significantly increased their motor performances at the last trial only at P45, whereas KO-k30 mice improved their performance by P55 but not at P45 (Supplemental Figure S6B).
      Finally, we evaluated breathing in awake, unrestrained mice using whole-body plethysmography at P30 and P55 (Figure 3A, B). The total respiratory cycle duration was measured for at least 230 cycles (mean 1303 ± 160 cycles) acquired during a period of quiet breathing. The count of apneas per minute (> 2 normal total respiratory cycle durations) was then quantified as previously described (
      • Zanella S.
      • Mebarek S.
      • Lajard A.M.
      • Picard N.
      • Dutschmann M.
      • Hilaire G.
      Oral treatment with desipramine improves breathing and life span in Rett syndrome mouse model.
      ). The number of apneas per minute dramatically increased in KO-v mice by P50 (
      • Zanella S.
      • Mebarek S.
      • Lajard A.M.
      • Picard N.
      • Dutschmann M.
      • Hilaire G.
      Oral treatment with desipramine improves breathing and life span in Rett syndrome mouse model.
      ,
      • Viemari J.-C.
      • Roux J.-C.
      • Tryba A.K.
      • Saywell V.
      • Burnet H.
      • Peña F.
      • et al.
      Mecp2 deficiency disrupts norepinephrine and respiratory systems in mice.
      ). Interestingly, ketamine treatment delayed the onset of apneas in the KO-k15 mice, while preventing worsening of apnea episodes when treatment started from P30 (Figure 3B).
      Figure thumbnail gr3
      Figure 3Ketamine 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 postnatal day (P)30 prevented the developmental increase of apneic episodes (Wilcoxon signed-rank test, *p < .05, **p ≤ .01. wild-type [WT] mice treated with vehicle [WT-v], n = 8; knockout [KO] mice treated with vehicle [KO-v], n = 11; KO mice treated with ketamine at P15 [KO-k15], n = 7; KO mice treated with ketamine at P30 [KO-k30], n = 8 mice). ns, not significant.
      Overall, these data suggest that both chronic ketamine treatments significantly ameliorate RTT neurological phenotypes without detrimental effects.

      Effect of Ketamine Treatment on Visual Cortical Processing

      We have previously demonstrated that the visual system is a reliable biomarker of regression in Mecp2-null mice (
      • Durand S.
      • Patrizi A.
      • Quast K.B.
      • Hachigian L.
      • Pavlyuk R.
      • Saxena A.
      • et al.
      NMDA receptor regulation prevents regression of visual cortical function in the absence of Mecp2.
      ). We therefore chose to investigate the effect of ketamine treatments on visual function using an optomotor (OPT) task to assess the response of unrestrained animals to high-contrast moving gratings. As previously shown (
      • Durand S.
      • Patrizi A.
      • Quast K.B.
      • Hachigian L.
      • Pavlyuk R.
      • Saxena A.
      • et al.
      NMDA receptor regulation prevents regression of visual cortical function in the absence of Mecp2.
      ), all mice reached adult acuity by P30, but then KO-v mice exhibited a rapid regression between P30 and P55. Consequently, at P45 and P55, the OPT visual acuity in KO-v mice was significantly lower than age-matched WT-v mice (Figure 4B). Interestingly, both ketamine treatments slowed down regression of visual acuity. At P45, the OPT visual acuity was not different between WT and KO treated mice. At P55, although KO-k mice displayed a lower OPT acuity than WT-v mice, it was significantly higher than in KO-v mice (Figure 4B).
      Figure thumbnail gr4
      Figure 4Ketamine delays regression of the visual acuity. (A) Example of stimulation used to measure visual acuity by optomotor task (OPT). (B) Average OPT visual in wild-type (WT) mice treated with vehicle (WT-v), (n = 14), knockout (KO) mice treated with vehicle (KO-v) (n = 20), KO mice treated with ketamine at postnatal day (P)15 (KO-k15) (n =11), and KO mice treated with ketamine at P30 (KO-k30) (n = 21). Visual acuity was significantly reduced in KO-v (two-way analysis of variance [ANOVA], **p ≤ .01, ***p ≤ .001, Bonferroni posttest), 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 ≤ .05, Bonferroni posttest) but still statistically lower than WT-v (two-way ANOVA, *p ≤ .05,**p ≤ .01, Bonferroni posttest).
      We then performed in vivo extracellular recordings of spontaneous and maximal-evoked responses of pyramidal cells in response to drifting sinusoidal gratings. At P55 to P60, both the maximal-evoked response and spontaneous activity were significantly reduced in KO-v mice, confirming that visual circuits are largely silent in Mecp2-null mice compared with WT littermates (Figure 5B, C) (
      • Durand S.
      • Patrizi A.
      • Quast K.B.
      • Hachigian L.
      • Pavlyuk R.
      • Saxena A.
      • et al.
      NMDA receptor regulation prevents regression of visual cortical function in the absence of Mecp2.
      ). In addition, neuronal responses exhibited higher variability to repeated presentation of the preferred stimulus (coefficient of variation; Figure 5D, E), suggesting less reliable signal processing and cognitive impairments (
      • Russell V.A.
      • Oades R.D.
      • Tannock R.
      • Killeen P.R.
      • Auerbach J.G.
      • Johansen E.B.
      • Sagvolden T.
      Response variability in attention-deficit/hyperactivity disorder: A neuronal and glial energetics hypothesis.
      ). Remarkably, both ketamine treatments increased the evoked and spontaneous activities to WT level. However, only the treatment starting at P30 was effective to renormalize the coefficient of variation to WT level (Figure 5E). Together, these results suggest that the treatment starting from P30 was more effective for renormalizing visual cortical processing in Mecp2-null mice.
      Figure thumbnail gr5
      Figure 5Ketamine increases neuronal activities and response reliability in visual cortex. (A) Representative spike trains and corresponding peristimulus time histogram in response to two oriented gratings or a uniform gray stimulus (eight 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 ≤ .05, ***p ≤ .001, Dunn’s posttest). (D, E) Coefficient of variation of the response to the preferred gratings. Only knockout (KO) mice treated with ketamine at postnatal day (P)30 (KO-k30) had a restored coefficient of variation value (Kolmogorov-Smirnov test, **p ≤ .01; ***p ≤ .001. Wild-type (WT) mice treated with vehicle at P15 [WT-v15], n = 74; KO mice treated with vehicle at P15 [KO-v15], n = 66; KO mice treated with ketamine at P15 [KO-k15], n = 44; WT mice treated with vehicle at P30 [WT-v30], n = 57; KO mice treated with vehicle at P30 [KO-v30], n = 60; KO-k30, n = 138 cells). KO-v, knockout mice treated with vehicle; ns, not significant; WT-v, wild-type mice treated with vehicle.

      Effect of Prolonged Ketamine on Parvalbumin Connectivity

      The spiking activity of cortical pyramidal cells is controlled by PV-positive inhibitory cells. Importantly, PV circuits are particularly sensitive to the inhibiting effects of ketamine and determine the overall disinhibition of cortical activity (
      • Kinney J.W.
      • Davis C.N.
      • Tabarean I.
      • Conti B.
      • Bartfai T.
      • Behrens M.M.
      A specific role for NR2A-containing NMDA receptors in the maintenance of parvalbumin and GAD67 immunoreactivity in cultured interneurons.
      ,
      • Behrens M.M.
      • Ali S.S.
      • Dao D.N.
      • Lucero J.
      • Shekhtman G.
      • Quick K.L.
      • Dugan L.L.
      Ketamine-induced loss of phenotype of fast-spiking interneurons is mediated by NADPH-oxidase.
      ,
      • Jeevakumar V.
      • Kroener S.
      Ketamine administration during the second postnatal week alters synaptic properties of fast-spiking interneurons in the medial prefrontal cortex of adult mice.
      ). In the absence of MECP2, PV circuits exhibit a significant increase in neurite complexity and number of axonal perisomatic boutons onto pyramidal cells starting as early as eye opening (P15), which contributes to the silencing of cortical circuits (
      • Durand S.
      • Patrizi A.
      • Quast K.B.
      • Hachigian L.
      • Pavlyuk R.
      • Saxena A.
      • et al.
      NMDA receptor regulation prevents regression of visual cortical function in the absence of Mecp2.
      ,
      • Dani V.S.
      • Chang Q.
      • Maffei A.
      • Turrigiano G.G.
      • Jaenisch R.
      • Nelson S.B.
      Reduced cortical activity due to a shift in the balance between excitation and inhibition in a mouse model of Rett syndrome.
      ,
      • Tomassy G.S.
      • Morello N.
      • Calcagno E.
      • Giustetto M.
      Developmental abnormalities of cortical interneurons precede symptoms onset in a mouse model of Rett syndrome.
      ). Consequently, we hypothesized that ketamine treatments may have restored the normal wiring of PV circuits, allowing WT level of cortical activity.
      PV immunofluorescence intensity was significantly upregulated in KO-v compared with WT-v age-matched mice (
      • Durand S.
      • Patrizi A.
      • Quast K.B.
      • Hachigian L.
      • Pavlyuk R.
      • Saxena A.
      • et al.
      NMDA receptor regulation prevents regression of visual cortical function in the absence of Mecp2.
      ) (Supplemental Figure S7). Both ketamine treatments normalized PV intensity similar to WT-v levels in adult Mecp2-null mice (Supplemental Figure S7). This decrease was not linked to a change in PV-cell density, as no change was observed between different genotypes and drug treatments despite a reduction in cortical thickness and soma volume (Supplemental Table S1). We then quantified the density of PV-positive perisomatic boutons onto pyramidal and PV-cell somata (
      • Hensch T.K.
      Critical period plasticity in local cortical circuits.
      ). While KO-v mice exhibited a significant increase of PV-positive boutons onto pyramidal but not on PV cells, both KO-k groups were indistinguishable from WT-v control mice (Figure 6). All together, these data demonstrate that both treatments were sufficient to rewire PV circuitry in the Mecp2-null visual cortex.
      Figure thumbnail gr6
      Figure 6Prolonged ketamine treatment restores parvalbumin-circuit inputs onto pyramidal cells. (A) Representative confocal high-magnification images showing parvalbumin (PV, red) and glutamic acid decarboxylase 65 (GAD65) (GAD, green) in wild-type (WT) mice treated with vehicle (WT-v), knockout (KO) mice treated with vehicle (KO-v), KO mice treated with ketamine at postnatal day (P)15 (KO-k15), and KO mice treated with ketamine at P30 (KO-k30). Scale bar = 10 μm. (B) PV-cell innervations of pyramidal cell somata were statistically increased in KO-v compared with WT-v (Kruskal-Wallis, **p ≤ .01, Dunn’s posttest). Both ketamine treatments reduced PV innervations toward WT levels. (C) PV-PV connections were not affected by the loss of MECP2 or by the ketamine treatments. (WT mice treated with vehicle at P15 [WT-v15], n = 3; KO mice treated with vehicle at P15 [KO-v15], n = 3; KO-k15, n = 4; WT mice treated with vehicle at P30 [WT-v30], n = 3; KO mice treated with vehicle at P30 [KO-v30], n = 4; KO-k30, n = 4 mice). Data are expressed as mean ± SEM. Ket, ketamine; Veh, vehicle.

      Discussion

      Our study provides the first in vivo evidence that prolonged treatment with a low dose of the NMDAR antagonist ketamine significantly extended life span and renormalized cortical circuits without detrimental side effects. Administration of ketamine slowed down the regression of optomotor visual acuity, prevented the silencing of neuronal cortical circuits, and rewired PV circuit connectivity to WT levels. Importantly, even a treatment starting at the onset of the regression (P30) was sufficient to rescue RTT phenotype.
      Ketamine’s mechanism of action is complex, affecting not only NMDAR but also neurotransmitter and neuromodulatory systems, particularly when administered at anesthetic concentrations (
      • Sleigh J.
      • Harvey M.
      • Voss L.
      • Denny B.
      Ketamine - more mechanisms of action than just NMDA blockade.
      ). Among them, ketamine and in particular one of its metabolites, dehydronorketamine, may act on ion channels such as the nicotinic acetylcholine receptor by inhibiting the receptor in a noncompetitive and voltage-dependent manner (
      • Moaddel R.
      • Abdrakhmanova G.
      • Kozak J.
      • Jozwiak K.
      • Toll L.
      • Jimenez L.
      • et al.
      Sub-anesthetic concentrations of (R,S)-ketamine metabolites inhibit acetylcholine-evoked currents in α7 nicotinic acetylcholine receptors.
      ,
      • Yamakura T.
      • Chavez-Noriega L.E.
      • Harris R.A.
      Subunit-dependent inhibition of human neuronal nicotinic acetylcholine receptors and other ligand-gated ion channels by dissociative anesthetics ketamine and dizocilpine.
      ). Although we cannot exclude this possible mechanism of action in our experiments, we believe that this is not a major contributor due to the fact our pharmacokinetic analysis revealed that norketamine and not dehydronorketamine was the major circulating metabolite at both ages (
      • Yamakura T.
      • Chavez-Noriega L.E.
      • Harris R.A.
      Subunit-dependent inhibition of human neuronal nicotinic acetylcholine receptors and other ligand-gated ion channels by dissociative anesthetics ketamine and dizocilpine.
      ,
      • Craft J.B.
      • Coaldrake L.A.
      • Yonekura M.L.
      • Dao S.D.
      • Co E.G.
      • Roizen M.F.
      • et al.
      Ketamine, catecholamines, and uterine tone in pregnant ewes.
      ) (Supplemental Figure S8) likely targeting the NMDAR (
      • Ebert B.
      • Mikkelsen S.
      • Thorkildsen C.
      • Borgbjerg F.M.
      Norketamine, the main metabolite of ketamine, is a non-competitive NMDA receptor antagonist in the rat cortex and spinal cord.
      ).
      As a use-dependent noncompetitive antagonist, ketamine binds within the open ion channel, blocking calcium influx and working preferentially at sites of excess NMDAR activation (
      • Mealing G.A.
      • Lanthorn T.H.
      • Murray C.L.
      • Small D.L.
      • Morley P.
      Differences in degree of trapping of low-affinity uncompetitive N-methyl-D-aspartic acid receptor antagonists with similar kinetics of block.
      ). Because PV cells are continuously depolarized and exhibit high levels of firing (
      • Tseng K.Y.
      • O’Donnell P.
      Dopamine modulation of prefrontal cortical interneurons changes during adolescence.
      ), their NMDARs are likely to escape magnesium blockade. Thus, a low dose of ketamine may be sufficient to dampen their firing activity (
      • Behrens M.M.
      • Ali S.S.
      • Dao D.N.
      • Lucero J.
      • Shekhtman G.
      • Quick K.L.
      • Dugan L.L.
      Ketamine-induced loss of phenotype of fast-spiking interneurons is mediated by NADPH-oxidase.
      ,
      • Homayoun H.
      • Moghaddam B.
      NMDA receptor hypofunction produces opposite effects on prefrontal cortex interneurons and pyramidal neurons.
      ), decrease overall inhibition of cortical circuits, and tip the balance of synaptic transmission toward excitation (
      • Homayoun H.
      • Moghaddam B.
      NMDA receptor hypofunction produces opposite effects on prefrontal cortex interneurons and pyramidal neurons.
      ) (Supplemental Figure S4A).
      In the absence of MECP2, PV circuits form exuberant somatic connections onto pyramidal neurons that increase throughout development and significantly contribute to the progressive silencing of cortical circuits (
      • Kron M.
      • Howell C.J.
      • Adams I.T.
      • Ransbottom M.
      • Christian D.
      • Ogier M.
      • Katz D.M.
      Brain activity mapping in Mecp2 mutant mice reveals functional deficits in forebrain circuits, including key nodes in the default mode network, that are reversed with ketamine treatment.
      ,
      • Durand S.
      • Patrizi A.
      • Quast K.B.
      • Hachigian L.
      • Pavlyuk R.
      • Saxena A.
      • et al.
      NMDA receptor regulation prevents regression of visual cortical function in the absence of Mecp2.
      ). PV circuits develop in an activity-dependent manner and sensory deprivation can halt such anatomical and functional maturation (
      • Chattopadhyaya B.
      • Di Cristo G.
      • Higashiyama H.
      • Knott G.W.
      • Kuhlman S.J.
      • Welker E.
      • Huang Z.J.
      Experience and activity-dependent maturation of perisomatic GABAergic innervation in primary visual cortex during a postnatal critical period.
      ). Similar to dark rearing from birth (
      • Durand S.
      • Patrizi A.
      • Quast K.B.
      • Hachigian L.
      • Pavlyuk R.
      • Saxena A.
      • et al.
      NMDA receptor regulation prevents regression of visual cortical function in the absence of Mecp2.
      ,
      • Sugiyama S.
      • Di Nardo A.A.
      • Aizawa S.
      • Matsuo I.
      • Volovitch M.
      • Prochiantz A.
      • Hensch T.K.
      Experience-dependent transfer of Otx2 homeoprotein into the visual cortex activates postnatal plasticity.
      ), prolonged ketamine treatment may reduce the excitatory drive onto PV cells. This results in a rapid loss of local inhibition, a long-lasting rewiring of PV connectivity onto pyramidal neurons, and the eventual restoration of cortical activity (Figure 5; Supplemental Figure S4) (
      • Kinney J.W.
      • Davis C.N.
      • Tabarean I.
      • Conti B.
      • Bartfai T.
      • Behrens M.M.
      A specific role for NR2A-containing NMDA receptors in the maintenance of parvalbumin and GAD67 immunoreactivity in cultured interneurons.
      ,
      • Behrens M.M.
      • Ali S.S.
      • Dao D.N.
      • Lucero J.
      • Shekhtman G.
      • Quick K.L.
      • Dugan L.L.
      Ketamine-induced loss of phenotype of fast-spiking interneurons is mediated by NADPH-oxidase.
      ,
      • Jeevakumar V.
      • Kroener S.
      Ketamine administration during the second postnatal week alters synaptic properties of fast-spiking interneurons in the medial prefrontal cortex of adult mice.
      ,
      • Homayoun H.
      • Moghaddam B.
      NMDA receptor hypofunction produces opposite effects on prefrontal cortex interneurons and pyramidal neurons.
      ).
      The indirect modulation of excitatory neuron firing rate may contribute to the activity-dependent release of brain-derived neurotrophic factor (BDNF) (
      • Kuczewski N.
      • Porcher C.
      • Gaiarsa J.-L.
      Activity-dependent dendritic secretion of brain-derived neurotrophic factor modulates synaptic plasticity.
      ). However, we cannot exclude the possibility that ketamine also acts directly on pyramidal cells. These two mechanisms are not mutually exclusive and could work together to exert a long-lasting effect. Indeed, the antidepressant action of ketamine occurs by blocking tonic activation of GLUN2B-containing NMDARs in pyramidal neurons (
      • Miller O.H.
      • Yang L.
      • Wang C.-C.
      • Hargroder E.A.
      • Zhang Y.
      • Delpire E.
      • Hall B.J.
      GluN2B-containing NMDA receptors regulate depression-like behavior and are critical for the rapid antidepressant actions of ketamine.
      ) and by modulating several downstream signaling components including mammalian target of rapamycin (mTOR) pathway, glycogen synthase kinase-3, and eukaryotic elongation factor 2 (
      • Naughton M.
      • Clarke G.
      • Oleary O.F.
      • Cryan J.F.
      • Dinan T.G.
      A review of ketamine in affective disorders: Current evidence of clinical efficacy, limitations of use and pre-clinical evidence on proposed mechanisms of action.
      ). Influencing each of these targets leads to an upregulation of protein synthesis including BDNF (
      • Autry A.E.
      • Adachi M.
      • Nosyreva E.
      • Na E.S.
      • Los M.F.
      • Cheng P.
      • et al.
      NMDA receptor blockade at rest triggers rapid behavioural antidepressant responses.
      ,
      • Duncan W.C.
      • Sarasso S.
      • Ferrarelli F.
      • Selter J.
      • Riedner B.A.
      • Hejazi N.S.
      • et al.
      Concomitant BDNF and sleep slow wave changes indicate ketamine-induced plasticity in major depressive disorder.
      ), which then contributes to strengthened synaptic plasticity (
      • Kavalali E.T.
      • Monteggia L.M.
      Synaptic mechanisms underlying rapid antidepressant action of ketamine.
      ). Indeed, we found that acute ketamine was sufficient to activate mTOR pathway by upregulating phosphorylated AKT/AKT ratio in both Mecp2 WT and KO visual cortex at P30 but not at P15 (Supplemental Figure S9A–D). Considering that in the absence of MECP2, both mTOR signaling (
      • Ricciardi S.
      • Boggio E.M.
      • Grosso S.
      • Lonetti G.
      • Forlani G.
      • Stefanelli G.
      • et al.
      Reduced AKT/mTOR signaling and protein synthesis dysregulation in a Rett syndrome animal model.
      ) (Supplemental Figure S9E, F) and BDNF expression are severely downregulated across brain regions as RTT progresses (
      • Li W.
      • Pozzo-Miller L.
      BDNF deregulation in Rett syndrome.
      ), ketamine treatment may alter this trajectory.
      The hypothesis that NMDAR modulation may have beneficial effects on the RTT phenotype has been proposed by multiple independent studies published in recent years. Memantine, a weak NMDAR blocker, partially reverses short-term plasticity in hippocampal slices of Mecp2-null mice but fails to halt the progression of RTT phenotypes (
      • Weng S.M.
      • McLeod F.
      • Bailey M.E.S.
      • Cobb S.R.
      Synaptic plasticity deficits in an experimental model of rett syndrome: Long-term potentiation saturation and its pharmacological reversal.
      ). It is noteworthy that there are several differences in the way memantine and ketamine interact with NMDAR, including their ability to change glutamate binding at rest (
      • Gideons E.S.
      • Kavalali E.T.
      • Monteggia L.M.
      Mechanisms underlying differential effectiveness of memantine and ketamine in rapid antidepressant responses.
      ), the trapping properties of the drugs (
      • Kotermanski S.E.
      • Wood J.T.
      • Johnson J.W.
      Memantine binding to a superficial site on NMDA receptors contributes to partial trapping.
      ), and their unique effects on synaptic and extrasynaptic receptors (
      • Zhou X.
      • Hollern D.
      • Liao J.
      • Andrechek E.
      • Wang H.
      NMDA receptor-mediated excitotoxicity depends on the coactivation of synaptic and extrasynaptic receptors.
      ). Moreover, ketamine and memantine activate different downstream intracellular pathways, as memantine does not inhibit the phosphorylation of eukaryotic elongation factor 2, nor does it augment subsequent expression of BDNF (
      • Gideons E.S.
      • Kavalali E.T.
      • Monteggia L.M.
      Mechanisms underlying differential effectiveness of memantine and ketamine in rapid antidepressant responses.
      ).
      Although reactivation of Mecp2 expression in fully symptomatic mice can reverse at least some of the deficits caused by the loss of gene function during development (
      • Guy J.
      • Gan J.
      • Selfridge J.
      • Cobb S.
      • Bird A.
      Reversal of neurological defects in a mouse model of Rett syndrome.
      ), the timing with which Mecp2 downstream signaling is altered could play a critical role in determining the effectiveness of potential therapeutic interventions. Overall, our results indicate that both treatments were effective in ameliorating multiple RTT phenotypes. However, the degree of efficacy varied depending on the features analyzed and most likely the neuronal circuit affected. The treatment starting at P15 was more effective in improving subcortical phenotype, while P30 treatment fully recovered normal cortical visual processing by increasing the reliability of visual responses in addition to the normal evoked and spontaneous activity (Figure 5C, E).
      A factor that may have contributed to these outcomes is represented by the higher brain exposure to ketamine at P15 compared with P30 and adulthood (Supplemental Figure S8). This may have impacted the maturation of excitatory cortical circuits by interfering with their synaptic development resulting in an incomplete reliability of the evoked neuronal responses (
      • Ko H.
      • Cossell L.
      • Baragli C.
      • Antolik J.
      • Clopath C.
      • Hofer S.B.
      • Mrsic-Flogel T.D.
      The emergence of functional microcircuits in visual cortex.
      ). Although brain circuit alterations in presymptomatic Mecp2-null mice have been identified (
      • Durand S.
      • Patrizi A.
      • Quast K.B.
      • Hachigian L.
      • Pavlyuk R.
      • Saxena A.
      • et al.
      NMDA receptor regulation prevents regression of visual cortical function in the absence of Mecp2.
      ,
      • Dani V.S.
      • Chang Q.
      • Maffei A.
      • Turrigiano G.G.
      • Jaenisch R.
      • Nelson S.B.
      Reduced cortical activity due to a shift in the balance between excitation and inhibition in a mouse model of Rett syndrome.
      ,
      • Gadalla K.K.E.
      • Ross P.D.
      • Riddell J.S.
      • Bailey M.E.S.
      • Cobb S.R.
      Gait analysis in a Mecp2 knockout mouse model of Rett syndrome reveals early-onset and progressive motor deficits.
      ), NMDAR subunit composition becomes disrupted in a cell-specific manner only at onset of regression (
      • Asaka Y.
      • Jugloff D.G.M.
      • Zhang L.
      • Eubanks J.H.
      • Fitzsimonds R.M.
      Hippocampal synaptic plasticity is impaired in the Mecp2-null mouse model of Rett syndrome.
      ,
      • Blue M.E.
      • Naidu S.
      • Johnston M.V.
      Altered development of glutamate and GABA receptors in the basal ganglia of girls with Rett syndrome.
      ,
      • Blue M.E.
      • Kaufmann W.E.
      • Bressler J.
      • Eyring C.
      • O’driscoll C.
      • Naidu S.
      • Johnston M.V.
      Temporal and regional alterations in NMDA receptor expression in Mecp2-null mice.
      ,
      • Mierau S.
      • Patrizi A.
      • Hensch T.
      • Fagiolini M.
      Cell-specific regulation of NMDA receptor maturation by MeCP2 in cortical circuits.
      ). By P30, pyramidal cells still exhibit immature GLUN2B-containing NMDARs, while PV cells have already completed their switch to GLUN2A and are hyperconnected onto pyramidal cells, actively silencing cortical circuits (
      • Mierau S.
      • Patrizi A.
      • Hensch T.
      • Fagiolini M.
      Cell-specific regulation of NMDA receptor maturation by MeCP2 in cortical circuits.
      ). A low dosage of ketamine at P30 may then be the ideal intervention to specifically target such differential GluN2 expression in cortical circuits with minimal or no off-target effects. The net result of these effects is a progressive renormalization of the wiring and function of neuronal circuits and RTT phenotype at large (Figure 7).
      Figure thumbnail gr7
      Figure 7Ketamine preferentially modulates parvalbumin cortical circuits leading to a rebalancing of cortical activity in Mecp2 knockout (KO) mice. Excitatory/inhibitory (E/I) imbalance underlies impairment in cortical processing. Increased parvalbumin (PV) puncta density onto pyramidal (Pyr) cells leads to a silent cortex. The N-methyl-D-aspartate receptor (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. WT, wild-type.
      In summary, our study demonstrated that NMDARs are new and innovative therapeutic targets for the treatment of RTT-related symptoms. Future preclinical studies are required to explore whether the therapeutic benefits of ketamine can extend to Mecp2 heterozygous female mice and eventually allow the design of effective and safe clinical trials in RTT patients.

      Acknowledgments and Disclosures

      This work was supported by the International Rett Syndrome Foundation, the Simons Foundation (Grant No. 24026 to MF), and Boston Children’s Hospital Translational Research Program.
      AP, NP, and MF conceived and designed the project and wrote the manuscript; NAA prepared pharmacokinetic samples; NP, AJS, GG, and EC performed behavioral studies; NP performed single-unit studies; AP performed immunofluorescence studies; and AP, NP, and GG analyzed the data.
      We thank G. Solinap and J. Ciarrusta for early contributions to the study, E. Chang for helping with Western blot experiments, A.D. Hill and Boston Children’s Hospital Intellectual and Developmental Disabilities Research Center imaging core (NIH-P30-HD-18655) for imaging software, L.M. Pereira and Quantitative Clinical Pharmacology and Pharmacokinetics Laboratory at Boston Children’s Hospital for performing pharmacokinetic analysis, Neurodevelopmental Behavioral Core (CHB IDDRC P30 HD18655) for behavioral analyses, and Dr. Hensch and members of the Fagiolini and Hensch labs for helpful discussions.
      All authors report no biomedical financial interests or potential conflicts of interest.

      Appendix A. Supplementary Materials

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      Linked Article

      • N-Methyl-D-Aspartate Receptors, Ketamine, and Rett Syndrome: Something Special on the Road to Treatments?
        Biological PsychiatryVol. 79Issue 9
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          In 2007, Adrian Bird and his colleagues at the University of Edinburgh published a groundbreaking study demonstrating that Rett syndrome (RTT), a severe genetic neurodevelopmental disorder, is reversible in mouse models of the disease (1). Using Cre-Lox technology, the Bird group engineered a mouse in which the disease-causing gene, Mecp2, could be reversibly inactivated. Animals born with the gene switched off—a condition that mimics the loss of MECP2 function that underlies the human disease—developed full-blown symptoms of murine RTT.
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