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Address correspondence to: Michela Fagiolini, Ph.D., Boston Children’s Hospital, Assistant Professor of Neurology, F.M. Kirby Neurobiology Center, Children’s Hospital (CLS 13036), 300 Longwood Avenue, Boston, MA 02115.
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.
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.
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.
Our findings provide strong evidence that targeting NMDA receptors can be a safe and effective treatment for RTT.
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 (
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) (
). 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 (
). 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 (
). 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 (
). 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 (
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 (
). 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
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.
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 (
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).
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).
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).
Animals were scored using the RTT phenotypic severity scoring system described previously (
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.
Visual threshold acuity was evaluated using the optomotor task (
). 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 (
). Cortical activity in the binocular zone of primary visual cortex was recorded using multichannel probes (A1x16-3mm50-177; Neuronexus Technologies, Ann Arbor, Michigan) (Supplement).
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).
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).
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).
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 (
). 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) (
) and was quickly eliminated (plasma elimination half-life = 1.1 ± .8 hours).
Table 1Mouse Pharmacokinetic Parameters After 8 mg/kg Intraperitoneal Administration of Ketamine
463 ± 87
115 ± 38
122 ± 26
.05 ± .03
.28 ± .17
157 ± 78
71 ± 12
14.7 ± 1
1760 ± 231
81 ± 12
296 ± 48
.09 ± .03
.25 ± .13
284 ± 86
30 ± 7.2
8.7 ± 3.7
1209 ± 152.5
69 ± 4
366 ± 59
.07 ± .01
1.11 ± .8
326 ± 51
48 ± 3
2.7 ± .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.
). 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 (
). 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) (
). 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 (
) (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).
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) (
). 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 (
). 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 (
). 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 (
), 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).
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) (
). 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 (
). 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.
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 (
). 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 (
) (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 (
). 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.
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 (
). 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 (
). 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 (
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 (
), 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) (
). 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 (
). 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 (
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 (
). 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 (
), 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 (
). 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 (
). 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).
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.
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.