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PLOS ONE logoLink to PLOS ONE
. 2021 Oct 21;16(10):e0258830. doi: 10.1371/journal.pone.0258830

Substantial acetylcholine reduction in multiple brain regions of Mecp2-deficient female rats and associated behavioral abnormalities

Hiroyasu Murasawa 1,2, Hiroyuki Kobayashi 1,2, Jun Imai 2, Takahiko Nagase 2, Hitomi Soumiya 1, Hidefumi Fukumitsu 1,*
Editor: Atsushi Asakura3
PMCID: PMC8530288  PMID: 34673817

Abstract

Rett syndrome (RTT) is a neurodevelopmental disorder with X-linked dominant inheritance caused mainly by mutations in the methyl-CpG-binding protein 2 (MECP2) gene. The effects of various Mecp2 mutations have been extensively assessed in mouse models, but none adequately mimic the symptoms and pathological changes of RTT. In this study, we assessed the effects of Mecp2 gene deletion on female rats (Mecp2+/−) and found severe impairments in social behavior [at 8 weeks (w), 12 w, and 23 w of age], motor function [at 16 w and 26 w], and spatial cognition [at 29 w] as well as lower plasma insulin-like growth factor (but not brain-derived neurotrophic factor) and markedly reduced acetylcholine (30%–50%) in multiple brain regions compared to female Mecp2+/+ rats [at 29 w]. Alternatively, changes in brain monoamine levels were relatively small, in contrast to reports on mouse Mecp2 mutants. Female Mecp2-deficient rats express phenotypes resembling RTT and so may provide a robust model for future research on RTT pathobiology and treatment.

Introduction

Rett syndrome (RTT) (OMIM 312750) is an X-linked progressive neurodevelopmental disorder with an incidence of about 1:10,000 among newborn females, making it the second most frequent cause of female mental retardation after Down syndrome [1]. It also shortens life expectancy by ~30‒40 years in females, while males usually demonstrate early onset and die shortly after birth. Mutations in the gene encoding transcription regulator methyl-CpG-binding protein 2 (MeCP2) are identified in more than 95% RTT patients [2, 3]. Females with RTT appear to develop normally until 6 to 18 months after birth, but then begin to exhibit deficits in cognitive function, motor function, and sociality, with continued developmental regression characterized by a loss of acquired communication and purposeful hand skills [4, 5].

Over 300 MECP2 mutations and genomic abnormalities have been documented in RTT patients as well as in other mental health disorders, including intellectual disability, autism spectrum disorder, bipolar disorder, and schizophrenia [6]. Based on these genetic studies, several mouse models with various Mecp2 mutations have been developed and studied over the past two decades [7, 8]. One of the most striking findings is that many phenotypes reported in Mecp2 gene null mutants [9] are also observed in mouse mutants generated using a post-mitotic neuron-specific knockout strategy [10], consistent with postnatal neurodevelopmental regression as a predominant symptom. Moreover, some of these phenotypes can be normalized by re-expression of the Mecp2 gene after birth [1113]. This potential postnatal reversibility of RTT phenotypes has prompted research on therapeutics to compensate for Mecp2 and associated deficiencies [14], but there has been no successful clinical translation of these findings.

While mouse models allow for convenient genetic manipulation and large-scale breeding, small body and brain sizes limit critical research methods such as region-specific in vivo electrophysiology and neurochemistry (e.g., microdialysis). Further, some complex and sophisticated behaviors are not observed in mice [15, 16], which may make it difficult to capture RTT-related regressive pathologies. Considering these shortcomings, a rat model was recently developed to investigate the effects of Mecp2 deficiency on brain development, cognition, and behavior [1719].

Since human RTT is limited to females, it is preferable to conduct all animal model experiments on females, although few studies have examined female mutant mice exclusively, likely because of the more delayed and complex phenotypic progression associated with cellular mosaicism [8]. To further explore the pathological progression associated with Mecp2 gene deficiency, we examined differences in spatial learning and memory, histopathology, and regional neurotransmitter levels between wild-type (Mecp2+/+) and Mecp2+/− female rats. These studies revealed progressive deficits in social behavior and motor function as well as impaired spatial learning and memory. Further, regional acetylcholine levels were substantially reduced, suggesting that disrupted cholinergic transmission or metabolism may contribute to the behavioral and cognitive abnormalities of RTT.

Materials and methods

All experimental protocols were approved by the Institutional Animal Care and Use Committee of Nihon Bioresearch Inc. (protocol number: 201601), and performed in compliance with the Guidelines for Management and Welfare of Experimental Animals of both Nihon Bioresearch and the National Institutes of Health. All efforts were made to minimize animal suffering and to reduce the number of animals used. We adopted the following protocol: if a rat showed any symptoms of suffering, such as abnormal breathing and pulse, decreased body temperature, lying down with less response to external stimuli, or showed rapid weight loss (>20% in several days), we would take euthanasia measures by exsanguination with the opening of the abdominal aorta under isoflurane anesthesia. None of the rats, however, showed any such symptoms. We therefore used all the datasets without any exclusion throughout this study.

Animals

Female Mecp2 gene mosaic heterozygotic Sprague Dawley (SD) rats (Mecp2+/−) were obtained from SAGE Labs (part of Horizon Discovery, UK). The genotype of these animals is guaranteed by SAGE Labs. The zinc-finger nuclease technology was used to establish an animal model for generating a 71-base pair deletion in exon 4 [18]. Female wild-type control SD rats (Mecp2+/+) were obtained from Charles River Laboratories (Yokohama, Japan) at 6 weeks (w) of age and housed under the same conditions as mutants (n = 6 for each genotype, respectively). Briefly, all animals were housed in an animal room with temperature maintained at 18.0°C to 28.0°C, relative humidity at 30.0%–80.0%, and a 12-hour/12-hour light/dark cycle (lighting: 6:00 a.m. to 6:00 p.m.) with filtered fresh air changes 12 times per hour. The animals were housed individually in plastic cages (W: 310 × D: 360 × H: 175 mm) lined with autoclaved paper bedding and allowed free access to food and water.

Study schedule

Behavioral test batteries are described in the order conducted. The social interaction test was performed at 8, 12, and 23 w of age, the locomotor activity test at 16 and 23W, rotarod performance test at 26W, and Morris water maze (MWM) test at 29 w (Fig 1). One week after the final MWM tests, blood was collected from the postcava into 1-mL disposable polypropylene syringe (Terumo Corporation, Tokyo, Japan) using a 23G needle (Terumo Corporation) under isoflurane anesthesia (Isoflurane Inhalation Solution [Pfizer], Mylan Inc., Osaka, Japan). The animals were then euthanized by bleeding, and the brain was collected. Half of the brain was fixed in 4% paraformaldehyde phosphate buffer solution and processed for immunohistology while the other half was used for measurement of brain neurotransmitters.

Fig 1. Experimental schedules.

Fig 1

This figure describes the experimental schedules for behavioral test batteries and tissue sample preparations.

Behavioral test batteries

Social interaction test

The social interaction test was performed in a clean plastic cage of the same type used for animal housing. Two female Mecp2+/−or Mecp2+/+ rats that had never been in contact were placed together in the cage, and social behaviors were observed for 20 minutes under interior light through a transparent cage top with air vents. Frequencies and accumulated durations of contact behaviors (genital investigation, sniffing, and social grooming) and independent self-grooming were measured separately with a counter and a stopwatch.

Locomotor activity test

Locomotor activity over 2 consecutive 24-periods with 12-hour light and dark cycles (48 h) was analyzed at 30-minute intervals in the home cage using a computerized system (Multi digital 32 port count system, Neuroscience, Inc. Osaka, Japan).

Rotarod test

To evaluate motor coordination, the animal was placed on a rotarod (Rota-rod, ENV-577, Med. Associates, Inc., St. Albans, VT, U.S.A). The rotation speed was gradually accelerated from 4 to 40 rpm and the time (in seconds) and speed at which the rat fell off were measured within 5 minutes.

Morris water maze (MWM) test

Spatial learning and memory were assessed in the MWM. The MWM apparatus consisted of a gray vinyl chloride circular pool (diameter: 148 cm, height: 44 cm) filled with water (17°C to 18°C) up to a height of about 32 cm so that a clear acrylic escape platform (12-cm in diameter) was submerged approximately 2 cm underneath the surface. The pool was divided onto four equal quadrants, with the escape platform located in the center of the fourth quadrant (see Fig 6A). In the acquisition phase, the animal was randomly placed in the water at a wall position between ‘a’ and ‘e’ (see Fig 6A) with the head facing the wall, and swimming behavior was recorded by a video camera set above the pool and displayed on a TV monitor. In acquisition trials, time to reach the platform (escape latency in seconds) and swimming distance (cm) were analyzed using a video tracking system (Etho Vision XT, Noldus Information Technology Inc., PA Wageningen, Netherlands) as indices of spatial learning. Acquisition trials were performed twice a day for 4 days (8 times in total). If the animal did not find the platform within 90 s, latency was recorded as 91 s and it was gently guided onto the platform and allowed to remain for 30 s. One day following the final acquisition trial, animals were subjected to a probe trial for spatial memory in which the escape platform was removed. The animal was released from c and allowed to swim freely for 90 s. Time in the former platform quadrant (quadrant 4) and number of platform position crossings were recorded as indices of spatial memory.

Fig 6. Impaired spatial learning and memory among female Mecp2+/− rats in the Morris water maze test.

Fig 6

The Morris water maze test was performed at 27 weeks of age. (A) Images of the Morris water maze indicating the release positions (a–h) and the first to fourth (target) quadrants. Acquisition trials for spatial learning with a hidden escape platform in quadrant 4 were performed twice per day for 4 days (8 times in total). Escape latency (B) and swimming distance (C) were measured. Graphs are mean ± standard error of the mean (SE) for the two daily trials (n = 6 rats per genotype). #p < 0.05 and ##p < 0.01 vs. female Mecp2+/+ rats on the same acquisition day, Friedman test). A probe trial was performed without the hidden platform for the confirmation of spatial memory on the day after completing acquisition trials. The frequency of entry into the platform area (D) and swimming time in the fourth (target) quadrant (E) were measured. Bar graphs are mean ± SE (n = 6 rats per genotype). *p < 0.05 vs. female Mecp2+/+ rats (unpaired two-tailed Mann–Whitney U-test).

High-performance liquid chromatography (HPLC) analysis of biogenic amine levels

Regional neurotransmitter levels were measured in excised brain tissue by isocratic HPLC separation and electrochemical detection. Briefly, the animals were euthanized by bleeding from the postcava under isoflurane anesthesia one week after the MWM test, and the brain tissues were collected in ice-cooled physiological saline. Using a metallic zinc brain matrix (ASI Instruments Inc. Warren, MI, U.S.A), each hemisphere was sliced into 2-mm thick coronal sections and divided into frontal cortex, amygdala, hippocampus, caudate nucleus, thalamus, hypothalamus, and medulla oblongata according to the rat brain atlas of Paxinos and Watson [20]. Each region was collected in a separate container, weighed, frozen in liquid nitrogen, and stored at −90°C to −70°C in a deep freezer (ULT-1186-3SI-A36, Thermo Electron Corporation Co., Ltd., Tokyo, Japan) until neurotransmitter measurements.

The frozen brain tissue was homogenized in 0.2 mol/L perchloric acid and centrifuged at 20227g and 4°C for 5 minutes. The following neurotransmitters and metabolites were measured from the supernatant using the HPLC-ECD system (Eicom Corporation, Kyoto, Japan): dopamine (DA) and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), serotonin (5-HT) and its metabolite 5-hydroxy indole-3-acetic acid (5-HIAA), norepinephrine (NE) and its metabolite 3-methoxyphenyl-4-hydroxy-glycol (MHPG), glutamate, γ-aminobutyric acid (GABA), and acetylcholine as follows.

The levels of monoamines (DA, 5-HT, and NE) and monoamine metabolites (DOPAC, HVA, 5-HIAA, and MHPG) in the brain tissue were estimated using an HPLC system equipped with a C18 reverse-phase column (Eicompak SC-5ODS, 150 mm × 3.0 ID, Eicom Corporation, Kyoto, Japan), guard column (PC-04, Eicom Corporation, Kyoto, Japan), and column thermostat (ATC-700) set to 25°C. Molecules were separated by a mixture of 0.1 mol/L acetic acid–citrate buffer (pH 3.5), methanol, 100 mg/mL sodium 1-octanesulfonate solution, and 5 mg/mL ethylenediaminetetraacetic acid (EDTA)-2Na solution (820:180:2.2:1) under the control of a pump system (EP-700) at a flow rate of 0.5 mL/min. For each measurement, 20 μL of the brain lysate supernatant was injected automatically using an automated sample injector (M-500). An electrochemical detector (ECD-700) with a graphite electrode (WE-3G), gasket, and Ag/AgCl reference electrode (RE-500) set to +750 mV was used for quantification.

Glutamate level was measured using an HPLC system equipped with a C18 reverse-phase column (Eicompak E-GEL, 150 mm × 4.6 ID, Eicom Corporation, Kyoto, Japan), enzyme column (E-EMZYMPAK, 4 mm × 3.0 ID, Eicom Corporation, Kyoto, Japan), guard column (PC-03, Eicom Corporation, Kyoto, Japan), and column thermostat (ATC-700) set to 33°C. A mixture of 60 mM ammonium chloride–ammonia solution (pH 7.2), hexadecyltrimethylammonium bromide, and 5 mg/mL EDTA-2Na solution (1000 mL:250 mg:0.01 mL) was used as the mobile phase under the control of the pump system (EP-700) at 0.37 mL/min. For each measurement, 20 μL of the brain lysate supernatant was injected automatically and the level measured using the electrochemical detector (ECD-700) with a platinum electrode (WE-PT), gasket, and Ag/AgCl reference electrode (RE-500) set at +450 mV.

GABA level was measured using an HPLC system equipped with a C18 reverse-phase column (Eicompak FA-3ODS, 50 mm × 3.0 ID, Eicom Corporation, Kyoto, Japan), guard column (PC-03, Eicom Corporation, Kyoto, Japan), and column thermostat (ATC-700) set to 40°C. A mixture of 0.1 mol/L phosphate buffer solution (pH 6.0), acetonitrile, and 5 mg/mL EDTA-2Na solution (500 mL:500 mL:1 mL) was used as the mobile phase under the control of the pump system (EP-700) at a flow rate of 0.5 mL/min. For each measurement, 20 μL sample was injected automatically using the automated sample injector (M-500) and the level measured using the electrochemical detector (ECD-700) with a graphite electrode (WE-GC), gasket, and Ag/AgCl reference electrode (RE-500) set at +600 mV.

Acetylcholine level was measured using an HPLC system equipped with a C18 reverse-phase column (Eicompak AC-GEL, 150 mm × 4.6 ID, Eicom Corporation, Kyoto, Japan), enzyme column (E-EMZYMPAK, 4 mm × 1.0 ID, Eicom Corporation, Kyoto, Japan), guard column (PC-03, Eicom Corporation, Kyoto, Japan), and column thermostat (ATC-700) set to 33°C. A mixture of 0.1 mol/L potassium bicarbonate solution, sodium decanesulfonate, and 5 mg/mL EDTA-2Na solution (1000 mL:400 mg:0.01 mL) was used as the mobile phase under the control of the pump system (EP-700) at a flow rate of 0.15 mL/min. For each measurement, 20 μL sample was injected automatically using the automated sample injector (M-500) and the level measured by the electrochemical detector (ECD-700) with a platinum electrode (WE-PT), gasket, and Ag/AgCl reference electrode (RE-500) set at +450 mV.

Tissue preparation and immunohistochemical analysis

The fixed brain tissue was embedded in paraffin using the routine method of our testing facility and cut into 5-μm thick sections. Every third section was processed for immunohistochemical staining with anti-glial fibrillary acidic protein (GFAP) antibody (×2000, Abcam, ab 7260). The three to five sections of choice were those closest to dorsal–ventral level interaural 3.20 mm (for frontal cortex) and -3.80 mm (for the hippocampus, hypothalamus, and amygdala) according to Paxinos and Watson [20]. Immunostained sections of the frontal cortex and hippocampus were photographed using an optical microscope with a digital camera, and GFAP-positive cells were counted using WinROOF V7.0 software.

Hematological examination

Plasma was collected by centrifugation of the collected blood (at 4°C and 2150×g for 15 minutes). Plasma insulin-like growth factor 1 (IGF-1) was estimated using the Quantikine® ELISA kit (Mouse/Rat IGF-I immunoassay, Cosmo Bio Co., Ltd., Tokyo, Japan) and plasma BDNF using the RayBio® Rat BDNF ELISA Kit (Ray Biotech. Inc., Peachtree Corners, GA, U.S.A).

Statistical analysis

Data are presented as the mean ± standard error of the mean (SE). All statistical analyses were performed using GraphPad Prism version 9.1.2 for Windows (GraphPad Software Inc., San Diego, CA, U.S.A, www.graphpad.com), according to Prism9 Statistics Guide (https://www.graphpad.com/guides/prism/latest/statistics/index.htm). All details of statistics have been indicated in figure legends. Because the sample size for each experiment was small, it was not appropriate to consider that the sample was normally distributed. Therefore, when comparing two independent groups, the unpaired two-tailed Mann–Whitney U-test was used. For the analysis of statistical significance in comparisons involving more than two groups, Kruskal–Wallis with Dunn’s post hoc multiple comparisons test was used. When comparing three or more matched groups, the Friedman test was used. In all cases, statistical significance was assessed with a 95% confidence interval; therefore, p < 0.05 was considered significant.

Results

Impaired social behaviors in Mecp2-deficient female rats

We first analyzed the effect of Mecp2 gene deficiency on rat social behaviors at 8, 12, and 23 w. The frequency and duration of contact behaviors gradually increased with age among female Mecp2+/+ rats but gradually declined with age among female Mecp2+/− rats, and both parameters were significantly lower compared to female Mecp2+/+ rats by 23 w (Fig 2A and 2B). We next analyzed self-grooming behavior during the social interaction test as higher frequency and longer duration are considered endophenotypic hyper-repetitive behaviors of autism spectrum disorder [2123] and male Mecp2 mutant mice [2426]. The frequencies (Fig 3A) of self-grooming were significantly reduced and the duration (Fig 3B) tended to be reduced but not significant in female Mecp2+/− rats. Thus, the deficiency in Mecp2 impaired social behaviors with reduced self-grooming.

Fig 2. Age-dependent alterations in social behaviors among female Mecp2+/− rats.

Fig 2

Female Mecp2+/− rats exhibited progressively less frequent social interactions with age, while social interactions increased with age among female Mecp2+/+ (wild-type) rats. Bar graphs indicate frequency of contact behaviors (A) and total duration of interactions (B) during a 20-min social interaction test (mean ± SE; n = 6 rats per genotype). #p < 0.05, and ***p < 0.001 vs. same genotyped female Mecp2+/− rats at 8 weeks (w) (Friedman test), and the age-matched female Mecp2+/+ rats at 23 w of age (Kruskal–Wallis with Dunn’s post hoc multiple comparisons test), respectively.

Fig 3. Age-dependent changes in self-grooming behavior among female Mecp2+/− rats.

Fig 3

Both frequency (A) and total time (B) of self-grooming tended to be lower in female Mecp2+/− rats compared to female Mecp2+/+ rats during the 20-min social interaction test at 8, 12, and 23 w of age (mean ± SE; n = 6 rats per genotype). *p < 0.05 vs. age-matched female Mecp2+/+ rats (Kruskal–Wallis with Dunn’s post hoc multiple comparisons test).

Impaired spontaneous locomotor activity and motor coordination in Mecp2-deficient female rats

To evaluate the effects of Mecp2 deficiency on motor function, we first analyzed spontaneous locomotor activity over 2 consecutive 24-periods with 12-h/12-h light/dark cycles at 16 w and 23 w. Spontaneous locomotor activity was significantly or tended to be lower among female Mecp2+/− rats compared to female Mecp2+/+ rats (Fig 4) during both light and dark phases. In addition, judging from the locomotor activity counts every 30 minutes, Mecp2 deficiency did not seem to affect circadian rhythms because female Mecp2+/- rat shows a tendency to decrease activity over the entire measurement time, not at any specific time point (S1 Fig). To assess motor coordination, animals were tested on the accelerating rotarod at 26 w. Both rotation time and speed at which the rat fell off were lower in the Mecp2+/− group compared with the Mecp2+/+ group (Fig 5A and 5B). Thus, based on the RTT symptoms, both spontaneous and task-evoked motor activity were impaired by Mecp2+/− deficiency.

Fig 4. Impaired locomotor activity among female Mecp2+/− rats during both light and dark phases at 16 and 23 weeks of age.

Fig 4

Bar graphs are mean ± SE (n = 6 rats per genotype). *p < 0.05, and **p < 0.01 vs. age-matched female Mecp2+/+ rats (Kruskal–Wallis with Dunn’s post hoc multiple comparisons test).

Fig 5. Impaired motor coordination among female Mecp2+/− rats at 26 weeks of age.

Fig 5

The animal was placed on a rotarod, which was accelerated from 4 to 40 rpm. The time at which the animal fell off (A) and the speed at which the animal fell off (B) were measured. Bar graphs are mean ± SE (n = 6 rats per genotype). *p < 0.05 vs. female Mecp2+/+ rats (unpaired two-tailed Mann–Whitney U-test).

Impaired spatial learning and memory in Mecp2-deficient female rats

To examine the effect of Mecp2 deficiency on spatial learning and memory, we compared escape latency and target memory between female Mecp2+/− and Mecp2+/+ rats in the MWM starting on 27 w (first acquisition trial). Compared to Mecp2+/+ rats, Mecp2+/− rats demonstrated significantly longer escape latencies (Fig 6B) and swimming distances (Fig 6C) over the four acquisition trial days. Further, Mecp2+/− rats made fewer crossing over the former platform location (Fig 6D) and tended to spend less time in the target (former platform) quadrant (Fig 6E). Thus, Mecp2 deficiency appears to impair spatial learning and memory.

Reduced acetylcholine but relatively normal biogenic amine levels in multiple brain regions of Mecp2-deficient rats

Several studies have reported significant downregulation of biogenic amines in the brains of Mecp2 mouse mutants [2731]. Surprisingly, however, biogenic amine and its metabolite levels (Table 1) and the glutamate/GABA ratio (Table 2) were also near normal in most measured regions of female Mecp2+/− rats. The genotypic differences in the biogenic amine/metabolite levels were observed in such brain regions as follows: DA and DOPAC levels were comparable to those in the caudate nucleus and medulla oblongata, although HVA level tended to be lower in the caudate nucleus and was slightly but significantly lower in the medulla oblongata of female Mecp2+/− rats. 5-HT level was lower in the thalamus and hypothalamus of female Mecp2+/− rats (Table 1). By contrast, the relative reductions in acetylcholine level were greater than those of other transmitters, including biogenic amines: acetylcholine level was substantially lower in the amygdala, hippocampus, caudate nucleus, and medulla oblongata (Table 3). These results suggest that impaired cholinergic signaling could severely affect the behavioral abnormalities observed in Mecp2+/− rats. Indeed, cholinergic inputs to the hippocampus are essential for spatial learning and memory [32, 33].

Table 1. Monoamine and monoamine metabolite levels in various brain regions of wild-type (Mecp2+/+) and Mecp2-deficient (Mecp2+/−) female rats.

Levels (ng/g wet weight)
NE MHPG DA DOPAC HVA 5-HT 5-HIAA
Frontal cortex Mecp2 +/+ 521.4 ± 69.2 281.9 ± 38.0 67.0 ± 7.5 23.3 ± 2.0 6.9 ± 0.9 193.8 ± 27.2 130.9 ± 16.3
Mecp2 +/− 400.2 ± 16.5 321.5 ± 34.5 83.2 ± 4.3 30.6 ± 2.7 6.7 ± 1.6 215.7 ± 23.6 162.0 ± 10.7
Amygdala Mecp2 +/+ 645.7 ± 55.9 239.4 ± 24.3 132.4 ± 14.8 23.1 ± 5.1 8.9 ± 2.7 263.9 ± 44.3 170.1 ± 22.7
Mecp2 +/− 590.7 ± 79.7 313.2 ± 45.8 103.1 ± 22.8 19.3 ± 2.1 5.8 ± 1.1 231.6 ± 61.3 190.9 ± 33.4
Hippocampus Mecp2 +/+ 741.6 ± 59.7 334.4 ± 17.9 23.0 ± 2.1 1.4 ± 0.4 1.1 ± 0.1 200.1 ± 15.9 243.5 ± 10.9
Mecp2 +/− 606.7 ± 43.0 368.2 ± 30.9 23.8 ± 1.7 1.6 ± 0.3 0.9 ± 0.0 175.5 ± 8.2 220.8 ± 9.1
Caudate nucleus Mecp2 +/+ 21.2 ± 0.9 194.8 ± 17.9 4106.8 ± 267.1 621.4 ± 62.6 183.2 ± 28.0 92.9 ± 8.0 207.5 ± 16.2
Mecp2 +/− 31.0 ± 5.2 263.4 ± 51.5 4188.6 ± 472.2 607.6 ± 82.7 125.4 ± 28.8 89.5 ± 14.8 216.0 ± 38.2
Thalamus Mecp2 +/+ 858.0 ± 32.8 324.6 ± 21.8 131.3 ± 3.3 16.7 ± 1.3 9.4 ± 1.6 512.0 ± 11.0 447.5 ± 18.0
Mecp2 +/− 847.9 ± 56.7 349.0 ± 26.5 114.7 ± 10.4 15.8 ± 1.9 7.3 ± 1.2 423.4 ± 22.5* 422.8 ± 27.9
Hypothalamus Mecp2 +/+ 2244.2 ± 160.9 286.9 ± 30.2 234.1 ± 44.0 30.4 ± 5.3 5.3 ± 0.9 439.3 ± 29.6 311.5 ± 27.7
Mecp2 +/− 2150.7 ± 286.9 328.5 ± 30.9 192.5 ± 20.7 31.4 ± 5.6 5.2 ± 1.1 322.2 ± 38.7* 266.7 ± 19.5
Medulla oblongata Mecp2 +/+ 940.1 ± 37.6 435.0 ± 34.1 39.8 ± 1.4 7.4 ± 0.6 5.6 ± 0.9 432.5 ± 14.2 219.9 ± 11.3
Mecp2 +/− 1004.4 ± 35.3 540.3 ± 37.6 38.5 ± 0.9 6.4 ± 0.4 3.2 ± 0.2* 434.3 ± 22.6 225.9 ± 12.7

NE: norepinephrine; MHPG: 3-methoxyphenyl-4-hydroxy-glycol hemipiperazium; DA: dopamine; DOPAC: 3,4-dihydroxyphenylacetic acid; HVA: homovanillic acid; 5-HT: 5-hydroxytryptamine (serotonin); 5-HIAA: 5-hydroxy indole-3-acetic acid.

Data was expressed as mean ± SE (n = 6 samples per region and genotype).

*p < 0.05 female Mecp2+/- vs. female Mecp2+/+ rats by unpaired two-tailed Mann–Whitney U-test.

Table 2. Glutamate/GABA ratios across brain regions.

Frontal cortex Amygdala Hippocampus Caudate nucleus Thalamus Hypothalamus Medulla oblongata
Mecp2 +/+ 7.9 ± 0.9 5.5 ± 0.3 6.5 ± 0.3 6.9 ± 0.4 2.3 ± 0.2 1.4 ± 0.2 4.7 ± 0.3
Mecp2 +/− 8.3 ± 0.8 6.1 ± 0.8 6.9 ± 0.4 5.0 ± 0.5 2.5 ± 0.1 1.5 ± 0.1 5.1 ± 0.6

Data expressed as mean ± SE (n = 6 for each genotype). Non significance female Mecp2+/- vs. Mecp2+/+ rats (unpaired two-tailed Mann–Whitney U-test).

Table 3. Acetylcholine levels across brain regions.

Frontal cortex Amygdala Hippocampus Caudate nucleus Thalamus Hypothalamus Medulla oblongata
Mecp2 +/+ 480.9 ± 129.6 1066.1 ± 139.1 949.0 ± 86.3 742.1 ± 77.4 771.5 ± 49.0 546.7 ± 80.8 776.1 ± 146.0
Mecp2 +/− 333.1 ± 38.5 475.7 ± 85.5** 649.4 ± 64.6* 478.3 ± 35.6* 675.8 ± 82.8 376.4 ± 81.9 401.8 ± 23.0**

Data expressed as mean ± SE (n = 6 for each genotype).

*p < 0.05 and

**p < 0.01 female Mecp2+/- vs. Mecp2+/+ rats (unpaired two-tailed Mann–Whitney U-test).

Changes in GFAP-positive astrocytes and serum IGF

To further examine neurodevelopment abnormalities associated with Mecp2 deficiency, we examined the number and morphological characteristics of GFAP-positive astrocytes. Astrocytes are known to support neuronal function through production and secretion of various extrinsic factors and by the uptake of neurotransmitters, and the number and morphological features are associated with neurodevelopmental abnormalities as well as neurodegeneration and neuroinflammation [34, 35]. Further, abnormal astrocyte morphology has been observed in RTT patients and models [36, 37]. Comparing with those of female Mecp2+/+ rats (hippocampus, Fig 7A, a-c; frontal cortex, Fig 7B, a and b; amygdala, Fig 8A, a and b; hypothalamus, Fig 8B, a and b; respectively), the somata of GFAP-positive cells were smaller and possessed less complex processes in the hippocampus (Fig 7A, d-f), frontal cortex (Fig 7B, c and d) and amygdala (Fig 8A, c and d) but not in the hypothalamus (Fig 8B, c and d) of female Mecp2+/− rats. In addition, cell numbers were significantly lower in frontal cortex, hippocampus and amygdala, but not in hypothalamus (Table 4). Finally, we also examined potential neurotrophin signaling deficits by measuring serum levels of IGF and BDNF [38]. Both neurotrophic factors play important roles in survival, differentiation and functional development for brain neuronal circuits. Compared to female Mecp2+/+ rats, plasma IGF-I was significantly reduced but plasma BDNF was normal in the female Mecp2+/− rats (Table 5).

Fig 7. Changes in the number of glial fibrillary acidic protein (GFAP)-positive cells (astrocytes) in the hippocampus and frontal cortex of a female Mecp2+/− rat brain.

Fig 7

The brain was collected one week after the Morris water maze test and immunostained for the astrocytic marker GFAP. Shown are the morphology and the number of GFAP-positive cells in the hippocampus (A: a-c: Mecp2+/+, b and c: higher magnification of square in a; d-f, Mecp2+/−, e and f: higher magnification of square in d), the frontal cortex (B: a and b: Mecp2+/+; c, d: Mecp2+/−; b and d: higher magnification of square in a and c, respectively). Scale bar = 300 μm; double bars = 100 μm, respectively.

Fig 8. Number of glial fibrillary acidic protein (GFAP)-positive cells (astrocytes) in the hypothalamus and amygdala of a female Mecp2+/− rat brain.

Fig 8

Shown are the morphology and number of GFAP-positive cells (A. amygdala, B. hypothalamus: a and b: Mecp2+/+; c and d: Mecp2+/−; b and d: higher magnification of square in a and c, respectively). Scale bar = 300 μm; double bars = 100 μm, respectively.

Table 4. Number of GFAP-positive cells across brain regions.

Number of GFAP-positive cells/slice
Mecp2 +/+ Mecp2 +/−
Frontal cortex 1108.2 ± 33.5 609.5 ± 66.0 **
Hippocampus 476.5 ± 15.4 347.3 ± 11.7 **
Amygdala 51.8 ± 1.5 49.5 ± 2.3**
Hypothalamus 39.8 ± 4.6 39.0 ± 2.7

Data expressed as mean ± SE (n = 6 for each genotype).

**p < 0.01 female Mecp2+/- vs. Mecp2+/+ rats (unpaired two-tailed Mann–Whitney U-test).

Table 5. Plasma IGF and BDNF levels.

Mecp2 +/+ Mecp2 +/−
IGF-1 (ng/mL) 1080.2 ± 70.3 769.4 ± 52.4 **
BDNF (pg/mL) 46.4 ± 5.9 51.9 ± 18.2

Data expressed as mean ± SE [IGF-1: n = 6 and BDNF: n = 2 (n = 4, under detection limit), for each genotype, respectively)].

**p < 0.01 female Mecp2+/- vs. Mecp2+/+ rats (unpaired two-tailed Mann–Whitney U-test).

Discussion

We demonstrated that Mecp2-deficient female rats (Mecp2+/−) exhibit multiple behavioral abnormalities resembling those of RTT, including progressively impaired social behavior with reduced self-grooming (at 8, 12, and 23 w, Figs 2 and 3), reduced spontaneous locomotor activity (at both 16 and 23 w, Fig 4, and S1 Fig), poor motor coordination (at 26 w, Fig 5), and deficient spatial cognition (at 27 w, Fig 6). Further, these animals exhibited substantially lower acetylcholine levels in multiple brain regions compared to wild types (Mecp2+/+), suggesting pervasive deficits in cholinergic signaling. The phenotype of this rat model basically recapitulates that of Mecp2-deficient mouse mutants [8, 17, 18] but in addition also suggests the importance of cholinergic signaling deficits to RTT pathogenesis or symptom expression. In fact, the regional reductions in acetylcholine were relatively larger than the reductions or elevations in other neurotransmitters and their metabolites, including the changes in monoamine levels. The relative stability of monoamine levels is in contrast to predominantly male Mecp2tm 1.1 Bird exon 3/4 null mutant mice [2730] and Mecp2tm 1.1 Jae exon 3 null mutant mice [31]. We suggest that these low acetylcholine levels across multiple brain regions may contribute to the postnatal behavioral abnormalities observed in Mecp2+/− rats and possibly to RTT [4, 5].

Progressive impairments in social behavior and abnormalities in motor coordination and spatial cognition

Patients with RTT have autistic symptoms such as social behavior abnormalities. While these symptoms have been reported to improve with age [39], some persist through life [40, 41]. Consistent with findings in female Mecp2tm1.1Bird mice [42], female Mecp2+/− rats demonstrated a lower frequency of social contact in the open field, and both frequency and duration of contact decreased with age (from 8 to 23 w). Certain abnormalities in social behavior have been observed among Mecp2+/− female rats even before 4 w [17, 18], so social deficits appear to be among the earliest onset and most severely progressive. However, the influence of progressive motor dysfunction on social behaviors cannot be excluded, especially at early ages [17, 18] (Figs 2 and 3). For instance, Adcock et al. reported no significant differences in social behavior between female Mecp2+/− and Mecp2+/+ rats following auditory discrimination training or skilled forelimb motor training, suggesting that poor perceptual and (or) motor skills may contribute to social deficits in untrained rats [43]. Further, environmental enrichment [44, 45], which is known to improve motor function and cognition, can partially mitigate the developmental symptoms in female Mecp2+/− rats. Despite such limitations, the influence of training for behavioral test battery and environment and so on, should be considered on the interpretation of results; the consistency and progressive nature of these social deficits may be useful for preclinical studies on treatments to improve autistic symptoms among RTT patients.

Poor spatial memory is also a critical feature of the rat model as severe intellectual disability is a hallmark of RTT [46, 47]. Although spatial learning and memory impairments are not included in the diagnostic criteria for RTT [3, 48, 49], spatial learning and memory are critical for rodent survival and thus impairments are sensitive indicators of brain maldevelopment and dysfunction, particularly hippocampal dysfunction. Further, hippocampal dysfunction is a shared cause of cognitive deficits among numerous neurodegenerative and neurodevelopmental diseases. In this study, we used MWM [50, 51] to demonstrate that spatial learning and memory are markedly impaired in adult (29W) female Mecp2+/− rats (Fig 6). The effects of Mecp2 gene mutations on spatial learning in the MWM are known to vary by mutation type, with impairments observed among Mecp2tm 1.1 Bird mice [43, 44] but not female Mecp2 tm 1.1 Jae mice [52, 53]. Alternatively, abnormalities in recognition memory have been observed across many mutations. Since the Mecp2+/− rat is an outbred strain, it may be useful for extracting phenotypes common across species and genotypes. Although further studies using inbred strains and rats with different Mecp2 mutations are needed, the differences in hippocampus-dependent cognition between mouse and rat models may provide important clues to the underlying neuroanatomical and neurochemical changes. For instance, our current findings suggest that poor MWM performance may stem from deficient cholinergic signaling in the hippocampus of female Mecp2+/− rats.

Relationships between cholinergic and behavioral abnormalities

Many neuroanatomical and neurochemical changes have been reported in RTT patients, but the molecular mechanisms through which MECP2 gene mutations lead to pathological alterations are unknown. Acetylcholine level was significantly reduced in multiple brain regions of female Mecp2-deficient rats and associated with impaired social behavior, motor activity, and spatial cognition (Table 3). In particular, the importance of cholinergic neurons to cognitive function and mood disorders is well established [32, 33]. Clinical studies have shown decreases in the cholinergic neuron number [54], acetylcholine biosynthetic enzymes choline acetyltransferase (ChAT) and vesicular acetylcholine transporter activities, and cholinergic receptor expression [55, 56] in the brains of patients with RTT, and the changes in cholinergic neuron markers are linked to the severity of symptoms [57, 58]. Abnormalities in the cholinergic system have also been reported in Mecp2 mutant mice, including reduced ChAT expression in basal forebrain and striatum [59, 60] and locus coeruleus (LC) neurons [61]. In addition, abnormalities in memory performance and social behavior among Mecp2 null mutants were reversed by administration of acetylcholine receptor agonists and the cholinesterase inhibitor donepezil [58, 62].

While acetylcholine level was markedly reduced in female Mecp2+/− rats, relative changes in monoamines, monoamine metabolites, and the glutamic acid/GABA ratio were smaller and observed in a more limited number of brain regions. This result was unexpected because the levels of many monoamine metabolites as well as expression levels of rate-limiting biosynthetic enzymes have been shown to decline with age in male Mecp2tm1.1Bird mice [27, 30]. The result was also unexpected because the disorders of involuntary movements in RTT (progressive rigidity, dyskinesia, and dystonia) have been suggested to be associated with the maldevelopment of the dopaminergic nervous system [6365]. It is possible that some compensatory mechanism may make it difficult to detect impairment in the monoaminergic nervous system because approximately half of the neurons in female Mecp2-mutant mouse brain maintain normal Mecp2 expression [31, 36]. Recently, Wong et al. reported that the density of dopamine D2 receptor was significantly decreased in the striatum of Mecp2tm 1.1 Bird null/heterozygous mutant mice at 7–10 w but not after 15 w. They pointed out that such developmental stage-dependent alteration could affect the measured density of D2 receptor in the RTT female brain postmortem [66]. Therefore, we could not exclude the possibility that some behavioral abnormalities observed in this study may be influenced by monoaminergic disturbances—particularly at earlier developmental stages. In fact, 5-HT levels in the thalamus and hypothalamus as well as the level of the dopamine metabolite HVA in the medulla oblongata were significantly reduced (Table 1), suggesting some abnormalities in the monoaminergic system development.

Self-grooming behavior in Mecp2+/− rats was reported to be equivalent to that of the control group (at 4 w [18]) and tended to be, or even was, significantly decreased in this study (at 8 w, 12 w, and 23 w, Fig 3). Excessive self-grooming behavior is known to be suppressed by acetylcholine antagonists [6769]. Therefore, progressive impairment in the cholinergic nervous system is likely to explain the self-grooming behavioral abnormalities. Excessive self-grooming behavior, however, is a typical symptom of autism model mice [2123] and has also been observed in Mecp2 null male mice [2426]. Monoamine reductions have been found not only in postmortem brain tissue but also in the cerebrospinal fluid (CSF) of patients with RTT [28, 70]; CSF measures during development should therefore be performed in the rat model also to clarify the relationship of disturbances in the cholinergic/monoaminergic system and behavioral changes. Such efforts, in future studies, would lead to an understanding of the complex pathological changes due to mutation in the MECP2 gene, leading to the development of optimal drug treatment strategies.

Value of the Mecp2+/− rat model for RTT diagnosis and treatment

We also observed reduced numbers of astrocytes (GFAP-positive cells) in the frontal cortex and hippocampus and as well as lower plasma IGF in female Mecp2+/− rats (Fig 7 and Tables 4 and 5), consistent with previous findings of stunted morphological and functional development of astrocytes [36, 37, 71] and reduced IGF signaling [38, 72, 73] in both RTT patients and Mecp2 mutant mice. Astrocytes regulate neuronal functions through production and secretion of growth factors, nutrients, and cytokines and by the uptake of transmitters and metabolites [74, 75], while IGF is involved in the differentiation and functional maintenance of neurons as a blood–brain barrier permeable neurotrophic factor [76, 77]. The identification of blood or CSF biomarkers associated with specific neuropathological processes may aid in RTT diagnosis and provide clues to pathogenic mechanisms, and the female Mecp2+/− rat model may be a powerful tool for this purpose. Candidate biomarkers for pathogenesis may include metabolites generated by astrocytes [78] and/or molecules associated with IGF signaling [79].

Supporting information

S1 Fig. Impaired locomotor activity among female Mecp2+/− rats during both light and dark phases at 16 and 23 weeks of age.

A-D: 24-h locomotor activity in female Mecp2+/+ (open symbols) and Mecp2+/- (filled symbols) rats. Data are the average ± SE of data collected at each half an hour from 16 w (A, B) or 23 w (C, D) old rats over 2 consecutive 24-h periods (A, C: day1; B, D: day2) (n = 6, for each geneotype). Non significance female Mecp2+/- vs. Mecp2+/+ rats (Kruskal–Wallis with Dunn’s post hoc multiple comparisons test).

(TIF)

Acknowledgments

We would like to thank Editage (www.editage.jp) for English language editing.

Abbreviations

BDNF

Brain-derived neurotrophic factor

ChAT

Choline acetyltransferase

CSF

Cerebrospinal fluid

DA

Dopamine

DBH

Dopamine β-hydroxylase

DOPAC

3,4-dihydroxyphenylacetic acid

EDTA

Ethylenediaminetetraacetic acid

ELISA

Enzyme-linked immunosorbent assay

5-HT

Serotonin

5-HIAA

5-hydroxy indole-3-acetic acid

GABA

γ-aminobutyric acid

GFAP

Glial fibrillary acidic protein

IGF-1

Insulin-like growth factor 1

LC

Locus coeruleus

MECP2

Methyl-CpG-binding protein 2

MHPG

3-methoxyphenyl-4-hydroxy-glycol

MWM

Morris water maze

NE

Norepinephrine

NMDA

N-methyl-D-aspartate

HPLC

High-performance liquid chromatography

HPLC-ECD

High-performance liquid chromatography with an electrochemical detector

HVA

Homovanillic acid

RTT

Rett syndrome

SD

Sprague Dawley

SE

Standard error of the mean

TH

Tyrosine hydroxylase

TPH

Tryptophan hydroxylase

w

Weeks

Data Availability

All relevant data are within the manuscript.

Funding Statement

The funder, Nihon Bioresearch Inc, provided support in the form of salaries for authors: HM, HK, JI, and TN, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study.

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Decision Letter 0

Atsushi Asakura

20 Apr 2021

PONE-D-21-05550

Substantial acetylcholine reduction in multiple brain regions of Mecp2-deficient female rats and associated behavioral abnormalities

PLOS ONE

Dear Dr. Fukumitsu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jun 04 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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We look forward to receiving your revised manuscript.

Kind regards,

Atsushi Asakura, Ph.D

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Review

March 19, 2021

PONE-D-21-05550

Substantial acetylcholine reduction in multiple brain regions of Mecp2-deficient female rats and associated behavioral abnormalities

The authors sought to assess the effects of Mecp2 gene deletion on female rats (Mecp2+/−) by examining social behavior, motor function, spatial cognition and other physiological parameters. The study was well-designed and well-constructed. This study represents major advances in the study of animal models of Rett syndrome. The manuscript will likely merit publication with revisions.

Financial Disclosure

Please include information about the funding for this study.

Include a Financial Disclosure.

Enter a financial disclosure statement that describes the sources of funding for the work included in this submission.

Materials and methods

Morris water maze test

Line 127

Since the second figure to be mentioned in the text occurs in this line, the figure should be numbered figure 2.

Statistical Analysis

Are the data normally distributed as demonstrated by the Kolmogorov-Smirnov or Wilks-Shapiro procedures? If the data are not normally distributed, then a nonparametric procedure such as Kruskal-Wallis is appropriate.

Results

Impaired social behaviors in Mecp2-deficient female rats

Page 9

Line 192

Change “disorders” to “disorder”

Table 1

Pages 13 to 15

Express the values of each row in a single line by using a smaller font.

Legend to figure 7

Line 316

The scale bar appears to be the same length in all panels. What is the magnification indicated by the two scale bars?

Relationships between cholinergic and behavioral abnormalities

Line 387-408

Include a discussion of the dopaminergic deficits observed as follows:

Wong DF, Blue ME, Brašić JR,, Nandi A, Valentine H, Stansfield KH, Rousset O, Bibat G, Yablonski ME, Johnston MV, Gjedde A, Naidu SB. Are dopamine receptor and transporter changes in Rett syndrome reflected in Mecp2-deficient mice? Exp Neurol. 2018; 307: 74-81. https://doi.org/10.1016/ j.expneurol.2018.05.019 PubMed PMID: 29782864.

Reference

Line 517

Remove the space between “30.” and “Panayotis”

Lines 549-550

Reference 41.

Include the original title of the article in German as follows:

Rett A. Uber ein bisher nicht bekanntes Krankheitsbild einer angeborenen Stoffwechselst örung [On an until now unknown disease of a congenital metabolic disorder]. Krankenschwester. 1966 Sep;19(9):121-2. German. PMID: 5179620.

Please add appendix of abbreviations.

Define all acronyms before their first use.

Supplemental Methods

The section in the supplemental methods belong in the body of the text.

There are no restrictions on word counts, number of figures, or amount of supporting information.

Reviewer #2: In the manuscript “Substantial acetylcholine reduction in multiple brain regions of Mecp2-deficient female rats and associated behavioral abnormalities” The authors assessed the effects of Mecp2 gene deletion on female rats (Mecp2 +/− ) and found severe impairments in social behavior, motor function, and spatial cognition and lower plasma IGF-1 (but not BDNF) and markedly reduced acetylcholine (30%–50%) in multiple brain regions compared to female Mecp2 +/+ rats.

This is a purely descriptive study which could be informative but some of the results appears to be disconnected. The correlation of behavioral changes with acetylcholine levels is appears far reaching based on the way the manuscript is organized.

Figure 7 images are of poor quality and images of amygdala and hypothalamus should be included.

Page 2, Line 25. “…..spatial cognition as well lower plasma” - the word “as” is missing.

Page 3, Line 45. “One of most striking findings” - the word “the” is missing

Page 9, Line 188. “….and the both” – remove “the”

Page 9, Line 193. The author report a decrease in repetitive grooming behavior. Veeraragavan et al. 2016 reported no significant changes in Mecp2-/+ mice, what do you attribute to the divergent results?

Page 22, Line 403: “….GABAA (GABAnergic….” – should be “GABAergic”

Figure 4 A and B. For consistency capitalize the first word on the y axis.

Figure 6 B and C. For consistency capitalize the words on the y axis “goal latency” and “swimming distance”.

**********

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Reviewer #1: Yes: James Brasic

Reviewer #2: No

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PLoS One. 2021 Oct 21;16(10):e0258830. doi: 10.1371/journal.pone.0258830.r002

Author response to Decision Letter 0


16 Jun 2021

Response to Reviewers:

We wish to express our appreciation to the Reviewers for their insightful comments, which have helped us improve our paper substantially.

Reply to Reviewer #1

Comments to authors

Point 1: Materials and methods Morris water maze test Line 127: Since the second figure to be mentioned in the text occurs in this line, the figure should be numbered figure 2.

Answer 1: Thank you for the correction. We have accordingly altered the order of the figure number.

Point 2: Materials and methods Statistical Analysis: Are the data normally distributed as demonstrated by the Kolmogorov-Smirnov or Wilks-Shapiro procedures? If the data are not normally distributed, then a nonparametric procedure such as Kruskal-Wallis is appropriate.

Answer 2: Thank you for the comment. Because sample size for each experiment was small, it was not appropriate to evaluate the equal distribution of the sample. We therefore changed all our statistical methods to nonparametric procedures, accordingly.

Point 3: Results Impaired social behaviors in Mecp2-deficient female rats: Page 9 Line 192 Change “disorders” to “disorder”.

Answer 3: Thank you for the correction. We have accordingly changed “disorders” to “disorder.”

Point 4: Table 1 Pages 13 to 15 Express the values of each row in a single line by using a smaller font.

Answer 4: Thank you for the correction. In accordance, we have corrected the table to express the values of each row in a single line using a smaller font.

Point 5: Legend to figure 7 Line 316 The scale bar appears to be the same length in all panels. What is the magnification indicated by the two scale bars?

Answer 5: Thank you for the correction. In accordance, we have corrected the scale bars.

Point 6: Discussion Relationships between cholinergic and behavioral abnormalities Line 387-408 Include a discussion of the dopaminergic deficits observed as follows: Wong DF, Blue ME, Brašić JR,, Nandi A, Valentine H, Stansfield KH, Rousset O, Bibat G, Yablonski ME, Johnston MV, Gjedde A, Naidu SB. Are dopamine receptor and transporter changes in Rett syndrome reflected in Mecp2-deficient mice? Exp Neurol. 2018; 307: 74-81. https://doi.org/10.1016/ j.expneurol.2018.05.019 PubMed PMID: 29782864.

Answer 6: Thank you for the comment and information. In accordance, we have thoroughly corrected the part of the Discussion section concerning the possible involvement of dopaminergic deficits in the behavioral abnormalities of Mecp2+/− rats and cited the article.

Point 7: Reference Line 517 Remove the space between “30.” and “Panayotis” 

Answer 7: Thank you for the correction. We have accordingly removed the space between “30.” and “Panayotis.”

Point 8: Lines 549-550 Reference 41. Include the original title of the article in German as follows: Rett A. Uber ein bisher nicht bekanntes Krankheitsbild einer angeborenen Stoffwechselst örung [On an until now unknown disease of a congenital metabolic disorder]. Krankenschwester. 1966 Sep;19(9):121-2. German. PMID: 5179620.

Answer 8: Thank you for the correction. In accordance, we have corrected the reference to include the original title of the article in German.

Point 9: Please add appendix of abbreviations. Define all acronyms before their first use.

Answer 9: Thank you for the correction. In accordance, we have added an appendix of abbreviations and defined all acronyms before their first use.

Point 10: Supplemental Methods The section in the supplemental methods belong in the body of the text. There are no restrictions on word counts, number of figures, or amount of supporting information.

Answer 10: Thank you for the comment. In accordance, we have moved the supplemental methods into the body of the text.

Reply to reviewer #2

Comments to authors

Point 1: The correlation of behavioral changes with acetylcholine levels is appears far reaching based on the way the manuscript is organized.

Answer 1: Thank you for the comment. Considering both reviewers’ comments, we have thoroughly corrected the part of the Discussion section concerning the possible involvement of monoaminergic dysfunctions in the behavioral abnormalities of the Mecp2+/− female rats.

Point 2: Figure 7 images are of poor quality and images of amygdala and hypothalamus should be included.

Answer 2: Thank you for the comment. In accordance, we have replaced the microscopic photographs and also inserted images of the amygdala and hypothalamus in Figure 7 and Figure 8.

Point 3: Page 2, Line 25. “…..spatial cognition as well lower plasma” - the word “as” is missing.

Answer 3: Thank you for the correction. In accordance, we have added the word “as.”

Point 4: Page 9, Line 193. The author report a decrease in repetitive grooming behavior. Veeraragavan et al. 2016 reported no significant changes in Mecp2-/+ mice, what do you attribute to the divergent results?

Answer 4: Thank you for the comments. Concerning self-grooming, we considered that the divergent results would come from the developmental impairment in the cholinergic system. Excessive self-grooming behavior, however, is a typical symptom of autism model mice and has also been observed in Mecp2 null male mice mutants. Therefore, further investigations are necessary to understand the meaning of this behavioral abnormality in the Mecp2-deficient female rat. We have added these limitations to the Discussion section.

Point 5: Page 22, Line 403: “….GABAA (GABAnergic….” – should be “GABAergic”

Answer 5: Thank you for the correction. In accordance, we have corrected the word to “GABAergic.”

Point 6: Figure 4 A and B. For consistency capitalize the first word on the y axis.

Answer 6: Thank you for the correction. In accordance, we have capitalized the first word on the y-axis.

Point 7: Figure 6 B and C. For consistency capitalize the words on the y axis “goal latency” and “swimming distance”.

Answer 7: Thank you for the correction. In accordance, we have capitalized the first word on the y-axis.

Attachment

Submitted filename: Response to Reviewer_210602.docx

Decision Letter 1

Atsushi Asakura

13 Aug 2021

PONE-D-21-05550R1

Substantial acetylcholine reduction in multiple brain regions of Mecp2-deficient female rats and associated behavioral abnormalities

PLOS ONE

Dear Dr. Fukumitsu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Sep 27 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Atsushi Asakura, Ph.D

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors compared and contrasted the neurochemical and behavioral aspects of wild type and Mecp2-deficient female rats utilizing a reasonable experimental design and analysis. They concluded that acetylcholine concentration was associated with behavior.

The study was well conducted. The discussion was reasonable.

Some readers may be unfamiliar with some technical details so explicitly documenting them will likely facilitate understanding. Some sections may be better worded and organized to help readers comprehend the key points.

The manuscript will likely merit publication with revisions.

Page 6

Line 131

Change “MWM test” to “Morris water maze (MWM) test”

Page 7

Line 157

Change “Friedman’s test” to “Friedman test”

Provide a reference for the Friedman test. What statistical program was used?

Line 161

Provide a reference for unpaired two-tailed Mann–Whitney U-test. What statistical program was used?

Page 11

Lines 245-246

Provide a reference for unpaired two-tailed Mann–Whitney U-test.

Line 247

Provide a reference for the Kruskal–Wallis with Dunn’s post hoc multiple comparisons test.

Line 248

Provide a reference for the Friedman test.

Page 12

Line 269

Provide a reference for the Friedman test.

Line 270

Provide a reference for the Kruskal–Wallis with Dunn’s post hoc multiple comparisons test.

Line 277

Provide a reference for the Kruskal–Wallis with Dunn’s post hoc multiple comparisons test.

Page 13

Lines 292-293

Provide a reference for the Kruskal–Wallis with Dunn’s post hoc multiple comparisons test.

Lines 298-299

Provide a reference for unpaired two-tailed Mann–Whitney U-test.

Line 308

Change “Fig-2D” to “Fig 2D”

Page 15

Line 327

Change “Mecp2-deficient.” to “Mecp2-deficient”

Page 16

None of the items utilize **.

Line 331

Change “*p < 0.05 and **p < 0.01 vs. female Mecp2+/+” to

“*p < 0.05 female Mecp2+/- vs. Mecp2+/+”

Lines 331-332

Provide a reference for unpaired two-tailed Mann–Whitney U-test.

Page 17

Table 2

None of the items utilize *.

Line 335

Change *p < 0.05 vs. female Mecp2+/+ rats” to

*Non significance female Mecp2+/- vs. Mecp2+/+ rats”

Lines 335-336

Provide a reference for unpaired two-tailed Mann–Whitney U-test.

Line 339

Change “vs. female Mecp2+/+ rats” to “female Mecp2+/- vs. Mecp2+/+ rats”

Provide a reference for unpaired two-tailed Mann–Whitney U-test.

Page 19

Line 361

Change **p <0.01 vs. female Mecp2+/+ rats” to **p <0.01 female Mecp2+/- vs. Mecp2+/+ rats”

Line 362

Provide a reference for unpaired two-tailed Mann–Whitney U-test.

Line 366

Change “**p < 0.01 vs. female Mecp2+/+ rats” to “**p < 0.01 female Mecp2+/- vs. Mecp2+/+ rats”

Page 20

Line 385

Change “demonstrate” to “demonstrated”

Abbreviations

Place the abbreviations in alphabetical order.

Reviewer #2: The authors have addressed the previous comments. Below are some additional minor comments.

Page 4, Line 78. Remove “on so on”

Page 7, Line 150. This is the Figure legend for figure 2, not sure why it is in the methods section.

Page 12, Line 260-261. “Both the frequency (Fig 4A) and duration 261 (Fig. 4B) of self-grooming behavior were significantly or tended to be reduced in female Mecp2+/−…” However, the graph in figure 4B does not show statistical significance for the duration. The current phrasing is not clear, authors should clarify that frequency was reduced and duration tended to be reduced but not significant.

Page 12, Line 283. “Spontaneous locomotor activity was significantly or tended to be lower among female Mecp2+/− rats compared to female Mecp2+/+ rats (Fig 5) during both light and dark phases.” Authors should clarify the specific timepoints of significance.

Page 12, Line 285. For consistency lower case w “29 w”

Page 13, Line 301. It is unclear why the result section “Impaired spatial learning and memory in Mecp2-deficient female rats” is Figure 2, following Figure 6.

Page 14, Line 314. “Surprising, however, biogenic amine and…” – Change to “Surprisingly”

Page 14, Line 323. “These results suggest that impaired cholinergic signaling would severely affect the behavioral abnormalities observed...” The word “would” should be changed to “could”.

Page 18, Line 344. ” Astrocytes are known to support neuronal function through production and secretion of various extrinsic factors and by the uptake of neurotransmitters, and the number and morphological features are associated with neurodevelopmental abnormalities as well as neurodegeneration and neuroinflammation.” A reference is needed for this sentence.

Page 18, Line 355. “Both the neurotrophic factors play important…” – Remove “the”

**********

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Reviewer #1: Yes: James Robert Brasic

Reviewer #2: No

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Attachment

Submitted filename: draft review 210728 2204 2147 PONE-D-21-05550R1.pdf

Attachment

Submitted filename: draft review 210728 2204 2147 PONE-D-21-05550R1.docx

PLoS One. 2021 Oct 21;16(10):e0258830. doi: 10.1371/journal.pone.0258830.r004

Author response to Decision Letter 1


30 Aug 2021

Response to Reviewers:

We wish to express our appreciation to the Reviewers for their insightful comments, which have helped us improve our paper substantially.

Reply to Reviewer #1

Point 1: Page 6

Line 131 Change “MWM test” to “Morris water maze (MWM) test”.

Answer 1: Thank you for the correction. We have accordingly spelled out “MWM test”.

Point 2: Page 7

Line 157 Change “Friedman’s test” to “Friedman test” Provide a reference for the Friedman test. What statistical program was used?

Line 161 Provide a reference for unpaired two-tailed Mann–Whitney U-test. What statistical program was used?

Answer 2: Thank you for the comment. We have accordingly changed “Friedman’s test” to “Friedman test” Concerning about statistical program, we had used GaphPad Prism (version 9.1.2 for Windows) for all statistical analyses (Friedman test, unpaired two-tailed Mann-Whitney U-test, and Kruskal-Wallis with Dunn’s post hoc multiple comparisons test), according to the software guide. So, we have described it in the revised manuscript.

Point 3: Page 13

Line 308 Change “Fig-2D” to “Fig 2D”

Answer 3: Thank you for the correction. We have accordingly changed “Fig-2D” to “Fig 2D”.

Point 4: Page 15

Line 327 Change “Mecp2-deficient.” to “Mecp2-deficient

Answer 4: Thank you for the correction. We have accordingly changed “Mecp2-deficient.” to “Mecp2-deficient”.

Point 5: Page 16

Table 1 None of the items utilize **.

Line 331 Change “*p < 0.05 and **p < 0.01 vs. female Mecp2+/+” to “*p < 0.05 female Mecp2+/- vs. Mecp2+/+”

Answer 5: Thank you for the correction. In accordance, we have changed “*p < 0.05 and **p < 0.01 vs. female Mecp2+/+” to “*p < 0.05 female Mecp2+/- vs. Mecp2+/+”.

Point 6: Page 17

Table 2 None of the items utilize *.

Line 335 Change *p < 0.05 vs. female Mecp2+/+ rats” to *Non significance female Mecp2+/- vs. Mecp2+/+ rats”.

Line 339 Change “vs. female Mecp2+/+ rats” to “female Mecp2+/- vs. Mecp2+/+ rats”

Answer 6: Thank you for the correction. In accordance, we have changed *p < 0.05 vs. female Mecp2+/+ rats” to *Non significance female Mecp2+/- vs. Mecp2+/+ rats”. We also have changed “vs. female Mecp2+/+ rats” to “female Mecp2+/- vs. Mecp2+/+ rats”.

Point 7: Page 19

Line 361 Change **p <0.01 vs. female Mecp2+/+ rats” to **p <0.01 female Mecp2+/- vs. Mecp2+/+ rats”

Line 366 Change “**p < 0.01 vs. female Mecp2+/+ rats” to “**p < 0.01 female Mecp2+/- vs. Mecp2+/+ rats”

Answer 7: Thank you for the correction. In accordance, we have changed **p <0.01 vs. female Mecp2+/+ rats” to **p <0.01 female Mecp2+/- vs. Mecp2+/+ rats”. We also have changed “**p < 0.01 vs. female Mecp2+/+ rats” to “**p < 0.01 female Mecp2+/- vs. Mecp2+/+ rats”.

Point 8: Page 20

Line 385 Change “demonstrate” to “demonstrated”.

Answer 8: Thank you for the correction. We have accordingly changed “demonstrate” to “demonstrated”.

Point 9: Abbreviations Place the abbreviations in alphabetical order.

Answer 9: Thank you for the correction. In accordance, we have replaced the abbreviations in alphabetical order.

Reply to reviewer #2

Comments to authors

Point 1: Page 4, Line 78. Remove “on so on”.

Answer 1: Thank you for the correction. In accordance, we have removed “on so on”.

Point 2: Page 7, Line 150. This is the Figure legend for figure 2, not sure why it is in the methods section.

Answer 2: Thank you for the comment. Since we had mentioned the figure in the materials and methods section “Morris water maze (MWM) test”, the reviewer#1 had advised the figure to be numbered figure 2 in the previous comments, according to the Plos One’s instructions. However, since the mismatch between the order of figures and results could confuse readers, we have replaced the Fig 2 after the result section “Impaired spatial learning and memory in Mecp2-deficient female rats” and altered the order of the figure number.

Point 3: Page 12, Line 260-261. “Both the frequency (Fig 4A) and duration 261 (Fig. 4B) of self-grooming behavior were significantly or tended to be reduced in female Mecp2+/−…” …… The current phrasing is not clear, authors should clarify that frequency was reduced and duration tended to be reduced but not significant.

Answer 3: Thank you for the correction. In accordance, we have revised the sentence to clarify that frequency was reduced and duration tended to be reduced but not significant.

Point 4: Page 12, Line 283. “Spontaneous locomotor activity was significantly or tended to be lower among female Mecp2+/− rats compared to female Mecp2+/+ rats (Fig 5) during both light and dark phases.” Authors should clarify the specific timepoints of significance.

Answer 4: Thank you for the comments. We have shown the locomotor activities at each 30-min from 16 w or 23 w old rats over 2 consecutive 24-periods as supplemental S1 Fig in the revised manuscript. After statistical analyses (Kruskal–Wallis with Dunn’s post hoc multiple comparisons test), we have found no significant difference between genotypes at any time point. These results indicated that the Mecp2 deficiency is not likely to affect circadian rhythms but locomotor activity itself. So, we have additionally described it in the revised manuscript.

Point 5: Page 12, Line 285. For consistency lower case w “29 w”

Answer 5: Thank you for the correction. We have accordingly changed “29W” to “29 w”.

Point 6: Page 13, Line 301. It is unclear why the result section “Impaired spatial learning and memory in Mecp2-deficient female rats” is Figure 2, following Figure 6.

Answer 6: Thank you for the correction. In accordance, we have revised this matter as described in Point 2.

Point 7: Page 14

Line 314. “Surprising, however, biogenic amine and…” – Change to “Surprisingly”

Line 323. “These results suggest that impaired cholinergic signaling would severely affect the behavioral abnormalities observed...” The word “would” should be changed to “could”.

Answer 7: Thank you for the correction. In accordance, we have changed “Surprising” to “Surprisingly”, and “would” to “could”.

Point 8: Page 18, Line 344.” Astrocytes are known to support neuronal function....” A reference is needed for this sentence.

Answer 7: Thank you for the correction. In accordance, we have added references for the sentence.

Point 9: Page 18, Line 355. “Both the neurotrophic factors play important…” – Remove “the”.

Answer 9: Thank you for the correction. In accordance, we have removed “the”.

Attachment

Submitted filename: Response to Reviewer_210830.docx

Decision Letter 2

Atsushi Asakura

7 Oct 2021

Substantial acetylcholine reduction in multiple brain regions of Mecp2-deficient female rats and associated behavioral abnormalities

PONE-D-21-05550R2

Dear Dr. Fukumitsu,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

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Kind regards,

Atsushi Asakura, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Acceptance letter

Atsushi Asakura

12 Oct 2021

PONE-D-21-05550R2

Substantial acetylcholine reduction in multiple brain regions of Mecp2-deficient female rats and associated behavioral abnormalities

Dear Dr. Fukumitsu:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Atsushi Asakura

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. Impaired locomotor activity among female Mecp2+/− rats during both light and dark phases at 16 and 23 weeks of age.

    A-D: 24-h locomotor activity in female Mecp2+/+ (open symbols) and Mecp2+/- (filled symbols) rats. Data are the average ± SE of data collected at each half an hour from 16 w (A, B) or 23 w (C, D) old rats over 2 consecutive 24-h periods (A, C: day1; B, D: day2) (n = 6, for each geneotype). Non significance female Mecp2+/- vs. Mecp2+/+ rats (Kruskal–Wallis with Dunn’s post hoc multiple comparisons test).

    (TIF)

    Attachment

    Submitted filename: Response to Reviewer_210602.docx

    Attachment

    Submitted filename: draft review 210728 2204 2147 PONE-D-21-05550R1.pdf

    Attachment

    Submitted filename: draft review 210728 2204 2147 PONE-D-21-05550R1.docx

    Attachment

    Submitted filename: Response to Reviewer_210830.docx

    Data Availability Statement

    All relevant data are within the manuscript.


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