Article

Daily Rhythmic Behaviors and Thermoregulatory Patterns Are Disrupted in Adult Female MeCP2-Deficient Mice

Division of Genetics and Development, Toronto Western Research Institute, University Health Network, Toronto, Ontario, Canada.
PLoS ONE (Impact Factor: 3.23). 04/2012; 7(4):e35396. DOI: 10.1371/journal.pone.0035396
Source: PubMed
ABSTRACT
Mutations in the X-linked gene encoding Methyl-CpG-binding protein 2 (MECP2) have been associated with neurodevelopmental and neuropsychiatric disorders including Rett Syndrome, X-linked mental retardation syndrome, severe neonatal encephalopathy, and Angelman syndrome. Although alterations in the performance of MeCP2-deficient mice in specific behavioral tasks have been documented, it remains unclear whether or not MeCP2 dysfunction affects patterns of periodic behavioral and electroencephalographic (EEG) activity. The aim of the current study was therefore to determine whether a deficiency in MeCP2 is sufficient to alter the normal daily rhythmic patterns of core body temperature, gross motor activity and cortical delta power. To address this, we monitored individual wild-type and MeCP2-deficient mice in their home cage environment via telemetric recording over 24 hour cycles. Our results show that the normal daily rhythmic behavioral patterning of cortical delta wave activity, core body temperature and mobility are disrupted in one-year old female MeCP2-deficient mice. Moreover, female MeCP2-deficient mice display diminished overall motor activity, lower average core body temperature, and significantly greater body temperature fluctuation than wild-type mice in their home-cage environment. Finally, we show that the epileptiform discharge activity in female MeCP2-deficient mice is more predominant during times of behavioral activity compared to inactivity. Collectively, these results indicate that MeCP2 deficiency is sufficient to disrupt the normal patterning of daily biological rhythmic activities.

Full-text

Available from: Berj Bardakjian
Daily Rhythmic Behaviors and Thermoregulatory
Patterns Are Disrupted in Adult Female MeCP2-Deficient
Mice
Robert G. Wither
1,4.
, Sinisa Colic
7.
, Chiping Wu
2,3
, Berj L. Bardakjian
3,7
, Liang Zhang
2,3,5
,
James H. Eubanks
1,3,4,6
*
1 Division of Genetics and Development, Toronto Western Research Institute, University Health Network, Toronto, Ontario, Canada, 2 Division of Fundamental
Neurobiology, Toronto Western Research Institute, University Health Network, Toronto, Ontario, Canada, 3 University of Toronto Epilepsy Research Program, University of
Toronto, Toronto, Ontario, Canada, 4 Department of Physiology, University of Toronto, Toronto, Ontario, Canada, 5 Department of Medicine (Neurology), University of
Toronto, Toronto, Ontario, Canada, 6 Department of Surgery (Neurosurgery), University of Toronto, Toronto, Ontario, Canada, 7 Department of Electri cal and Computer
Engineering, University of Toronto, Toronto, Ontario, Canada
Abstract
Mutations in the X-linked gene encoding Methyl-CpG-binding protein 2 (MECP2) have been associated with
neurodevelopmental and neuropsychiatric disorders including Rett Syndrome, X-linked mental retardation syndrome,
severe neonatal encephalopathy, and Angelman syndrome. Although alterations in the performance of MeCP2-deficient
mice in specific behavioral tasks have been documented, it remains unclear whether or not MeCP2 dysfunction affects
patterns of periodic behavioral and electroencephalographic (EEG) activity. The aim of the current study was therefore to
determine whether a deficiency in MeCP2 is sufficient to alter the normal daily rhythmic patterns of core body temperature,
gross motor activity and cortical delta power. To address this, we monitored individual wild-type and MeCP2-deficient mice
in their home cage environment via telemetric recording over 24 hour cycles. Our results show that the normal daily
rhythmic behavioral patterning of cortical delta wave activity, core body temperature and mobility are disrupted in one-year
old female MeCP2-deficient mice. Moreover, female MeCP2-deficient mice display diminished overall motor activity, lower
average core body temperature, and significantly greater body temperature fluctuation than wild-type mice in their home-
cage environment. Finally, we show that the epileptiform discharge activity in female MeCP2-deficient mice is more
predominant during times of behavioral activity compared to inactivity. Collectively, these results indicate that MeCP2
deficiency is sufficient to disrupt the normal patterning of daily biological rhythmic activities.
Citation: Wither RG, Colic S, Wu C, Bardakjian BL, Zhang L, et al. (2012) Daily Rhythmic Behaviors and Thermoregulatory Patterns Are Disrupted in Adult Female
MeCP2-Deficient Mice. PLoS ONE 7(4): e35396. doi:10.1371/journal.pone.0035396
Editor: Nicoletta Landsberger, University of Insubria, Italy
Received Decembe r 2, 2011; Accepted March 15, 2012; Published April 16, 2012
Copyright: ß 2012 Wither et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Funding provided by an operating grant awarded by the Canadian Institutes of Health Research to JHE (MOP-106481), and by a Canadian Institutes of
Health Research to BLJ (MOP-20158). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interest s: The authors have declared that no competing interests exist.
* E-mail: jeubanks@uhnres.utoronto.ca
. These authors contributed equally to this work.
Introduction
Mutations in the X-linked gene encoding Methyl-CPG-binding
protein 2 (MECP2) cause the neurodevelopmental disorder Rett
syndrome [1], and MECP2 mutations and duplications have been
documented in several other neurodevelopmental and neuropsy-
chiatric disorders, such as X-linked mental retardation syndrome,
severe neonatal encephalopathy, Angelman’s syndrome, and in
some cases of idiopathic autism [2–4]. Further, diminished levels
of MeCP2 have been noted in the autistic brain [5], and in cases of
nonspecific neuropsychiatric disorder [6]. These observations
highlight the essential role played by MeCP2 in establishing and
maintaining neural homeostasis, and illustrate that modest
alterations in its prevalence are sufficient to induce neurological
impairments.
To better elucidate how MeCP2 regulates neural development
and neural function, and to allow for preclinical translational
studies, several mutant mouse models have been developed that
either lack MeCP2 or express a clinically relevant mutant form of
MeCP2 [7–11]. Studies in these mice have confirmed that MeCP2
deficiency alters normal brain development, synaptic communi-
cation, and neural network activities [12–13], and several
behavioral impairments have been identified that likely stem
from these neural deficiencies. To date, however, the primary
behavioral parameters examined tend to rely on tests that take the
subjects out of their cages, and transiently expose them to a new
environment for assay. While these tasks have high value for
assessing specific behavioral endpoints, the daily cyclic behavioral
performance of the mutant mice in their home environment is not
typically assessed, and electrographic brain wave activity patterns
that are known to correlate with specific behaviors in wild-type
subjects remain largely uninvestigated in MeCP2-deficient mice.
Given the apparent link between impaired MeCP2 function and
altered behavioral state rhythmicity in Rett syndrome patients [14]
and in Mecp2
308/y
mice [15], we sought to determine whether
impaired MeCP2 function would be sufficient to alter the daily
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EEG behavior, thermoregulatory, and/or periodic ambulatory
cycles of mice. Here, we provide the first report of how these daily
cyclic activity patterns are affected by a heterozygous deficiency of
Mecp2 in female mice.
Materials and Methods
Ethics Statement
All animal experimentation was conducted in accordance with
the guidelines of the Canadian Council of Animal Care, and
thoroughly reviewed and approved before implementation by the
Toronto General and Western animal care committee (Protocol
1321.7). All surgery was performed under general anesthesia, and
every effort was made to minimize suffering.
Animal Subjects
Two strains of MeCP2-deficient mice ( Mecp2
tm1.1Bird
[7] and
Mecp2
tm2Bird
[16], obtained from Jackson Laboratories, Bar
Harbor, ME) were used in this study. The Mecp2
tm1.1Bird
(n = 7),
Mecp2
tm2Bird
(n = 4), and wild-type mice were all female, aged
between 300 and 400 days, and maintained on a pure C57Bl/6
background. Although different in molecular design, the Mecp2
gene is disrupted in each of these lines, and each displays common
phenotypic progression [16]. Genotyping was done via polymerase
chain reaction (PCR) as described previously [16–17]. All animals
were housed in a vivarium that was maintained at 22–23uC with a
standard 12-hour light on/off cycle commencing at 6:00. For this
study, Zeitgeber time of 0 refers to the 6:00 lights-on daily time.
Implantation Surgery
Experimental mice were implanted with a mouse-specific
wireless telemetry probe (TA11ETA-F10; Data Sciences Interna-
tional (DSI), St. Paul, MN) for recording of body temperature,
general activity and EEG. The surgical implantation procedure
was as described previously [18] with minor modifications. Briefly,
mice were anesthetized with 2% isoflurane and the wireless
transmitter placed into their peritoneal cavity. Silicone elastomer
insulated sensing and reference wires connecting the transmitter
were orientated rostrally toward the head via a subcutaneous
route. The sensing wire was soldered to an intracranial EEG
polyimide-insulated stainless steel electrode with an outside
diameter of 125
mm, and placed in the parietal cortex region
(bregma 20.6 mm, lateral 1.5 mm, and depth 1.5 mm) with the
reference wire placed at bregma 25 mm, lateral 1 mm, and depth
1.5 mm. The implantation surgery caused no apparent abnor-
malities in the mice, and average body weights of both Mecp2
2/+
and wild-type mice returned to pre-operative values within 2
weeks post-surgery (32.3 g versus 32.9 g and 26.8 g versus 27.0 g
for Mecp2
2/+
(n = 11) and wild-type (n = 8) respectively).
Electrophysiology Data Collection
Body temperature, activity, and EEG waveforms were collected
from the implanted mice for continuous 24-hour periods.
Waveform data was transmitted from the TA11ETA-F10
telemetry probes to a wireless receiver (RPC-1, DSI), which
passes the data through a data exchange matrix serving as a
multiplexer (DSI), and was analyzed using DataQuest A.R.T.
(DSI). Body temperature was acquired using the TA11ETA-F10’s
thermosensor from the peritoneal cavity. Gross locomotive activity
was determined by assessing the standard deviation of the wireless
signal strength of the transmitter in relationship to two receiving
antennae arranged perpendicularly in the RPC-1 wireless receiver.
This method and arrangement has been used previously to track
and measure locomotive activity in mice [19–20]. The accuracy of
the system to detect ambulatory movement was further validated
by visually comparing the activity output of the system with
movement revealed by synchronized video recordings. Analysis of
random 10 minute segments from these video data revealed that
the collection program detected all of the ambulatory movements
in the mice and conversely that .95% of the activity identified by
the program was accompanied by visible gross movement by the
mouse (n = 5 mice, Video S1). Both temperature and motor
activity data were transmitted at a rate of 50 Hz, using a sampling
frequency (analog to digital) of 250 Hz. The EEG waveform was
transmitted at 200 Hz and sampled at 1 kHz.
Characterization of cortical epileptiform discharge events
24 hour EEG traces were visually inspected to confirm and
quantify the presence of discharge activity as described previously
[20–21]. In brief, a discharge event was defined as having am-
plitudes of at least 1.5-fold background, durations of at least
0.4 seconds, and a frequency of between 6 and 10 Hz. Two
genotype-blinded investigators independently assessed EEG activ-
ity, and the individual counts averaged. The overall concordance
between these individuals was 86.4%, and these differences were
averaged for final analysis of discharge incidence rate and the
times of discharge occurrence over the 24-hour cycle. Having
confirmed the presence of discharge activity using established
manual criteria [20–21], we then developed an automated method
to characterize the duration and frequency components of the
discharges. For this, a 6–10 Hz FIR band pass filter was applied
to specifically isolate the frequency band associated with the
discharges. The envelope of the filtered signal was produced by
convolution of the square of the filtered data with a Gaussian
kernel of 200-point aperture [22]. This envelope peaks whenever
strong 6–10 Hz activity is present. As normal cortical EEG signals
rarely display high-amplitude rhythmic spiking within this fre-
quency, the envelop peak reflects discharge events (Figure S1). To
determine discharge durations, the left and right inflection points
of detected events were used to find the start and end points
respectively. The inflection points were computed by convolving
the envelope with the derivative of the Gaussian kernel as above.
The DataQuest A.R.T. program (DSI) was used to generate total
spectral plots over the 24-hour period for individual mice. Time-
frequency analysis was conducted using the continuous wavelet
transform (CWT) found in the Matlab digital signal processing
toolbox. The basis function used in the CWT analysis was the
Morlet mother wavelet [23–24], which is commonly used in EEG
analyses [23]. To minimize the issue of scaling, the analysis was
divided into low frequencies (0.5–30 Hz) and high frequencies
(30–80 Hz), with 0.5 Hz step size.
Recognition of periodic variations in EEG, gross motor
activity, and core body temperature
EEG signals within the delta band (0.5–4 Hz) [25] were
extracted by applying a series of steps. First, the data were pre-
processed by removing segments indicative of movement artifacts
(characterized by voltages higher than 0.5 V), and the 0.25 second
time period preceding and succeeding these events. Then, a FIR
band pass filter with an order of 1000 was applied to isolate
specifically the delta band. Delta power was obtained by squaring
the delta band signal, and then averaging these values over
30 second intervals so the resulting value aligns with the
movement activity and temperature signals (which were also
recorded at 30 second sampling periods) by the data acquisition
system. The Pearson’s product-moment correlations between delta
power, motor activity, and core body temperature were conducted
using smoothed versions of these raw 30 second interval data. The
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smoothing function employed was the 50-point Fast Fourier
Transformation (FFT) in OriginPro 6.1 (OriginLab Corporation,
Northampton, MA). To then discern the daily patterning of these
three signals, each was normalized to have 0 mean and variance of
1, and a Gaussian-based kernel with aperture 50 was applied on all
three signals generating an envelope of the signals. A threshold of 0
was then applied to discretize the signals into two different states
(Figure S2). The delta power parameter was discretized into delta
and non-delta states with a ‘complete delta cycle’ being defined as
a state of delta followed by state of non-delta, where each
individual state has a duration of at least 15 minutes. Similarly,
‘mobility cycles’ and ‘body temperature cycles’ were defined as
the combination of a consecutive active and an inactive state, or
consecutive high body temperature and a low body temperature
states, respectively.
Statistical analysis
Student’s t-tests were used for direct comparisons between
two groups. For comparisons between multiple groups, one-way
ANOVA with Bonferroni post hoc correction for multiple com-
parisons was utilized. F-tests were used to compare the equality
of two variances between groups. For comparing the correlative
strength between two groups, Pearson’s product moment correla-
tion coefficient, a determiner of linear dependency between two
variables, was employed. Significance was set at p,0.05. Mean and
standard error of the mean are presented throughout the text and
figures.
Results
The general properties of cortical EEG activity are
preserved in Mecp2
2/+
mice
Consistent with our previous observations [21], the general
neocortical EEG signals in adult Mecp2
2/+
mice did not display
overt differences from adult female wild-type mice. Waveforms
with elevated amplitude and slow frequency (0.5–4 Hz, delta
band) were evident during immobile and sleep-like behavior, while
lower amplitude higher frequency activity was seen during periods
of movement or exploration. CWT time-frequency analyses [23]
of these cortical EEG activities (excluding periods of EEG
discharge activity, see below) revealed no qualitative differences
in the frequency powers of wild-type and Mecp2
2/+
mice within
the 0.5–80 Hz spectrum during either the active or inactive states
of behavior (Figure 1A–D). Additionally, time-frequency analysis
of Mecp2
2/+
and wild-type 24-hour EEG waveforms using a Fast
Fourier Transformation revealed the overall power spectrum
distributions between the two groups was preserved (Figure 1E, F),
and analysis of specific waveforms, e.g., the delta band (0.5–4 Hz),
alpha band (8–12 Hz) and beta band (15–30 Hz), across the full
24-hour day also revealed no significant differences in overall
power between the groups (Figure 1G).
Mecp2
2/+
mice display alterations in their daily pattern of
cortical delta wave activity
The presence of delta slow wave cortical EEG waveforms is
often used as an indicator of sleep in wild-type rodents [26].
Analysis of smoothed cortical delta power (as derived from the
EEG waveforms, Figure S3A–D) in wild-type mice revealed clearly
defined patterns of rhythmicity over the 24-hour period
(Figure 2A). In contrast, the daily patterns of delta power in
Mecp2
2/+
mice were more erratic in periodicity and duration
(Figure 2B). Comparison of wild-type and Mecp2
2/+
mice revealed
a significant decrease in the average number of delta cycles over a
24-hour period (12 hours light, 12 hours dark, Figure 2C).
Mecp2
2/+
mice displayed an average of 7.660.6 delta cycles
compared to 11.360.7 in controls (p = 0.001). This overall decease
was equivalently diminished in both phases of the 24-hour day
(p,0.01, for each respectively, Figure 2C). Further, the average
duration of the non-delta state of each cycle period was
significantly longer in Mecp2
2/+
mice than wild-type mice
(1.560.3 hours versus 0.7560.1 hours, p,0.05, Figure 2D). This
increase in non-delta state duration was present during both the
light and dark phases of the 24-hour day, and consistent with the
preferential nocturnal behavior of mice, both Mecp2
2/+
and wild-
type mice displayed greater non-delta time durations in the dark
phase of the 24-hour day.
Mecp2
2/+
mice display alterations in daily cyclic mobility
patterns
In contrast to the alterations in delta power periodic patterning,
examination of smoothed mobility patterns (Figure S3E–H) failed
to reveal differences in cycle number between wild-type (Figure 3A)
and Mecp2
2/+
mice (Figure 3B) over the 24-hour day. Mecp2
2/+
mice displayed an average of 9.060.8 total mobility cycles over the
24-hour day, while wild-type mice displayed an average of
9.060.7 cycles (Figure 3C). However, although the number of
mobility cycles was preserved, the distribution of time in the active
phase versus the inactive phase of these cycles differed between
Mecp2
2/+
and wild-type mice. Specifically, the average duration
of the active state of a cycle was significantly decreased in
Mecp2
2/+
mice over the 24-hour day (0.5260.03 hours versus
0.6660.04 hours, p,0.05, Figure 3D), with the difference being
predominant in the dark phase of the 24-hour cycle. Wild-type
mice showed longer active state durations in the dark relative to
the light phases (0.7860.06 hours versus 0.5460.02 hours in dark
and light respectively, p,0.005), while Mecp2
2/+
mice exhibited
similar active state durations in both light and dark phases
(0.5260.04 hours versus 0.5060.05 hours in dark and light
respectively, Figure 3D).
Mecp2
2/+
mice possess altered home cage mobility
profiles
The reduced active state duration in Mecp2
2/+
mice suggests
that they spend more time in the awake-immobile state than the
awake-active state relative to wild-type mice. Analysis of the raw
movement profiles revealed a significant reduction in the total
amount of mobility (as deduced by changes in strength of the
telemetry signal at the receiver) between Mecp2
2/+
mice and wild-
type mice (12167 versus 204620 mobility counts, respectively
p,0.005). Further, the overall time spent by Mecp2
2/+
mice
moving in their home cages over a 24-hour period was sig-
nificantly reduced compared to age-matched wild-type mice
(694631 versus 931643 segments containing mobility, p,0.001,
Figure 3E, F). In addition to total mobility differences, the average
rate of movement by the Mecp2
2/+
mice was also diminished
relative to wild-type (0.1760.01 versus 0.2260.01 mobility
counts/sec, p,0.05), and this effect was the most pronounced in
the dark phase of the day (Figure 3G).
The inverse correlation of delta power and behavioral
activity is disrupted in Mecp2
2/+
mice
In wild-type mice, there was a strong inverse correlation
between mobility and cortical delta power (Figure 4A). This was
not the case in Mecp2
2/+
mice (Figure 4B). The strength of the
Pearson’s product-moment correlation coefficient (a measure of
linear dependency) revealed a strong inverse correlation (average
r=20.75) between delta power and movement in wild-type mice,
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consistent with delta power serving as a good predictor for
sleep/immobility in wild-type mice [26]. However, the Pearson’s
correlation coefficient for delta power and movement in Mecp2
2/+
mice was significantly weaker (average r = 20.42), indicating that
delta power is not a good predictor for immobile or sleep states
in Mecp2
2/+
mice (Figure 4C). In fact, as shown in Figure 4B,
instances of high delta power concomitant with mobility were
frequently observed in Mecp2
2/+
mice.
Mecp2
2/+
mice display impaired body temperature
patterning and regulation
The patterns of cyclic body temperature fluctuations (derived
from smoothed raw data, Figure S3I–L) also revealed differences
between wild-type (Figure 5A) and Mecp2
2/+
mice (Figure 5B).
Mecp2
2/+
mice displayed fewer temperature cycles per day than
wild-type mice (6.860.5 versus 9.860.7, Figure 5C), and an
increase in the average duration of time spent in the high phase of
their temperature cycle relative to wild-type mice (1.4760.13
versus 0.9560.09 hours, respectively, Figure 5D). In addition to
having impaired periodic rhythmic patterns, the average daily
minimal temperature, and the average daily maximal temperature
of Mecp2
2/+
mice were each significantly lower than wild-type
(33.960.7uC versus 35.660.2uC respectively, for minimum,
p,0.05; and 37.960.2uC versus 38.560.1uC respectively, for
maximum, p,0.05). Consistently, the core body temperature
range of Mecp2
2/+
mice had higher variance than wild-type mice
over the 24-hour day (4.06uC
2
range versus 0.31uC
2
range,
respectively, p,0.005, Figure 6A). Moreover, during periods of
mobility and inactivity specifically, the temperature of Mecp2
2/+
mice was significantly lower than that of wild-type mice
(36.660.2uC versus 37.460.1uC for mobile states and
35.860.3uC versus 36.860.1uC for inactive states, p,0.005 and
p,0.05, Figure 6B, C), and the correlation coefficient between
movement and body temperature in the Mecp2
2/+
mice was
Figure 1. The general EEG waveform properties of the
Mecp2
2/+
mouse cortex are similar to wild-type. Panels A–D: Representative
examples of a 1 minute segment of raw EEG activity (i) taken from wild-type (A and C) and Mecp2
2/+
mice (B and D) during mobility (A and B) and
during inactivity (C and D). Shown below each raw EEG trace is the corresponding wavelet transformation showing the spectrum of frequency power
for the 0.5–30 Hz range (ii) and for the 30–80 Hz range (iii). Panels E and F: Average normalized time-frequency power spectrum plots of wild-type (E)
and Mecp2
2/+
(F) mice. Panel G: Average power of the delta band (0.5–4 Hz), the alpha band (8–12 Hz) and beta band (15–30 Hz) in wild-type and
Mecp2
2/+
mice normalized to peak power for each respective mouse. Histograms are plotted as mean 6 SEM. Asterisks denote statistical significance
p,0.05, student’s unpaired t-test for n = 11 Mecp2
2/+
mice and n = 8 wild-type mice.
doi:10.1371/journal.pone.0035396.g001
Figure 2.
Mecp2
2/+
mice display altered daily delta cycle periodicity and duration. Panels A and B: Representative traces of delta power
patterning over 24 hours in a wild-type (A) and a Mecp2
2/+
(B) mouse. The dark phase (ZT 12–24) is shaded in these plots. Panels C and D: Histograms
showing the average number of delta cycles (C), and the average duration of time spent in a non-delta state per delta cycle (D). For panels C and D,
the total 24-hour data set is shown with the data for either the light (ZT 0–12) or dark (ZT 12–24) phases specifically. Shown are the mean 6 SEM for
n=11Mecp2
2/+
and n = 8 wild-type mice. Asterisks denote statistical significance at p,0.05, one-way ANOVA with Bonferroni post hoc correction for
multiple comparisons.
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substantially weaker than that of the wild-type mice (0.76 versus
0.54, respectively, p = 0.001, Figure 6D). Collectively, these results
indicate Mecp2
2/+
mice display an overall reduction in core body
temperature throughout the day, and that their homeostatic
regulation of body temperature is impaired.
Mecp2
2/+
mice display spontaneous cortical epileptiform
discharge activity
Raw EEG waveform data was examined from Mecp2
2/+
mice
to determine the prevalence and distribution of epileptiform
discharges throughout the 24-hour period. For these assessments,
a discharge event was defined as a high amplitude rhythmic
waveform lasting at least 0.4 seconds with a frequency between 6
and 10 Hz (Figure 7A). No discharge activity was detected in any
of the wild-type mice examined (n = 8). Cortical EEG discharges
were observed in 8 of 11 Mecp2
2/+
mice. In these mutants, the
average number of cortical epileptiform discharges per hour over a
24-hour period was 10.761.6 (Figure 7B). The average duration
of the discharge events was 0.7660.01 seconds, and the average
frequency of the discharges was 8.660.02 Hz (Figure 7C, D).
Figure 3.
Mecp2
2/+
mice display abnormal cycles of daily activity and mobility deficits in their home cage environment. Panels A and
B: Representative traces of activity patterning over 24 hours in a wild-type (A) and a Mecp2
2/+
(B) mouse. The dark phase is shaded. Panels C and D:
Histograms showing the average number of mobility cycles (C), and the average duration of time spent in an active state per mobility cycle (D). For
panels C and D, the total 24-hour data set is shown with the results specific for either the light or dark phases. Panels E–G: Histograms showing
the home-cage activity parameters of Mecp2
2/+
and wild-type mice over the full 24 hours, and for the light and dark phases of the day specifically.
Panel E shows the total amount of mobility of mice over 24 hours. Panel F shows the number of 30-second segments throughout the day in which
Mecp2
2/+
and wild-type mice displayed mobility. Panel G shows the average rate of movement (magnitude of movement per second) performed by
Mecp2
2/+
and wild-type mice. Shown are the mean 6 SEM for n = 11 Mecp2
2/+
and n = 8 wild-type mice. Asterisks denote statistical significance at
p,0.05, one-way ANOVA with Bonferroni post hoc correction for multiple comparisons.
doi:10.1371/journal.pone.0035396.g003
Figure 4. The normal correlation between cortical delta power and mobility is altered in
Mecp2
2/+
mice. Panels A and B: Representative
traces of activity and delta power parameters over a 6 hour period in the light phase of the day for a wild-type (A) and a Mecp2
2/+
(B) mouse. For
each, the solid black line denotes mobility while the grey dotted line denotes delta power. Panel C: Scatter plots showing the Pearson’s product-
moment correlation coefficients for delta power compared to activity in Mecp2
2/+
and wild-type mice. Each point represents the daily correlative
strength for a single subject. The bar on the scatter plot indicates the mean for each set. Asterisks denote statistical significance (p,0.05) between
the indicated groups (student’s unpaired t-test). n = 11 Mecp2
2/+
and n = 7 wild-type mice, respectively.
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While spontaneous convulsions were not observed in any of the
Mecp2
2/+
mice, cortical discharge activity was associated with
behavioral freezing, which often lasted longer than the duration of
the discharge (data not shown). Analysis of discharge activity
during the light and dark phases of the day failed to reveal any
significant differences: the incidence rate of the discharges, their
average duration, and their average frequency did not significantly
differ during the light or dark phases.
Cortical epileptiform discharge activity predominates
during the active state of a mobility cycle
To assess whether cortical discharge activity occurred randomly
throughout the day, or was preferentially seen during certain
behavioral states, we compared discharge activity across active
and inactive states, and in periods of high and low core body
temperature. These assessments revealed that significantly higher
discharge activity was found in Mecp2
2/+
mice during the active
phase of their behavioral mobility cycle during the entire day
(22.064.0 versus 5.461.0 discharges, p,0.005, Figure 8A, B), and
during the light and dark phases of the day, specifically. In
contrast, no significant association between core body temperature
and discharge activity was seen. For this, we compared discharge
activity in mice during times when their core body temperature
was within the top or bottom 25% range of the full 24-hour day.
No significant differences in discharge rate were observed between
these periods of high and low core body temperature either during
the entire day (13.263.5 versus 11.263.0 discharges per hour,
respectively p = 0.63, Figure 8C, D), or during the light or dark
phases specifically.
Discussion
In this study, we examined the daily periodic cortical EEG
waveform activity, body temperature, and movement activity
parameters of Mecp2
2/+
mice in their home-cage setting. Five
principal observations emerge from our work. First, the normal
daily pattern of cyclic EEG delta wave activity is altered in
Mecp2
2/+
mice. These mutants display a decreased number of
daily delta cycles, and spend longer periods than normal in a low
delta power state. Second, Mecp2
2/+
mice display significantly less
movement in their home-cage environment, particularly during
the nocturnal phase, and display significantly more time in an
awake-but-inactive state. Third, the daily minimum, maximum,
and overall average temperature of Mecp2
2/+
mice is lower than
that of wild-type mice. Fourth, Mecp2
2/+
mice display spontaneous
cortical epileptiform discharges, and this discharge activity is most
pronounced when the mouse is in an active behavioral state. Fifth,
the daily rhythmic and correlative patterns of delta power,
movement activity, and body temperature are significantly altered
in Mecp2
2/+
mice. Collectively, these investigations identify novel
behavioral deficits associated with MeCP2 deficiency, and provide
Figure 5. The normal pattern of body temperature cycling is altered in
Mecp2
2/+
mice. Panels A and B: Representative traces of daily body
temperature over 24 hours in a wild-type (A) and a Mecp2
2/+
(B) mouse. The dark phase of the day is shaded on each panel. Panels C and D:
Histograms showing the average number of body temperature cycles (C), and the average duration of time spent in the top half of the full
temperature range for each mouse across the day (D). For panels C and D, the total 24-hour data set is shown along with the results specific for either
the light or dark phases. Shown are the mean 6 SEM for n = 11 Mecp2
2/+
and n = 8 wild-type mice. Asterisks denote statistical significance at p,0.05,
one-way ANOVA with Bonferroni post hoc correction for multiple comparisons.
doi:10.1371/journal.pone.0035396.g005
Altered Periodic Behavior in MeCP2-Deficient Mice
PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e35396
Page 8
a new investigative procedure that can be employed for trans-
lational studies.
Although there is clear evidence for disrupted sleep-wake cycles
in Rett syndrome patients [27–28], there have been few
assessments of whether normal biological patterning is altered in
MeCP2-deficient mice. Our data show that Mecp2
2/+
mice display
significantly disrupted daily behavioral patterns compared to age
and gender-matched wild-type mice. Specifically, Mecp2
2/+
mice
display reduced numbers of normal cortical delta activity and body
temperature cycles over a 24-hour period. High delta power has
been used as an index for determining sleep and awake times in
wild-type animals [26] [29]. Consistent with this, we found a
strong correlation between periods of high delta power and
periods of low activity in wild-type mice. Intriguingly, though, this
correlation was not observed in Mecp2
2/+
mice, where high delta
power was often observed during periods of high activity. This
suggests that the normal homeostatic balance of neural circuits is
disrupted in the Mecp2
2/+
brain. However, we cannot exclude the
possibility that a movement artifact caused by the slow ambulatory
patterns of MeCP2
2/+
mice may have contributed to this signal.
Irrespective of origin, the clear difference in delta power and
activity correlational strength between wild-type and MeCP2
2/+
mice illustrates a phenotypic difference that arises from the
MeCP2 deficiency.
The observation of disrupted daily rhythmic patterning in
Mecp2
2/+
mice is consistent with the recent studies that found
Mecp2 mRNA to be a direct target of the microRNA miR-132
[30–31]. miR-132 expression is robustly induced within neurons of
the suprachiasmatic nucleus (SCN) by light stimulation [32], and
miR-132 negatively regulates MeCP2 protein levels in these
neurons. This regulation of MeCP2 expression is one component
of the system regulating the expression of Period genes and thus
contributes to clock entrainment [30]. Given the strong evidence
that disruption of clock gene regulation in SCN is sufficient to alter
cortical delta periodicity and power [33], the altered delta patterns
we observe in Mecp2
2/+
mice are in line with MeCP2 playing a
significant role in circadian regulation, as suggested by Alverez-
Savaadra et al. [30]. Based on these results and our findings, it
Figure 6. Core body temperature regulation is altered in
Mecp2
2/+
mice. Panel A: Scatter plot showing the range of core body temperature
in Mecp2
2/+
and wild-type mice. Each point represents the absolute range (min to max during the 24-hour day) of core body temperature for an
individual mouse. On Panel A, # denotes p,0.05 as determined using an F-test for the equality of two variances. Panel B and C: Histograms showing
the mean 6 SEM of the active body temperature of Mecp2
2/+
and wild-type mice (B), and their average inactive body temperature (C) throughout the
day, or specifically during the light or dark phases. Panel D: Scatter plot showing the Pearson’s product-moment correlation coefficients for mobil ity
and temperature in Mecp2
2/+
and wild-type mice. Each point represents the daily correlative strength between mobility and temperature for a single
subject. The bar on the scatter plot indicates the mean for each set. Asterisks denote statistical significance (p,0.05) between the indicated groups
(student’s unpaired t-test). n = 11 Mecp2
2/+
mice and n = 7 wild-type mice.
doi:10.1371/journal.pone.0035396.g006
Altered Periodic Behavior in MeCP2-Deficient Mice
PLoS ONE | www.plosone.org 9 April 2012 | Volume 7 | Issue 4 | e35396
Page 9
would be of interest to further explore whether alterations of
cortical delta activity patterns occur in Rett syndrome patients
and/or in patients with other MeCP2-related neural disorders.
In addition to disrupted rhythmic behavioral patterning,
Mecp2
2/+
mice displayed diminished overall movement in their
home-cage setting. Consistent with previous results from Mecp2
308/y
male mice [17] [34], the activity of female Mecp2
2/+
mice was
reduced similarly during the light and dark phases of the diurnal
cycle. Analysis of the home-cage body temperature also revealed
alterations in daily temperature cycling patterns in Mecp2
2/+
mice.
Mecp2
2/+
mice showed significant decreases in both their peak
minimum and maximum body temperature over the day, and
collectively showed an overall decrease in their average body
temperature both throughout the day, and also during periods of
activity and inactivity specifically. These observations confirm and
extend from those of a recent report in which the basal body
temperature of male MeCP2
2/y
mice was found to be reduced
compared to wild-type mice [35]. In addition to showing a decrease
in average daily temperatures, though, our results also show that the
range of normal body temperature fluctuation over the day is
significantly greater in Mecp2
2/+
mice, and that Mecp2
2/+
mice have
a poorer ability than wild-type mice to regulate body temperature.
Collectively, these results are consistent with impaired autonomic
nervous system function, which is a cardinal phenotype of clinical
Rett syndrome.
In agreement with our previous acute study [21], we observed
the presence of abnormal epileptiform-like discharges in the
somatosensory cortex of Mecp2
2/+
mice. Our examination of the
distribution of discharges throughout the 24-hour diurnal cycle
revealed no differences in discharge incidence between the light
and dark phases of the day. However, the incidence rate for
discharge activity did correlate with times when the mutant mice
were in specific behavioral states. Significantly more discharge
activity was observed in the mutants during times of activity and/
or movement compared to times of immobility. Perhaps
surprisingly, however, no differences in discharge rate were seen
in the mice when their body temperature was in the upper or lower
25% of their daily range. This result was somewhat unexpected, as
lower temperature tends to slow metabolic processes, and has been
linked to an attenuation of seizure rates [36]. The most likely
Figure 7. Properties of epileptiform discharges in
Mecp2
2/+
mice. Panel A: Representative example of a 10 second segment of raw EEG
activity (i) illustrating a typical discharge event in a Mecp2
2/+
mouse, and the corresponding wavelet transformation showing the spectrum of
frequency power for the 0.5–30 Hz range (ii) and for the 30–80 Hz range (iii). Panels B–D: Histograms showing the mean 6 SEM of the discharge rate
per hour (B), the average discharge duration (C), and the average frequency component of all the discharges (D) in Mecp2
2/+
mice. Presented on each
histogram is the total over the 24-hour period, and the data stratified for specifically light and dark phases. No statistically significant differences in
discharge activity, duration, or frequency were seen between the light and day phases (student’s paired t-test, n = 8 Mecp2
2/+
mice).
doi:10.1371/journal.pone.0035396.g007
Altered Periodic Behavior in MeCP2-Deficient Mice
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Page 10
explanation for this is that although lower, the decreased core
body temperature is not sufficient to have a major effect on neural
activity, and thus the hyper-excitability of the MeCP2-deficient
circuits is not diminished.
In summary, in this study we conducted the first concurrent
examination of 24-hour cortical EEG waveforms, movement
activity, and body temperature profiles in Mecp2
2/+
mice in their
home-cage environment. Our results indicate that in addition to
attenuating home-cage movement activity, MeCP2 deficiency is
sufficient to alter the normal daily cyclic patterns of cortical delta
wave activity and body temperature. Further, we characterize the
average incidence, frequency, and duration of epileptiform
discharges in Mecp2
2/+
mice over a 24-hour period, and show
that there is a relationship between their behavioral state and the
prevalence of cortical discharge activity.
Figure 8. Epileptiform discharge activity in
Mecp2
2/+
mice differs between behavioral states. Panels A and B: Incidence rate of cortical
discharge activity during either the mobile or inactive behavioral states. The histogram (A) shows the mean 6 SEM of the discharge rate per hour,
normalized to the time spent in each behavioral state as above. Panel B shows a representative plot of epileptiform discharge distribution over the
light (i) and dark (ii) phases of a 24-hour day. Red spikes represent individual discharge events and the shaded regions denote times in which mobility
was present. Panels C and D: Incidence rate of cortical discharge activity when core body temperature for the Mecp2
2/+
mice was within the top 25%
(high) or the lowest 25% (low) of the mean value for their 24-hour cycle. The histogram (C) shows the mean 6 SEM of the discharge rate per hour,
normalized for time spent in each temperature category as above. Panel D shows a representative plot in the same similar format as Panels B, except
dark shading reflects times when temperature was in the upper 25% and light shading reflects time when temperature was in the lower 25% of the
daily range. Times spend in the intermediate temperature range show no shading. Asterisks denote statistical significance (p,0.05) as determined
using a Student’s paired t-test, for n = 8 Mecp2
2/+
mice.
doi:10.1371/journal.pone.0035396.g008
Altered Periodic Behavior in MeCP2-Deficient Mice
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Page 11
Supporting Information
Video S1 Synchronized video recording and activity
output of a MeCP2
2/+
mouse. This video shows a 1 minute
segment of a MeCP2
2/+
mouse in its home cage environment.
Shown below the video is the activity output plot generated by the
DSI analysis program that is synchronized to the video recording.
Note the concordance of the ambulation activity of the mouse with
the peaks depicted in the plot.
(MOV)
Figure S1 Automated detection of epileptiform dis-
charges. Panel A: Raw 10-second EEG waveform segment
collected from a representative MeCP2
2/+
mouse displaying 2
epileptiform discharges as determined and confirmed by visual
inspection (Red lines represent the start and end of the respective
discharge event). Panel B: Resulting envelope of the EEG
waveform in Panel A after band pass filtering the signal through
a 6–10 Hz FIR filter and then convoluting the square of this
filtered data with a Gaussian kernel of 200 point aperture (Red
lines represent the start and end of the respective discharge events,
the green line represents the envelope of the black 6–10 Hz FIR
band pass filtered signal). Panel C: Resulting derivative of the
convolved envelope signal presented in Panel B used to determine
the start and end of the discharge event. The red lines denote the
left and right inflection points used to determine the start and end
of the discharges, respectively.
(DOC)
Figure S2 Recognition of periodic variations in EEG,
gross motor activity, and core body temperature. Panels
A and B: Representative traces of cortical delta power patterning
over the light (i) and dark (ii) phases of a 24 hour day in a wild-type
(A) and a MeCP2
2/+
(B) mouse. Shaded regions denote areas
classified as high delta states and non-shaded regions denote areas
classified as low (non) delta states. Panels C and D: Representative
traces of mobility patterning over a 24 hour day in a wild-type (C)
and a MeCP2
2/+
(D) mouse. Shaded regions denote areas
classified as mobile behavioral states whereas non-shaded regions
denote areas classified as inactive behavioral states. Panels E and
F: Representative traces of core body temperature patterning over
a 24 hour day in a wild-type (E) and a MeCP2
2/+
(F) mouse.
Shaded regions denote areas where body temperature was above
the daily mean value, whereas non-shaded regions denote areas
where body temperature was below the mean.
(DOC)
Figure S3 Illustration of smoothed data generated from
raw delta power, mobility, and body temperature
traces. Panels A–D: Representative traces of raw cortical delta
power (grey line) and the resulting smoothed data (black line) as
generated using the 50-point Fast Fourier Transformation (FFT)
smoothing function in OriginPro 6.1 (OriginLab Corporation,
Northampton, MA) for a wild-type (A and B) and a MeCP2
2/+
(C
and D) mouse during the day (A and C) and night (B and D)
phases of the 24-hour day. Panels E–H: Representative traces of
raw mobility (grey line) and the resulting smoothed data (black
line) as generated above for a wild-type (E and F) and a MeCP2
2/+
(G and H) mouse during the day (E and G) and night (F and H)
phases of a 24-hour day. Panels I–L: Representative traces of raw
core body temperature (grey line) and the resulting smoothed data
(black line) as generated above for a wild-type (I and J) and a
MeCP2
2/+
(K and L) mouse during the day (I and K) and night (J
and L) phases of a 24-hour day.
(DOC)
Acknowledgments
We thank members of the Eubanks, Zhang, and Bardakjian labs for helpful
discussions and manuscript review, and Dr. John Christodoulou for helpful
comments. We also thank Ms. Alyssa Gagnon for technical assistance.
Author Contributions
Conceived and designed the experiments: JHE LZ BLB. Performed the
experiments: RGW SC CW LZ. Analyzed the data: JHE RGW SC BLB
LZ. Contributed reagents/materials/analysis tools: JHE CW BLB LZ.
Wrote the paper: JHE RGW SC LZ BLB.
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  • Source
    • "These recordings can be performed in freely behaving animals for extended periods of time, thus allowing the measurement of brain activity in the rest and activity phases of the wake-sleep cycle [2, 15, 16]. Among EEG techniques, those using telemetric wireless devices represent a very attractive methodology, in order to reduce the stress in the animal and make fully free its movements in the cage [16] . For this reason, EEG research may play a fundamental role in the study of characteristic biomarkers in wild-type and transgenic animal models of several diseases in the framework of a genuine back-translation model of research [17, 18, 19]. "
    [Show abstract] [Hide abstract] ABSTRACT: The gray mouse lemur (Microcebus murinus) is considered a useful primate model for translational research. In the framework of IMI PharmaCog project (Grant Agreement n°115009, www.pharmacog.org), we tested the hypothesis that spectral electroencephalographic (EEG) markers of motor and locomotor activity in gray mouse lemurs reflect typical movement-related desynchronization of alpha rhythms (about 8-12 Hz) in humans. To this aim, EEG (bipolar electrodes in frontal cortex) and electromyographic (EMG; bipolar electrodes sutured in neck muscles) data were recorded in 13 male adult (about 3 years) lemurs. Artifact-free EEG segments during active state (gross movements, exploratory movements or locomotor activity) and awake passive state (no sleep) were selected on the basis of instrumental measures of animal behavior, and were used as an input for EEG power density analysis. Results showed a clear peak of EEG power density at alpha range (7-9 Hz) during passive state. During active state, there was a reduction in alpha power density (8-12 Hz) and an increase of power density at slow frequencies (1-4 Hz). Relative EMG activity was related to EEG power density at 2-4 Hz (positive correlation) and at 8-12 Hz (negative correlation). These results suggest for the first time that the primate gray mouse lemurs and humans may share basic neurophysiologic mechanisms of synchronization of frontal alpha rhythms in awake passive state and their desynchronization during motor and locomotor activity. These EEG markers may be an ideal experimental model for translational basic (motor science) and applied (pharmacological and non-pharmacological interventions) research in Neurophysiology.
    Full-text · Article · Nov 2015 · PLoS ONE
  • Source
    • "An important goal of this study was to evaluate the translational value of insights gained from qEEG studies in the RTT animal model to provide a better understanding of the impairments in EEGs from girls with RTT. We applied algorithms developed to quantitate sleep dysfunction in 24h EEGs from a Mecp2 KO mouse model Mecp2tm1.1Bird of RTT [26,41] to the present human EEG data sets. As anticipated from previous reports, significantly lower SWS percent was detected in RTT overnight EEGs similar to the animal model studies. "
    [Show abstract] [Hide abstract] ABSTRACT: Sleep problems are commonly reported in Rett syndrome (RTT); however the electroencephalographic (EEG) biomarkers underlying sleep dysfunction are poorly understood. The aim of this study was to analyze the temporal evolution of quantitative EEG (qEEG) biomarkers in overnight EEGs recorded from girls (2-9 yrs. old) diagnosed with RTT using a non-traditional automated protocol. In this study, EEG spectral analysis identified high delta power cycles representing slow wave sleep (SWS) in 8-9h overnight sleep EEGs from the frontal, central and occipital leads (AP axis), comparing age-matched girls with and without RTT. Automated algorithms quantitated the area under the curve (AUC) within identified SWS cycles for each spectral frequency wave form. Both age-matched RTT and control EEGs showed similar increasing trends for recorded delta wave power in the EEG leads along the antero-posterior (AP). RTT EEGs had significantly fewer numbers of SWS sleep cycles; therefore, the overall time spent in SWS was also significantly lower in RTT. In contrast, the AUC for delta power within each SWS cycle was significantly heightened in RTT and remained heightened over consecutive cycles unlike control EEGs that showed an overnight decrement of delta power in consecutive cycles. Gamma wave power associated with these SWS cycles was similar to controls. However, the negative correlation of gamma power with age (r = -.59; p<0.01) detected in controls (2-5 yrs. vs. 6-9 yrs.) was lost in RTT. Poor % SWS (i.e., time spent in SWS overnight) in RTT was also driven by the younger age-group. Incidence of seizures in RTT was associated with significantly lower number of SWS cycles. Therefore, qEEG biomarkers of SWS in RTT evolved temporally and correlated significantly with clinical severity.
    Full-text · Article · Oct 2015 · PLoS ONE
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    • "We did not see a decrease in amplitude in either MeCP2 patient line but did see a change in phase of peak expression in the R106W cells. rhythms have been shown in adult female MeCP2-deficient mice (Wither et al., 2012). Taken together with our data, we conclude that normal MeCP2 expression is essential for normal temporal patterning of daily activity. "
    [Show abstract] [Hide abstract] ABSTRACT: Disturbances in the sleep/wake cycle are prevalent in patients with Rett Syndrome (RTT). We sought to determine whether the circadian system is disrupted in a RTT model, Mecp2(-/y) mice. We found that MeCP2 mutants showed decreased strength and precision of daily rhythms of activity coupled with extremely fragmented sleep. The central circadian clock (suprachiasmatic nucleus) exhibited significant reduction in the number of neurons expressing vasoactive intestinal peptide (VIP) as well as compromised spontaneous neural activity. The molecular clockwork was disrupted both centrally in the SCN and in peripheral organs, indicating a general disorganization of the circadian system. Disruption of the molecular clockwork was observed in fibroblasts of RTT patients. Finally, MeCP2 mutant mice were vulnerable to circadian disruption as chronic jet lag accelerated mortality. Our findings suggest an integral role for MeCP2 in the circadian timing system, provides a possible mechanistic explanation for the sleep/wake disturbances observed in RTT patients. The work raises the possibility that RTT patients may benefit from a temporally structured environment. Copyright © 2015. Published by Elsevier Inc.
    Full-text · Article · Mar 2015 · Neurobiology of Disease
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