Reduction of EEG Theta Power and Changes in Motor
Activity in Rats Treated with Ceftriaxone
Michele Bellesi1, Vladyslav V. Vyazovskiy2, Giulio Tononi2, Chiara Cirelli2, Fiorenzo Conti1,3*
1Department of Experimental and Clinical Medicine, Section of Neuroscience and Cell Biology, Universita ` Politecnica delle Marche, Ancona, Italy, 2Department of
Psychiatry, University of Wisconsin at Madison, Madison, Wisconsin, United States of America, 3Fondazione di Medicina Molecolare, Universita ` Politecnica delle Marche,
The glutamate transporter GLT-1 is responsible for the largest proportion of total glutamate transport. Recently, it has been
demonstrated that ceftriaxone (CEF) robustly increases GLT-1 expression. In addition, physiological studies have shown that
GLT-1 up-regulation strongly affects synaptic plasticity, and leads to an impairment of the prepulse inhibition, a simple form
of information processing, thus suggesting that GLT-1 over-expression may lead to dysfunctions of large populations of
neurons. To test this possibility, we assessed whether CEF affects cortical electrical activity by using chronic
electroencephalographic (EEG) recordings in male WKY rats. Spectral analysis showed that 8 days of CEF treatment
resulted in a delayed reduction in EEG theta power (7–9 Hz) in both frontal and parietal derivations. This decrease peaked at
day 10, i.e., 2 days after the end of treatment, and disappeared by day 16. In addition, we found that the same CEF
treatment increased motor activity, especially when EEG changes are more prominent. Taken together, these data indicate
that GLT-1 up-regulation, by modulating glutamatergic transmission, impairs the activity of widespread neural circuits. In
addition, the increased motor activity and prepulse inhibition alterations previously described suggest that neural circuits
involved in sensorimotor control are particularly sensitive to GLT-1 up-regulation.
Citation: Bellesi M, Vyazovskiy VV, Tononi G, Cirelli C, Conti F (2012) Reduction of EEG Theta Power and Changes in Motor Activity in Rats Treated with
Ceftriaxone. PLoS ONE 7(3): e34139. doi:10.1371/journal.pone.0034139
Editor: Miguel Maravall, Instituto de Neurociencias de Alicante UMH-CSIC, Spain
Received October 18, 2011; Accepted February 22, 2012; Published March 30, 2012
Copyright: ? 2012 Bellesi 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: This work was supported by funds from Universita ` Politecnica delle Marche. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
The amino acid L-glutamate (Glu) is the major excitatory
neurotransmitter in the mammalian central nervous system, and is
involved in most aspects of normal brain function, including fast
excitatory signaling, synaptogenesis, and synaptic plasticity [1,2].
Extracellular Glu levels are regulated by a group of Glu
transporters (GluTs) that take up Glu from extracellular space,
preventing its accumulation. Five GluTs have been characterized
in the mammalian central nervous system: GLAST (EAAT1;
SLC1A1), EAAT4 (SLC1A6) and EAAT5 (SLC1A7); of these,
GLT-1 exhibits the highest level of expression, is responsible for
the largest proportion of total Glu transport and its functional
inactivation raises extracellular Glu to toxic levels [2–9]. GLT-1 is
expressed by astrocytes [10–14], and, albeit at lower levels, by
neurons [13–17]. In both astrocytic processes and axon terminals,
most GLT-1a is perisynaptic, i.e. in the plasma membrane region
extending 200–250 nm from the edge of the active zone , a
position suitable for modulating Glu concentration in the cleft.
Due to its localization, GLT-1 controls the glutamatergic
transmission by regulating the activation of the receptors mainly
expressed at perisynaptic sites, thus playing an important role in
synaptic physiology and pathophysiology [9,18]. Several diseases
indeed have been associated to changes of GLT-1 expression
[1,19–21], and more recent observations suggest that GLT-1
could be an ideal pharmacological target to prevent those
conditions characterized by increased levels of extracellular Glu
Rothstein and colleagues have recently shown that ceftriaxone
(CEF) increases robustly and specifically GLT-1 expression and
function . Using this tool, we recently characterized GLT-1
up-regulation in different brain regions, and showed that CEF
robustly increases GLT-1 expression in neocortex, hippocampus,
striatum and thalamus. In addition, physiological studies have
shown that GLT-1 up-regulation strongly affects the efficacy of the
glutamatergic transmission , and leads to an impairment of the
prepulse inhibition, a simple form of information processing
[25,26]. Altogether, these data suggest that CEF-induced GLT-1
over-expression has widespread effects on brain’s functions
involving large populations of neurons. To test this possibility,
we assessed whether CEF treatment affects cortical activity by
performing chronic electroencephalographic (EEG) recordings
coupled with videorecordings in rats before and after CEF
Ceftriaxone reduces theta (7–9 Hz) power
Analysis of EEG traces did not show pathological elements (e.g.,
epileptic discharges or gross signal modifications) after CEF
treatment (Figure 1). Power spectra analysis carried out on waking
epochs at different time points showed that CEF administration
was associated to a reduction (211.461.2% frontal, 210.961.2%
PLoS ONE | www.plosone.org1March 2012 | Volume 7 | Issue 3 | e34139
parietal) in theta power (7–9 Hz) (Figure 2A). The analysis was
performed by dividing the EEG spectrum in 200 bins (1–200,
frequency range 0.25–50 Hz, resolution 0.25 Hz) and comparing
each bin across the different time points with a repeated-measure
ANOVA. Statistically significant bins were further compared to
the respective baseline value (day 0) by Dunnett’s post-hoc test. The
analysis showed that no significant differences were present at day
1, indicating that CEF did not affect EEG after a single injection.
However, a significant cluster of bins corresponding to frequencies
ranging between 7.5 Hz and 8.5 Hz was evident in both frontal
and parietal channels at day 10 (p,0.05), i.e., two days after CEF
withdrawal. Other frequency bands were not affected. At day 16,
the same analysis did not show any significant difference,
indicating that the effect of CEF on EEG was reversible
(Figure 2B). Extending the analysis to different time points and
taking into account a specific band (7–9 Hz), we showed that CEF
treatment had a significant effect on EEG [F(5,25)=4.6, p,0.01
frontal channel, F(5,25)=3.8, p,0.05 parietal channel]. Post-hoc
analysis of single time points compared to the baseline showed that
theta power reduction started at day 8, even if not significantly,
peaked at day 10, persisted for a few days (days 10 and 12 both
p,0.05), and then faded at day 16 (Figure 2C and D). Although
our study was mainly focused on waking, we also analyzed the
EEG power spectra during sleep, and found significant differences
for NREM and REM sleep at day 10. Power spectrum analysis,
performed on frontal and parietal channels, showed a decrease in
power for frequencies ranging between 7 and 13 Hz for NREM
sleep, whereas a theta reduction, similar to the one demonstrated
for waking, was documented for REM sleep (Figure 2E and F).
Ceftriaxone treatment is associated to an increase of
Since changes in theta activity are strongly associated to changes
in motor behavior in rats [27–29], we investigated whether our
animals showed motor abnormalities due to CEF treatment.
Firstly, we determined whether CEF treatment modifies the time
spent in waking and sleep, in order to rule out that changes in
motor activity were simply a consequence of changes in wake
duration. To this aim, we performed a quantitative analysis of the
total amount of waking, NREM and REM sleep epochs scored,
and found no significant differences, neither during the 10 days of
treatment nor afterwards [waking: F(5,25)=0.98, ns; NREM:
F(5,25)=1.2, ns; REM: F(5,25)=0.26, ns; Figure 3A].
Since the amount of waking and sleep time did not change with
CEF treatment, we next evaluated whether the type or ‘‘intensity’’
of motor activity have been affected by CEF treatment. We
therefore analyzed both EMG traces and video recordings before
and after treatment, and quantified EMG and Motion activity,
respectively. By setting a threshold equal to 95thpercentile of
EMG or Motion activity during all NREM sleep episodes (see
Methods), we assessed the amount of motor activity occurring
during active and quiet waking. For active waking, time-course
analysis of EMG activity during the light period showed a
significant effect of time [F(5,25)=5.8; p,0.01], and post-hoc
comparisons showed a significant increase on day 10 and 12
compared to baseline (p,0.01 for each time points). In addition,
analysis of EMG activity during the dark period showed a similar
significant effect of time [F(5,25)=7.4; p,0.001] with a significant
increase on day 10 compared to baseline (p,0.01) at post-hoc
comparisons. For quiet waking, the same analysis carried out for
both dark and light period resulted not significant [light:
F(5,25)=1.08, ns; dark F(5,25)=1.04, ns] (Figure 3C). EMG data
were confirmed by video recordings analysis. For active waking,
motion activity measured on the same animals was increased after
the end of treatment for both light and dark periods. Time-course
analysis showed an overall effect of time [F(5,25)=4.1 p,0.01 for
light period; F(5,25)=3.93 p,0.01 for dark period] and
subsequent comparisons confirmed a significant increase of
activity at day 10 (p,0.01 for dark period, p,0.05 for light
period) compared to the baseline. On the contrary, motion activity
measured for quiet waking did not change through the course of
the experiment [dark: F(5,25)=0.93, ns; light: F(5,25)=1.01, ns]
(Figure 3D). Taken together, these data show that CEF treatment
is associated to a time-limited increase of motor activity after
treatment withdrawal with a peak at day 10. Next, we verified
whether the time course of the increase of motor activity and theta
power reduction were correlated. We found that both EMG and
Motion activity were negatively correlated to theta power decrease
(r=20.89, p,0.05; r=20.94, p,0.01, respectively, Figure 3E
The main result reported here is that one week CEF treatment
resulted in a delayed reduction in EEG theta power (7–9 Hz) in
both frontal and parietal derivations. This decrease peaked at day
10, 2 days after the end of treatment, and disappeared by day 16.
In addition, we found that the same CEF treatment increased
motor activity, especially during those experimental days in which
the EEG changes are more prominent.
Since EEG is sensitive to different environmental conditions and
animal manipulations , we assessed the effects of CEF by using
a within-subjects design, comparing data before and after CEF
treatment. To rule out the possibility that EEG changes reflected
the chronic effect of i.p. injections and/or treatment withdrawal
rather than the actual effect of CEF, we continued to treat the
animals with daily saline i.p. injections until the end of the
experiment. In this way, the animals were exposed every day to the
same manipulation for the entire length of the experiment,
minimizing possible interferences of animal handling on the EEG
Although it has been well documented that CEF treatment can
affect the expression of the major Glu transporter GLT-1 in
rodents [22,25,31,32], we did not measure GLT-1 levels in treated
animals because of our experimental design. Nonetheless, using
the same treatment schedule, we previously showed that CEF-
induced GLT-1 modifications persist for at least four days after the
end of treatment, and return to baseline level eight days after the
end of treatment . The delayed emergence of electrophysio-
logical and behavioral changes relative to GLT-1 up-regulation,
which can be documented at day 8 , may indicate that all the
synaptic modifications observed and their functional consequences
[18,26] require time to affect the dynamics of large neuronal
populations and lead to behavioral changes.
In rodents, theta oscillations are easily observed in the
hippocampus, but can also be detected in other cortical and
subcortical brain structures [33–35]. Historically, they have been
associated to learning and memory and voluntary movements [36–
40]. Lesions at different level of the hippocampal formation
correlate to sizeable reductions of the hippocampal theta rhythm
and to impairments of memory tasks [38,41–43]. Parallel
augmentation of long-term synaptic potentiation and theta activity
was described by Maren and collaborators in a contextual fear-
conditioning task , and more recent electrophysiological
studies showed an increase of theta rhythm associated with
induction of long-term potentiation , thereby suggesting a
close relationship between theta oscillations and long-term
synaptic plasticity . Recently, Omrani and colleagues
Effect of GLT-1 Up-Regulation on EEG Activity
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demonstrated that CEF-induced GLT-1 up-regulation at CA3
synapses impairs long-term plasticity, by preventing perisynaptic
metabotropic receptors activation . It is worth noting that our
results are in line with these findings, and it is therefore
conceivable to hypothesize that changes in synaptic plasticity
can interfere with the normal function of the hippocampus, which
in turn can generate a lower degree of synchronization, visible as a
reduction in EEG theta power. Omrani and colleagues also
showed that baseline fEPSPs were not affected by CEF treatment,
indicating that the glutamatergic function is altered by GLT-1 up-
regulation only when synapses undergo long-term plasticity . It
is therefore conceivable that AMPA receptors activation at the
glutamatergic synapses, even in a condition of reduced synaptic
plasticity, could ensure a normal EEG signal. This might explain
why the observed decrease in theta activity was not associated with
an overall decrease in EEG power over the entire range of
It is worth noting that we also found modifications of NREM
and REM sleep power spectrum at day 10. Specifically, the
analysis of NREM power spectrum showed a significant reduction
of a broad band ranging between 7 and 13 Hz, which includes the
spindle frequency band, suggesting that other pattern generators
beside the hippocampus might be affected by CEF treatment.
Interestingly, in a previous study we found that the GLT-1 is up-
regulated by CEF in several brain regions, including thalamus and
cerebral cortex, brain regions crucial in spindles formation and
propagation . REM sleep normally is characterized by a
robust theta activity in rodents, and several lines of evidence
indicate that modifications in waking theta activity can be reflected
in REM theta activity [48–50]. Along this line, we found a
decrease in theta power during REM sleep resembling the one
observed during waking, although less prominent. The combined
theta reduction in waking and in REM sleep induced by CEF
could be ascribable to an impairment of a common circuit
promoting theta formation in the two behavioral states.
Rats treated with CEF showed an increase of motor activity
occurring after the end of treatment, i.e., when EEG alterations
were clearly evident. Reportedly, theta is the electrical sign of
activity in a forebrain mechanism that is organizing higher
voluntary motor acts [27,51,52]. Evidence suggests that there are
two distinct types of hippocampal theta rhythm in behaving
animals: the first (4–7 Hz) appears when animals are immobile or
during repetitive acts such as sniffing or whiskers movements
[53,54]; the second one is more directly linked to voluntary motor
behaviors (i.e., walking, running, rearing etc) and it is character-
ized by higher frequency (7–10 Hz) . In physiological
Figure 1. Lack of pathological elements after ceftriaxone treatment. A. Schematic description of electrodes location. B. Saline and CEF
Treatment schedule. Day 0 represents the baseline. C. Waking absolute spectra, raw EEG and EMG signals of baseline, day 10 and day 16. Signals
appeared stable across the entire length of the experiment and the signal quality was not affected by CEF treatment.
Effect of GLT-1 Up-Regulation on EEG Activity
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conditions, motion speed is associated to the frequency of theta
rhythm, as indicated by studies reporting that the higher is the
speed, the faster is frequency [28,55,56]. Moreover, electrophys-
iological studies demonstrated that theta activity recorded while
the animal was running is larger than during walking, thus
suggesting that the amplitude of theta rhythm increases as the
intensity of movement increases . On the contrary, our results
show a reduction of EEG theta power when motor activity is
increased. By assessing separately motor activity during active and
quiet waking we found that only active waking (mostly
characterized by exploratory behavior) was affected by CEF
treatment, thereby revealing a dissociation between theta power
and motor activity. Of note ketamine, another drug that affects
glutamatergic transmission, when administered to mimic patho-
logical states (i.e., schizophrenia) causes both an increase in motor
activity and a decrease in EEG theta power [57–59]. Thus, it
appears that the reduction of theta power, at least in non-
physiological, drug-induced conditions, can be associated with an
increase in motor activity. In addition, drug-induced motor
activity is often mediated by aberrant activation of basal ganglia
and cerebral cortex . It is therefore possible that glutamatergic
alterations also affecting those structures [25,26] may contribute to
the increase of motor activity we observed. In support to the
possible co-occurrence of theta activity reduction and increased
motor activity, we also asked whether the time courses of those
alterations were correlated. We found indeed a significant
correlation between the EEG changes and the motor modifica-
tions, supporting a link between them, although a causal
relationship still remains to be established.
In addition, there is evidence that theta oscillations play a role in
integrating sensorimotor information [52,61]. It is well known that
a simple measure to evaluate sensorimotor integration in animals
Figure 2. Power spectra analysis following ceftriaxone administration. A. Reduction in theta power at day 10. Waking mean absolute power
spectra of Day 0 and Day 10 for frontal (above) and parietal (below) EEG channel. B. Power spectra analysis relative to the baseline illustrating a
reduction of theta power at Day 10 (shown in detail for single animals in the small inset) and a return to the baseline eight days after CEF withdrawal
(Day 16) in frontal (above) and parietal (below) EEG channels. Statistical significance (p,0.05) is represented by black dots. Values are mean 6 sem. C.
Time course analysis of relative spectra (7–9 Hz frequency band) showing a significant reduction of theta power for frontal and parietal channels two
days (Day 10) and four days (Day 12) after CEF withdrawal compared to the baseline (Day 0). *p,0.05. Values are mean 6 sem. D. Example of power
spectrum relative to the baseline for a representative animal during the entire length of the experiment. E. Frontal and parietal relative spectra
showing a broad band (7–13 Hz) reduction in power during NREM sleep at day 10. Statistical significance (p,0.05) is represented by black dots.
Values are mean 6 sem. F. Frontal and parietal relative spectra showing a reduction in theta power during REM sleep at day 10. Statistical
significance (p,0.05) is represented by black dots. Values are mean 6 sem.
Effect of GLT-1 Up-Regulation on EEG Activity
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and in humans is prepulse inhibition of the startle reflex [62,63],
which reflects the ability of the brain to temporarily adapt to a
strong sensory stimulus when a preceding weaker signal is given to
warn the organism [62,63]. PPI is a neurological phenomenon
regulated by a complex interplay between several brain structures,
including cortical and subcortical sites . Interestingly, we
recently demonstrated that the PPI was impaired in rats treated
with CEF and that this deficit was blocked by dihydrokainate, a
selective GLT-1 inhibitor , thus suggesting that sensorimotor
integration is affected by GLT-1 up-regulation.
Taken together, these data indicate that GLT-1 up-regulation,
by modifying the efficacy of the glutamatergic transmission,
impairs the activity of widespread neural circuits and that the
reduction in EEG theta power could be its electrophysiological
signature. In addition, the increased motor activity and PPI
alterations previously described suggest that neural circuits
involved in sensorimotor control are particularly sensitive to
changes in the efficacy of glutamatergic transmission induced by
Materials and Methods
Animal protocols followed the National Institutes of Health
Guide for the Care and Use of Laboratory Animals, in accordance
with institutional guidelines. They were reviewed and approved by
the IACUC of the University of Wisconsin-Madison, and were
inspected and accredited by AAALAC (Protocol M2006).
Animals, surgery and treatment
Male WKY rats (n=6, Harlan, 11–12 weeks old at time of
surgery) were used. Under deep isoflurane anesthesia (1.5–2%
volume), rats were implanted bilaterally with epidural screw
electrodes over the frontal (B: +2 mm, L: 2 mm) and parietal
cortex (B: 22 mm, L: 4 mm) and cerebellum (reference electrode
and ground) for chronic EEG recordings. Electrodes were fixed to
the skull with dental cement. Two stainless steel wires (diameter
0.4 mm) were inserted into neck muscles to record electromyo-
gram (EMG) (Figure 1A).
After surgery, all rats were housed individually in transparent
plexiglas cages (36.5625646 cm), and kept in sound-proof
recording boxes for the duration of the experiment. Light and
temperature were kept constant (LD 12:12, light on at 10 am,
2361uC; food and water were available ad libitum and replaced
daily at 10 am). About seven days were allowed for recovery after
surgery, and experiments were started only after animals were fully
recovered. Animal protocols followed the National Institutes of
Health Guide for the Care and Use of Laboratory Animals and
were in accordance with institutional guidelines.
Rats were connected by a flexible cable to a commutator
(Airflyte, Bayonne, NJ) and recorded continuously for 4 weeks
Figure 3. Effects of ceftriaxone treatment on motor activity. A. Time-course of the amount of time expressed in 4s epochs spent in waking,
NREM and REM sleep. Values are expressed as mean 6 sem. B. Example of EMG activity during the entire length of the experiments. Note the intense
activity after the end of CEF treatment. The grey line represents the threshold above and below which the motor activity is identified as active waking
or quiet waking, respectively. C–D. Quantitative analysis of EMG activity (C) and Motion activity (D) during active and quiet waking for light and dark
periods. Values are relative to the baseline (day 0) and expressed as mean 6 sem. * (p,0.05), ** (p,0.01). E–F. Negative correlation between the
time-course of relative theta power and the EMG (E) or Motion activity (F).
Effect of GLT-1 Up-Regulation on EEG Activity
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using the Multichannel Neurophysiology Recording and Stimula-
tion System (Tucker-Davis Technologies Inc., TDT).
Rats were treated intraperitoneally (i.p.) for two days with saline
(day 1 was for habituation, day 2 was the baseline), followed by
CEF (dissolved in saline [,0.2 ml] and administered i.p. at a dose
of 200 mg/kg; ) for 8 days, and then with saline i.p. for 8 days.
All injections were administered at 10 am (Figure 1B).
Data acquisition and analysis
EEG and EMG.
filtered as follows: EEG: high-pass filter at 0.1 Hz; low-pass filter at
100 Hz; EMG: high-pass filter at 10 Hz; low-pass filter at 100 Hz.
All signals were sampled and stored at 256 Hz resolution. EEG
power spectra were computed by a Fast Fourier Transform
routine for 4-s epochs (0.25 Hz resolution). For staging, signals
were loaded with custom-made Matlab programs using standard
TDT routines, and subsequently transformed into the EDF
(European Data Format) with Neurotraces software. Waking,
NREM sleep, and REM sleep were manually scored off-line
(SleepSign, Kissei COMTEC, Matsumoto, Japan) in 4-s epochs
according to standard criteria. Epochs containing artifacts,
predominantly during active waking, were excluded from
spectral analysis. Vigilance state could always be determined.
EEG channels presenting artifacts or abnormal reduction of signal
amplitude during the entire duration of the experiment were not
included in the analysis. At the end, all animals had at least one
valid channel from frontal and parietal cortices. Absolute spectra
revealed a normal pattern in all the animals recorded before and
after CEF treatment (Figure 1C).
EMG signals were loaded with custom-made Matlab programs
and rectified amplitudes were calculated over 4-s epochs and used
to quantify the activity of the animals. The magnitude of this index
corresponded to the amount of motor activity that occurred during
that 4-sec epoch. A threshold, below and above which all waking
epochs were classified as quiet waking and active waking,
respectively, was determined by calculating the 95thpercentile of
EMG activity during all NREM sleep episodes . This method
accurately identified periods of relative inactivity during waking, in
which the animals were quiet or performed little movements (such
EEG and EMG signals were amplified and
as head movements or postural adjustments), but staying at the
same place, and more active periods in which animals were
engaged in explorative activities.
continuously with infrared cameras (OptiView Technologies,
Inc. Potomac Falls, VA) and stored in real time (AVerMedia
Technologies, Inc. Milpitas, CA). A custom-made Matlab script
was used for analysis. The program detected animal motion every
second within a previously set monitored area (corresponding to
the cage area), by calculating the numbers of pixels whose intensity
changed over time. Specifically, it compared the last image with
the following one and defined a value in percent of changes
occurring every second. These values and the relative time were
then daily saved in a txt report file and subsequently loaded with
custom-made Matlab programs. Percentage values (Motion
activity) for light and dark periods were processed separately,
because cameras showed a different sensibility in detecting motion
in different light conditions.
A threshold corresponding to the mean amount of pixels
changing during NREM sleep was set to classify active versus quiet
waking as for EMG studies.
For EEG analysis, mean absolute EEG
spectrum values for each state were normalized by dividing each
0.25 Hz bin value by the total mean value. For time-course
studies, EEG, EMG and Motion activity, all values at different
time points during the experimental days were compared to the
corresponding baseline (Day 0) values. Comparisons were
performed by using a repeated-measure ANOVA, with time as a
within factor, follow by Dunnett’s post-hoc test or a paired t-test in
presence of multiple or single comparisons, respectively. Pearson’s
correlation coefficient was calculated for correlative studies
between EMG or motion activity and theta relative power.
Alpha was set at 0.05.
Conceived and designed the experiments: MB VV GT CC FC. Performed
the experiments: MB VV CC. Analyzed the data: MB VV GT CC FC.
Contributed reagents/materials/analysis tools: GT CC FC. Wrote the
paper: MB VV GT CC FC.
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