used reverse-microdialysis, electrophysiology, pharmacological, and histological methods to determine how changes in glutamatergic
unable to reverse REM atonia. We conclude that an endogenous glutamatergic drive onto somatic motoneurons contributes to the
Skeletal muscle tone is modulated in a stereotypical pattern
by showing that muscle tone is maximal in alert waking, reduced
in quiet waking, further reduced in non-rapid eye movement
(NREM) sleep, and potently suppressed in rapid eye movement
(REM) sleep (Jouvet, 1962, 1967). Since this initial description,
considerable attention has been paid to deciphering the mecha-
nisms controlling muscle tone during the sleep cycle because
including REM sleep behavior disorder, narcolepsy/cataplexy,
and obstructive sleep apnea. Although considerable progress has
tone suppression in REM sleep (Kubin et al., 1998; Chase and
cle tone regulation in waking and NREM sleep.
transmitter in the mammalian CNS (Collingridge and Lester,
sible for controlling motoneuron excitability and for mediating
motor behaviors such as involuntary and rhythmic movements
as well as afferent reflexes (Rekling et al., 2000). Motoneurons
motor control (Rekling et al., 2000). The discharge pattern of
neurons in the medial medulla, in which glutamate-containing
maximal in waking, minimal in NREM sleep, and either silent or
episodically active in REM sleep (Siegel et al., 1983, 1992). De-
spite this evidence, the role of glutamate in regulating levels of
motoneuron excitability and muscle tone during natural sleep–
wake motor behaviors has never been tested.
Therefore, we used reverse-microdialysis, electrophysiology,
pharmacological, and histological methods to examine how glu-
tamatergic neurotransmission within the trigeminal motoneu-
ron pool contributes to basal levels of masseter muscle tone dur-
ing sleep and waking in freely behaving rats. The masseteric
of Veterans Affairs, National Institutes of Health Grant NS14610, and United States Public Health Service Grant
TheJournalofNeuroscience,April30,2008 • 28(18):4649–4660 • 4649
motor system was the focus of this study because trigeminal mo-
disorders, including REM sleep behavior disorder, obstructive
sleep apnea, cataplexy/narcolepsy, and bruxism (Guilleminault,
1994; Horner, 1996; Kato et al., 2003). In addition, the glutama-
tergic control of trigeminal motoneurons has been well docu-
mented (Chandler, 1989; Kolta, 1997).
tamatergic neurotransmission at the trigeminal motor pool are
responsible for the sleep–wake pattern of masseter muscle tone.
First, we identified an endogenous glutamatergic drive at the tri-
geminal motor pool during waking and in phasic REM sleep, by
antagonizing ionotropic glutamate receptors on trigeminal mo-
toneurons. Then, we determined that the motor suppression of
NREM, but not tonic REM, sleep could be restored to waking
levels by exogenously replacing the glutamatergic drive that is
normally withdrawn in sleep. We conclude that an endogenous
jor neurochemical cue controlling the stereotypical pattern of
sic REM sleep but not tonic REM sleep.
Rats were housed individually and maintained on a 12 h light/dark cycle
(lights on at 7:00 A.M. and off at 7:00 P.M.), and both food and water
were available ad libitum. All procedures and experimental protocols
were approved by the University of Toronto animal care committee and
were in accordance with the Canadian Council on Animal Care.
Surgical preparation for sleep and microdialysis studies
Studies were performed using 30 male Sprague Dawley rats (average
mass, 340 ? 8.2 g). To implant electroencephalogram (EEG) and elec-
was performed under anesthesia induced with intraperitoneal ketamine
(85 mg/kg) and xylazine (15 mg/kg) and maintained with additional
anesthesia given by inhalation (isoflurane, 0.5–2%). Effective depth of
anesthesia was determined by the abolishment of the pedal withdrawal
and blink reflexes. Body temperature was monitored with a rectal probe
(CWE, Ardmore, PA) and maintained at 37 ? 1°C.
Three insulated, multi-stranded stainless steel wire EMG electrodes
(Cooner Wire, Chatsworth, CA) were implanted into the left and right
inserted into the nuchal muscle. Four stainless steel screws (JI Morris
Company, Southbridge, MA), attached to insulated 34 gauge wire
(Cooner Wire), were implanted in the skull for recording cortical EEG;
their coordinates were 2 mm rostral and 2 mm to the left and right of
bregma, and 3 mm caudal and 2 mm to the left and right of bregma. An
was 9.4 mm caudal and 0.5 mm lateral to bregma.
To implant a microdialysis probe into the left trigeminal motor nu-
to bregma (Paxinos and Watson, 1998). A microdialysis guide probe
(CMA/Microdialysis, Solna, Sweden) was then lowered 8.2 mm below
Dental, Wheeling, IL) secured the probe in place, and, after the cement
was dry, EEG and EMG electrodes were connected to pins (Allied Elec-
tronics, Bristol, PA) and inserted into a custom-made head plug (Allied
Electronics) that was affixed to the skull with dental cement.
After surgery, rats were given an intraperitoneal injection of 0.03
mg/kg buprenorphin and kept warmed by a heating pad. They were also
given a dietary supplement (i.e., Nutri-Cal) and soft food for the follow-
ing 2 d. Rats recovered for at least 7–10 d before experimental testing
Experimental procedures for sleep and microdialysis studies
Recording environment. During experiments, animals were housed in
a movement-responsive caging system eliminating the need for a com-
mutator or liquid swivel. This caging system was housed inside a sound-
attenuated, ventilated, and illuminated (lights on, 110 lux) chamber.
by attaching a lightweight cable to a plug on the rat’s head, which was
connected to a Super-Z head-stage amplifier and BMA-400 alternating
times and bandpass filtered between 1 and 100 Hz. EMG signals were
amplified between 500 and 1000 times and bandpass filtered between 30
Hz and 30 kHz. All electrophysiological signals were digitized at 250 Hz
(Spike 2 Software, 1401 Interface; Cambridge Electronic Design, Cam-
bridge, UK) and monitored and stored on a computer.
Microdialysis probe. A microdialysis probe was used to exogenously
perfuse glutamate antagonists and agonists into the trigeminal motor
nucleus. The microdialysis probe (CMA/Microdialysis) (34 kDa cutoff;
membrane length and diameter, 1 mm ? 250 ?M) was lowered into the
FEP Teflon tubing (inside diameter, 0.12 mm; Eicom, Moraine, OH),
which was connected to a 1 ml gastight syringe via a liquid switch (BAS
Bioanalytical Systems). The probe was continually perfused with filtered
(0.2 ?m nylon; Thermo Fisher Scientific, Waltham, MA) artificial CSF
(aCSF) (in mM: 125 NaCl, 5 KCl, 1.25 KH2PO4, 24 NaHCO3, 2.5 CaCl2,
1.25 MgSO2, and 20 D-glucose) at a flow rate of 2 ?l/min using a syringe
pump (BAS Bioanalytical Systems).
Drug preparation. All drugs were dissolved in aCSF at the beginning
of each experimental day. Glutamatergic antagonists 6-cyano-7-
nitroquinoxaline-2,3-dione (CNQX) [molecular weight (MW), 232.16;
nist, and D-2-amino-5-phosphonopentanoate (D-AP-5) (MW, 197.13;
Tocris Bioscience), a selective NMDA antagonist, were used to block
acid (glutamate; MW, 147.13; Tocris Bioscience), AMPA (MW, 186.17;
Tocris Bioscience), and NMDA (MW, 147.13; Tocris Bioscience) were
used to activate glutamate receptors.
Each experiment took 2 d to complete. On the first day at 8:00 A.M. to
least 1 h to habituate before they were connected to the electrical tether.
They were then given a minimum of 3 h to habituate to this before
recordings began. Baseline recordings (without the microdialysis probe
4:00 P.M. The microdialysis probe was inserted between 5:00 P.M. and
7:00 P.M., and aCSF was perfused throughout the night. Probes were
inserted the night before experiments began because previous studies
release and local neuronal activation (Di Chiara, 1990; Kodama et al.,
On the second day of experimentation, perfusion of candidate drugs
began at 8:00 A.M. to 9:00 A.M. Drug treatments were randomized, and
washout period of at least 2 h followed every drug treatment.
inal motoneuron excitability during natural behavior, NMDA and non-
NMDA receptors were antagonized using (1) application of 0.5 mM
CNQX and 5.0 mM D-AP-5 in combination, (2) application of 0.5 mM
because previous in vivo studies demonstrate that they effectively block
glutamate neurotransmission onto somatic motoneurons (Steenland et
al., 2006). Each drug was applied onto the motor nucleus for 2–4 h; this
typically allowed sufficient time for the animal to pass through at least
is, drug administration into the motor pool began regardless of the ani-
mal’s arousal state.
4650 • J.Neurosci.,April30,2008 • 28(18):4649–4660 Burgessetal.•GlutamatergicControlofSomaticMotoneuronsinSleep
Study 2: agonism of glutamate receptors. To determine whether addi-
glutamate; (2) 0.1 mM AMPA; or (3) 0.1 mM NMDA. We used 25 mM
glutamate because previous studies show that only 10% of glutamate
diffuses across the microdialysis membrane (Alessandri et al., 1996).
Therefore, based on this observation, it is estimated that only 2.5 mM
glutamate will diffuse onto trigeminal motoneurons; this concentration
approximates glutamate levels at the mammalian synaptic cleft (Clem-
rons (Steenland et al., 2006), and we found that they potently activated
masseter muscle tone in waking.
Verification of microdialysis probe location
Two procedures were used to demonstrate that microdialysis probes
neck EMG activity. This result verified that trigeminal motoneurons
were viable and able to respond to glutamatergic activation, that micro-
dialysis probes were functional at the end of each experiment, and that
probes were located in the trigeminal motor nucleus. We also used post-
mortem histological analysis to demonstrate that microdialysis probes
were physically located in the trigeminal nucleus.
Histology. Under deep anesthesia (ketamine at 85 mg/kg and xylazine
at 15 mg/kg, i.p.), rats were decapitated, and their brains removed and
placed in chilled 4% paraformaldehyde (in 0.1 M PBS) for 24 h. Brains
then frozen in dry ice and transversely sectioned in 30 ?m slices using a
microtome (Leica, Wetzlar, Germany). Brain sections were mounted,
dried, and stained with Neutral Red. Tissue sections span-
ning regions rostral and caudal to the trigeminal motor
pool were viewed using a light microscope (Olympus, To-
on standardized brain maps (Paxinos and Watson, 1998)
to verify probe location. These data are summarized in
Behavioral state. We identified and classified four behav-
ioral states. Alert wake (AW) was characterized by high-
els of EMG activity (i.e., chewing, grooming, and
drinking) (see Fig. 1A). Quiet wake (QW) was character-
absence of overt motor activity. NREM sleep was charac-
minimal EMG activity. REM sleep was characterized by
low-amplitude, high-frequency theta-like EEG activity
Sleep states were visually identified and analyzed in 5 s
epochs using the Sleepscore version 1.01 script (Cam-
bridge Electronic Design).
EMG analysis. Raw EMG signals were full-wave rec-
tified, integrated, and quantified in arbitrary units. Av-
erage EMG activity for left and right masseter and neck
muscle activity was quantified in 5 s epochs for each
behavioral state. When glutamatergic agents were ap-
plied onto the left trigeminal motor pool, EMG data
were not analyzed for the first 30 min of perfusion be-
cause the latency from the syringe pump to the micro-
dialysis probe was ?15 min. We analyzed and quanti-
fied levels of EMG activity (i.e., left and right masseter
and neck muscle) for all behavioral states (i.e., AW,
QW, NREM, and REM) occurring across the 2–4 h per-
fusion period (i.e., aCSF and candidate drugs).
EMG analysis in REM sleep. REM sleep consists of both tonic and
phasic motor events. The stereotypical periods of motor atonia occur
ments) (Aserinsky and Kleitman, 1953; Jouvet, 1967). Because a major
mission in modulating motor activity in REM sleep, we developed an
objective method for identifying and quantifying the phasic (i.e., muscle
twitches) and tonic (REM atonia) periods of REM sleep. To quantify
of EMG activity during the first 5 s of each REM period because muscle
twitches are conspicuously absent during this time (Lu et al., 2005;
Brooks and Peever, 2008). The muscle twitches that define phasic REM
sleep were classified as motor events that exceeded the 99th percentile of
EMG activity during the first 5 s of REM; and conversely, REM sleep
atonia was classified as any period in which muscle activity was equal to
or less than the 99th percentile of EMG activity during the first 5 s of
REM. In each rat, REM atonia and muscle twitches were quantified for
into the trigeminal motor pool.
function and subjected to a fast Fourier transform to yield the power
spectrum. The power within four frequency bands was recorded as ab-
calculated over each 5 s epoch. The band limits used were as follows:
The statistical tests used for analysis are included in Results. Compari-
sons between treatments for mean basal muscle tone in all behavioral
states were made using a two-way repeated-measures (RM) ANOVA
with post hoc Tukey’s tests to infer statistical significance. Comparisons
between treatments for both the amplitude and number of muscle
follow a stereotypical pattern of activity across the sleep–wake cycle, with muscle tone being significantly sup-
pressed during both NREM and REM sleep ( p ? 0.001). Traces were taken during baseline conditions, before a
Burgessetal.•GlutamatergicControlofSomaticMotoneuronsinSleepJ.Neurosci.,April30,2008 • 28(18):4649–4660 • 4651
twitches per REM episode were made using paired t tests. All statistical
analyses used SigmaStat (SPSS, Chicago, IL) and applied a critical two-
tailed ? value of p ? 0.05. Data are presented as means ? SEM.
to characterize how masseter muscle tone changes as a function
activity across the natural sleep–wake cycle under baseline con-
ditions (i.e., before probe insertion) and compared it with neck
muscle activity. A total of 61.8 h of EMG activity were analyzed,
with 21.1 h of it spent in AW, 21.8 h in QW, 14.5 h in NREM
sleep, and 4.4 h in REM sleep. We found that masseter muscle
EMG activity is significantly affected by different sleep–wake
a typical example illustrating that basal levels of masseter muscle
tone are highest in AW, reduced in QW, and minimal in NREM
ride the background of motor atonia.
We found that inserting a probe into the left trigeminal nucleus
This intervention had no affect on either right masseter or neck
dialysis of 0.1 mM AMPA into the left trigeminal motor pool
during the last 15 min of each experiment significantly increased
without altering levels of either the right masseter ( p ? 0.574)
(Fig. 3C,D) or neck muscle activity ( p ? 0.431; data not shown).
This procedure verified that probes were located within the left
trigeminal nucleus and that microdialysis probes were func-
tional, and that motoneurons were viable and able to respond to
compounds that manipulate glutamatergic transmission. In 28
rats, we confirmed by postmortem histology that microdialysis
probes were located within or immediately adjacent to the left
trigeminal motor nucleus (Fig. 3A,B). However, in two rats, left
masseter EMG activity was unaffected by either probe insertion
or AMPA application; histology confirmed that probes were lo-
cated outside of the trigeminal motor nucleus (Fig. 3B). These
two animals were not included in this study.
To demonstrate that inserting a microdialysis probe into the tri-
geminal motor pool had no affect on the sleep–wake pattern of
masseter muscle activity, we compared levels of masseter muscle
EMG activity before and 1 h after probe placement (with aCSF
of masseter muscle tone before compared with after probe inser-
tion ( p ? 0.252 for all states, two-way RM-ANOVA) (Fig. 2C)
and conclude that the sleep–wake pattern of masseter muscle
tone is unaffected by probe placement.
Because the trigeminal motor pool is located in close proximity to
pontine regions that regulate sleep (e.g., locus ceruleus and sublat-
tergic neurotransmission in the trigeminal nucleus did not affect
sleep–wake behaviors. We found that, under baseline conditions,
ing period in AW, 30% in QW, 27% in NREM, and 9% in REM
sleep. There was no significant difference in the amount of time
spent in each state when baseline and agonist/antagonist treatment
percentage beta/percentage delta) before and after drug treatment
activity. B, Group data demonstrating that left masseter muscle tone significantly increased
Insertion of a microdialysis probe into the trigeminal motor pool has transient
4652 • J.Neurosci.,April30,2008 • 28(18):4649–4660Burgessetal.•GlutamatergicControlofSomaticMotoneuronsinSleep
Combined non-NMDA and NMDA receptor antagonism
To determine how endogenous glutamate release contributes to
mediating basal levels of masseter muscle tone during different
waking and sleeping states, we antagonized non-NMDA and
NMDA receptors on trigeminal motoneurons using 0.5 mM
CNQX and 5.0 mM D-AP-5 while monitoring masseter muscle
3.1 ? 0.15 h, neither right masseter muscle activity ( p ? 0.921,
two-way RM-ANOVA) (Fig. 4) nor neck muscle activity ( p ?
0.199, two-way RM-ANOVA; data not shown) changed (relative
to baseline) during any behavioral state.
Antagonism of non-NMDA and NMDA receptors resulted a
significant suppression of left masseter muscle tone during both
alert and quiet waking ( p ? 0.005, two-way RM-ANOVA) (Fig.
5). Compared with left masseter muscle activity under baseline
conditions, application of both CNQX and D-AP-5 markedly re-
duced basal levels of left masseter activity during AW by 79%
( p ? 0.001) and during QW by 58% ( p ? 0.03) (Fig. 5B). This
reduction was so potent that it reduced waking masseter muscle
tone to levels that were no longer significantly different from
(Fig. 6A), indicating that withdrawal of a wake-related glutama-
tergic drive contributes to the suppression of motor tone in
NREM sleep. However, antagonism of non-NMDA and NMDA
REM sleep values ( p ? 0.001) (Fig. 6A), indicating that with-
drawal of glutamatergic inputs is not suf-
ficient for inducing REM sleep atonia.
effect on basal masseter tone during either
NREM ( p ? 0.879) or tonic REM ( p ?
0.939) sleep. However, this intervention
significantly reduced the number of mus-
cle twitches during phasic REM sleep by
90% of baseline conditions ( p ? 0.03,
paired t test) (Fig. 5C); it also reduced the
mean amplitude of the remaining muscle
twitches by 67% of baseline levels ( p ?
0.001) (Fig. 5D).
NMDA and non-NMDA
Having demonstrated that there is an en-
dogenous glutamatergic drive on trigemi-
nal motoneurons during wakefulness and
which of the two major ionotropic gluta-
mate receptors (i.e., non-NMDA or
or NMDA receptors by applying either 0.5
recording left masseter muscle EMG
Antagonism of non-NMDA receptors
caused a potent suppression of left masse-
ter muscle EMG activity during both wak-
baseline conditions, CNQX application
reduced basal levels of left masseter activity during waking by
antagonism significantly reduced the number of muscle twitches
during phasic REM sleep by 86% of baseline conditions ( p ?
0.036, paired t test) (Fig. 7B). The amplitude of muscle twitches
that persisted during CNQX application were significantly re-
duced by 85% of baseline levels ( p ? 0.001) (Fig. 7C).
The reduction in masseter muscle activity during blockade of
non-NMDA receptors was comparable with that observed when
both non-NMDA and NMDA receptors were both inactivated.
We found that simultaneous application of both CNQX and
D-AP-5 decreased left masseter muscle activity during AW by
alone decreased muscle tone by the same magnitude, decreasing
it by 81% in AW and by 86% in phasic REM sleep. There was no
significant difference between the magnitude of masseter tone
suppression during combined antagonism versus non-NMDA
non-NMDA receptors mediate glutamatergic drive onto trigem-
However, we found that antagonism of NMDA receptors
alone also decreased left masseter EMG activity but only during
waking. Application of D-AP-5 into the left trigeminal nucleus
reduced left masseter tone during waking by 49% ( p ? 0.034,
two-way RM ANOVA) (Fig. 7D); however, this intervention had
no affect on masseter tone during either NREM ( p ? 0.896) or
tonic REM ( p ? 0.974) sleep, nor did it affect muscle twitches
potentincreaseinleft( p?0.012)butnotrightmassetertone( p?0.574).Allvaluesaremeans?SEM;*p?0.05.A.U.,
Burgessetal.•GlutamatergicControlofSomaticMotoneuronsinSleepJ.Neurosci.,April30,2008 • 28(18):4649–4660 • 4653
during phasic REM sleep. Application of
D-AP-5 did not affect (relative to baseline)
(Fig. 7E) or amplitude ( p ? 0.197) (Fig.
7F) of muscle twitches during REM sleep.
an endogenous excitatory drive on trigem-
inal motoneurons during waking and pha-
or tonic REM sleep, we wanted to deter-
mine whether exogenous application of
glutamate during sleep could restore basal
did this by applying 25 mM glutamate onto
trigeminal motoneurons while measuring
levels of masseter tone during sleep and
waking. In six rats, glutamate was micro-
for 3.0 ? 0.21 h, over which time it had a
potent excitatory effect on trigeminal mo-
toneurons resulting in an increase in left
masseter tone (Fig. 8A).
During glutamate perfusion, masseter
EMG activity increased (relative to base-
line) by 128% in AW ( p ? 0.001, two-way
RM ANOVA) (Fig. 8B), by 332% in QW
( p ? 0.001), and by 556% in NREM sleep
( p ? 0.002). The excitatory actions of glu-
tamate were maintained with equipotency
(i.e., receptors did not desensitize to the
stimulus) across the application period;
basal levels of masseter muscle tone were
first 30 s vs last 30 s NREM sleep).
Glutamate application during NREM
sleep increased masseter muscle activity
during this state to levels that were not sig-
nificantly different from those during wak-
ing under baseline conditions ( p ? 0.713)
(Fig. 6B), suggesting that sleeping levels of
muscle tone can be restored to waking lev-
els by replacing the glutamate that is natu-
rons during NREM sleep. However, this
same excitatory stimulus had no effect on
0.916) (Fig. 8B). Figure 9A depicts a typical example illustrating
that the stimulatory effects of glutamate during NREM sleep are
rapidly abolished during entry into REM sleep.
To demonstrate that the mechanism responsible for blocking
64 REM periods analyzed (mean REM duration, 65.4 ? 13.5 s),
we found that glutamate increased masseter tone (relative to
REM (i.e., waking; p ? 0.001) periods (Fig. 9B). However, there
of motor atonia despite the continued presence of glutamate at
the trigeminal motor pool (Fig. 9A,B). This nullifying effect was
immediately reversed during entrance into post-REM waking,
therefore demonstrating that the excitatory actions of glutamate
are rapidly regained during entry into waking.
left but not right masseter tone. A, A typical example demonstrating that application of CNQX and D-AP-5 into the left
sleep, this intervention had no affect on right masseter muscle tone during any behavioral state. These observations
4654 • J.Neurosci.,April30,2008 • 28(18):4649–4660Burgessetal.•GlutamatergicControlofSomaticMotoneuronsinSleep
phasic REM sleep. Compared with baseline levels, the number of
muscle twitches increased by 200% during phasic REM ( p ?
0.035, paired t test) (Fig. 8C), with the amplitude of muscle
twitches being unaffected by glutamate perfusion ( p ? 0.099)
AMPA and NMDA application
during tonic REM sleep was not attributable to an insufficient
dosage of glutamate, we also activated trigeminal motoneurons
with doses of non-NMDA and NMDA re-
ceptor agonists that have been demon-
strated previously to potently excite mo-
toneurons (Steenland et al., 2006). We
applied either 0.1 mM AMPA or 0.1 mM
NMDA into the left trigeminal motor pool
during both sleep and wakefulness.
Compared with baseline, AMPA in-
in AW (n ? 6; p ? 0.001, two-way RM-
ANOVA) (Fig. 10A) and by 934% in
NREM sleep ( p ? 0.002). The excitatory
effects of AMPA were rendered completely
ineffective during tonic REM sleep ( p ?
0.879). The number of phasic muscle
twitches was significantly increased with
AMPA application ( p ? 0.001, paired t
test) (Fig. 10B), but the amplitude of pha-
sic activity was unchanged ( p ? 0.978)
(Fig. 10C). There was a rapid loss of the
excitatory actions of AMPA at the transi-
tion of NREM to REM sleep ( p ? 0.012)
that resulted in the persistence of atonia,
but the stimulatory effects of AMPA were
immediately reinstated during entrance
into post-REM waking ( p ? 0.032).
Activation of NMDA receptors on tri-
geminal motoneurons also increased mas-
seter tone during AW by 95% (n ? 6; p ?
0.001, two-way RM-ANOVA) (Fig. 10D)
and in NREM sleep by 267% ( p ? 0.003)
but not during tonic REM sleep ( p ?
0.895). There was no significant change in
either the amount of phasic activity during
REM sleep ( p ? 0.192, paired t test) (Fig.
10E) or the amplitude of muscle twitches
( p ? 0.688) (Fig. 10F) during NMDA ap-
plication. There was a rapid loss of the ex-
citatory effects of NMDA as soon as tonic
REM sleep began ( p ? 0.024), and these
effects were rapidly regained during post-
REM waking ( p ? 0.022).
Skeletal muscle tone is regulated across
the sleep–wake cycle: it is maximal dur-
ing alert waking, suppressed at sleep on-
set and during NREM sleep, and minimal
or absent during REM sleep except for
periodic muscle twitches. Here we pro-
vide the first evidence that an endoge-
nous excitatory glutamatergic drive onto
motoneurons is a contributing factor
controlling the stereotypical pattern of muscle tone during
wakefulness, NREM sleep, and phasic REM sleep but not dur-
ing tonic REM sleep.
We demonstrate that a functional glutamatergic drive con-
tributes to motoneuron activity during natural motor behav-
iors. We show that antagonism of ionotropic glutamate recep-
REM sleep. B, Group data demonstrating that application of CNQX and D-AP-5 caused a significant reduction in left masseter
Burgessetal.•GlutamatergicControlofSomaticMotoneuronsinSleep J.Neurosci.,April30,2008 • 28(18):4649–4660 • 4655
tors on trigeminal motoneurons significantly suppresses
masseter tone during waking, indicating that a glutamatergic
drive plays an important role in controlling motoneuron ex-
citability and motor tone during waking behaviors. This wak-
NMDA receptors. However, non-NMDA receptors play the
predominant role because their blockade reduced waking
masseter tone by 82%, whereas blockade of NMDA receptors
only reduced masseter tone by 49%. This observation
confirms previous findings that non-NMDA receptors trans-
duce the majority of the excitatory effects of glutamate on
motoneurons (Funk et al., 1993; Del Negro and Chandler,
Additional excitatory neuromodulators also contribute to
waking levels of muscle tone because non-NMDA and NMDA
antagonism did not eliminate basal muscle tone. Possible
sources of excitatory wake-related drives to motoneurons in-
clude inputs from orexinergic (hypocretinergic), serotoner-
gic, and noradrenergic cell groups, which not only project to
and facilitate motoneuron excitation (Peever et al., 2003;
Yamuy et al., 2004; Fenik et al., 2005; Lee et al., 2005), but also
discharge maximally during wakefulness (McGinty and
Harper, 1976; Horvath et al., 1999; Mileykovskiy et al., 2005).
We report that withdrawal of a wake-related glutamatergic
drive during NREM sleep is, at least in part, responsible for
the reduction in basal muscle tone observed during this state.
We found that antagonism of ionotropic glutamate receptors
on trigeminal motoneurons had no effect on muscle tone dur-
ing NREM sleep, indicating that there is negligible glutama-
tergic excitation during this state. We also found that gluta-
mate receptor antagonism reduced waking muscle tone to
NREM sleep levels, thus suggesting that withdrawal of this
excitatory drive contributes to muscle tone suppression in
Although withdrawal of glutamatergic excitation may be
the primary neurochemical responsible for reducing motor
tone in NREM sleep, other transmitters may also regulate mo-
levels during baseline conditions (downward arrow; p ? 0.001); however, this intervention was
ditions( p?0.001);however,itdidnotincreasemuscletoneduringtonicREMsleep( p?0.916).All
strating that antagonism of non-NMDA receptors by perfusion of 0.5 mM CNQX into the left
noeffectonlevelsofmassetertoneduringeitherNREM( p?0.939)ortonicREM( p?0.985)
duringREMsleep( p?0.231)ortheamplitudeofmuscletwitches( p?0.197).Allvaluesare
4656 • J.Neurosci.,April30,2008 • 28(18):4649–4660Burgessetal.•GlutamatergicControlofSomaticMotoneuronsinSleep
toneuron activity during this state. Indeed, we recently iden-
tified the presence of functional excitatory noradrenergic and
inhibitory glycinergic drives that contribute to muscle activity
during NREM sleep (Mir et al., 2006; Brooks and Peever,
2008); similar NREM drives have also been identified in the
hypoglossal motor pool (Morrison et al., 2003; Chan et al.,
pared with either waking or NREM sleep
because it is characterized by flurries of pe-
riodic muscle twitches that occur on a
background of motor atonia. We demon-
strate that an endogenous glutamatergic
drive is sufficient for generating muscle
twitches without affecting REM atonia.
However, antagonism of NMDA receptors
sleep; this may be related to the fact that
voltage-dependent NMDA receptors are
rendered inactive in REM sleep when mo-
toneurons are hyperpolarized (Chase and
Morales, 1983; Mayer et al., 1984). These
findings suggest that muscle twitches are
mediated by glutamate release and con-
firms intracellular studies in cats showing
that trigeminal motoneurons receive non-
NMDA-mediated glutamatergic excitation
Two recent studies suggest that inspira-
suppressed during REM sleep because nor-
adrenergic excitation of hypoglossal mo-
toneurons is withdrawn during this state
of an excitatory noradrenergic drive at
nonrespiratory motor pools is also respon-
ing REM sleep, then pharmacologically ac-
tivating trigeminal motoneurons should
reverse REM atonia. Our results show that,
despite potent glutamatergic excitation of
trigeminal motoneurons during REM
sleep, we were unable to override REM
atonia. We conclude that reduced excita-
tion of motoneurons does not trigger REM
underlie the suppression of inspiratory ac-
tivity during REM sleep. This contention is
supported by the demonstration that
norepinephrine release is decreased within
the hypoglossal motor
stimulation-induced REM atonia (Lai et
The loss of glutamatergic excitation
during tonic REM sleep is in striking con-
trast to the excitatory response that gluta-
mate evokes on muscle tone during all
other behavioral states. We show that the
excitatory effects of glutamate are rapidly lost on entrance into
ings also report that the excitatory actions that neuromodulators
(e.g., norepinephrine) have on trigeminal (Mir et al., 2006) and
hypoglossal motoneuron activity are also nullified during REM
sleep (Chan et al., 2006). We therefore conclude that motor
atonia is mediated by a powerful REM-specific inhibitory mech-
muscletoneinwakingandNREMsleep( p?0.002),withoutchanginglevelsofmotortoneduringtonicREMsleep( p?0.916).
Burgessetal.•GlutamatergicControlofSomaticMotoneuronsinSleepJ.Neurosci.,April30,2008 • 28(18):4649–4660 • 4657
strengthened by the fact that glutamatergic activation during
effect probably occurs because motoneurons are maximally hy-
perpolarized during periods of phasic REM sleep (Chase et al.,
toneurons, thereby limiting muscle twitch magnitude.
Intracellular studies show that somatic motoneurons are bom-
barded by inhibitory glycinergic and GABAergic potentials during
natural REM sleep (Nakamura et al., 1978; Soja et al., 1987), and
microdialysis studies demonstrate that glycine and GABA release
onto motoneurons is increased during pharmacologically induced
muscle atonia (Kodama et al., 2003). However, neither glycinergic
nor GABAergic inhibition are sufficient to induce motor suppres-
either the trigeminal or hypoglossal motor pools does not reverse
REM atonia (Morrison et al., 2003; Brooks and Peever, 2008). This
Determining the neural substrate responsible for inducing
REM atonia is of major clinical significance. For example, if a
reproduced, it could serve as powerful therapeutic tool to sup-
press the pathological movements associated with motor disor-
ders such as Parkinson’s disease. Indeed, De Cock et al. (2007)
recently demonstrated that parkinsonian symptoms such as
NREM sleep but abolished during REM sleep in Parkinson’s pa-
tients (De Cock et al., 2007). This observation is consistent with
our findings that glutamate-induced motor excitation is blocked
only in REM sleep.
The major implication of this study is that a functional glutama-
tergic drive onto motoneurons regulates levels of muscle tone
during wakefulness and phasic REM sleep. A possible source of
this drive could be the medial reticular formation, a brainstem
region that plays a pivotal role in coupling arousal state and mo-
al., 2007); most cells in this region also discharge maximally in
fusion into the left trigeminal motor pool causes a potent activation of left masseter muscle
(LM) activity during NREM sleep, but this excitatory effect is immediately abolished during
post-REM waking (glutamate vs baseline, p ? 0.001). All values are means ? SEM; A.U.,
( p ? 0.895). E, F, During phasic REM sleep, NMDA application did not increase either the
number( p?0.192)oramplitudeofmuscletwitches( p?0.688).Allvaluesaremeans?
4658 • J.Neurosci.,April30,2008 • 28(18):4649–4660 Burgessetal.•GlutamatergicControlofSomaticMotoneuronsinSleep
(Siegel et al., 1983, 1992). These cells could therefore provide the
Another potential and very potent source of a waking gluta-
matergic drive could be from the dopaminergic, noradrenergic,
and serotonergic cell groups that promote arousal and facilitate
These monoaminergic cells not only exhibit a wake–active dis-
charge pattern, project to and excite motoneurons, but they also
synthesize and corelease glutamate (for example, 86% of norad-
renergic cells in the locus ceruleus coexpress glutamate) (Liu et
al., 1995). Therefore, in addition to providing an endogenous
waking drive (e.g., from norepinephrine), monoaminergic cells
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2003). In fact, several lines of evidence demonstrate that motor
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actions on motoneurons primarily by glutamatergic excitation;
drive is responsible for controlling levels of motor tone during
waking. That glutamate plays the dominate role in regulating
basal motor tone may explain why pharmacological strategies
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