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
(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
may also be a major source of glutamatergic input to motoneu-
rons during wakefulness (Fung et al., 1994; Bouryi and Lewis,
2003). In fact, several lines of evidence demonstrate that motor
activation produced by monoaminergic stimulation is primarily
mediated by glutamate, with monoamine activity playing a mi-
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
rotonin reuptake inhibitors) have limited success at increasing
motor tone during sleep and cataplexy (Berry et al., 1999; Sonka
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