J Appl Physiol 102: 2240 2250, 2007.
First published March 1, 2007; doi:10.1152/japplphysiol.01229.2006.
Genioglossus muscle activity and serotonergic modulation of hypoglossal
motor output in obese Zucker rats
Sandeep Sood, Xia Liu, Hattie Liu, and Richard L. Horner
Departments of Medicine and Physiology, University of Toronto, Toronto, Canada
Submitted 30 October 2006; accepted in final form 26 February 2007
Sood S, Liu X, Liu H, Horner RL. Genioglossus muscle activity
and serotonergic modulation of hypoglossal motor output in obese
Zucker rats. J Appl Physiol 102: 2240 2250, 2007. First published
March 1, 2007; doi:10.1152/japplphysiol.01229.2006.Obese Zucker
rats have a narrower and more collapsible upper airway compared
with lean controls, similar to obstructive sleep apnea (OSA) patients.
Genioglossus (GG) muscle activity is augmented in awake OSA
patients to compensate for airway narrowing, but the neural control of
GG activity in obese Zucker rats has not been investigated to deter-
mine whether such neuromuscular compensation also occurs. This
study tests the hypotheses that GG activity is augmented in obese
Zucker rats compared with lean controls and that endogenous 5-hy-
droxytryptamine (5-HT) contributes to GG activation. Seven obese
and seven lean Zucker rats were implanted with electroencephalogram
and neck muscle electrodes to record sleep-wake states, and they were
implanted with GG and diaphragm wires for respiratory muscle
recordings. Microdialysis probes were implanted into the hypoglossal
motor nucleus for perfusion of artificial cerebrospinal fluid and the
5-HT receptor antagonist mianserin (100 M). Compared with lean
controls, respiratory rates were increased in obese rats across sleep-
wake states (P 0.048) because of reduced expiratory durations (P
0.007); diaphragm activation was similar between lean and obese
animals (P 0.632). Respiratory-related, tonic, and peak GG activ-
ities were also similar between obese and lean rats (P 0.139). There
was no reduction in GG activity with mianserin at the hypoglossal
motor nucleus, consistent with recent observations of a minimal
contribution of endogenous 5-HT to GG activity. These results sug-
gest that despite the upper airway narrowing in obese Zucker rats,
these animals have a sufficiently stable airway such that pharyngeal
muscle activity is normal across sleep-wake states.
sleep; hypoglossal motor nucleus; genioglossus muscle; serotonin;
obesity; obstructive sleep apnea
OBESITY IS ASSOCIATED WITH upper airway narrowing in humans
(17, 40, 42) and is a known risk factor for obstructive sleep
apnea (OSA) (37). The Zucker rat is an established model of
obesity (8), and these animals exhibit a narrower upper airway
compared with lean littermates (5, 24). The upper airway of
obese Zucker rats is also more collapsible compared with lean
controls as judged by more positive critical closing pressures
(32, 35), similar to observations made in OSA patients (41, 45).
Although measures of upper airway size and mechanics in
obese Zucker rats have similarities to OSA patients, studies of
the neural control of pharyngeal muscle activity in obese and
lean Zucker rats have not yet been performed. Such studies are
important if this obese model is to be valuable to evaluate
neural as well as anatomical factors in upper airway collaps-
ibility relevant to OSA.
Address for reprint requests and other correspondence: R. L. Horner, Rm.
6368, Medical Sciences Bldg., 1 Kings College Circle, Toronto, ON, Canada,
M5S 1A8 (e-mail: richard.horner@utoronto.ca).
2240 8750-7587/07 $8.00 Copyright 2007 the American Physiological Society
Pharyngeal muscle tone is augmented in awake patients with
OSA, a compensatory response thought to be relevant to
maintaining airway patency in these individuals with narrow
airways (26, 50). However, suppression of this compensatory
response in sleep predisposes to OSA (25). Similar increases in
pharyngeal dilator muscle activity occur in English bulldogs
(15), a breed also with a narrow airway and sleep-disordered
breathing (14). Only one study has reported genioglossus (GG)
muscle activity in obese Zucker rats and compared this activity
with that of lean controls (32). Those recordings, however,
were obtained in only two rats that were studied under anes-
thesia and in the supine position, and those conditions may
have contributed to the increased GG activity observed (32).
Magnetic resonance imaging showed smaller changes in oro-
pharyngeal size during inspiration in obese, compared with
lean, Zucker rats, but there were no differences in pharyngeal
wall tissue strain as indicated by noninvasive tissue tagging
(5). The results from this study may indicate normal, rather
than enhanced, pharyngeal muscle tone in obese Zucker rats,
but this has not yet been determined. Accordingly, the first aim
of the present study was to record GG muscle activity across
sleep-wake states in freely behaving obese Zucker rats to
determine if GG activity is augmented compared with lean
littermates.
The increased GG activity observed in two anesthetized,
supine, and obese Zucker rats was diminished by intravenous
ritanserin, a serotonin (5-hydroxytryptamine; 5-HT) receptor
antagonist (32). Similar effects in awake bulldogs (53) impli-
cates a 5-HT neural component to the augmentation of pha-
ryngeal motor tone in these animals with compromised upper
airways. Nevertheless, endogenous 5-HT receptor mechanisms
at the hypoglossal motor nucleus (HMN), the source of motor
outflow to the GG muscle of the tongue, play a minimal role in
the normal modulation of GG activity in intact lean rats in
wakefulness and natural sleep (47, 48). Taken together, these
results suggest that a normally low endogenous 5-HT tone
operates at the HMN in intact lean animals but that this role of
5-HT may be increased in obese animals, for example, by
reflex compensation for a narrow airway.
In obese Zucker rats, however, the notion that an enhanced
5-HT-mediated excitatory drive augments pharyngeal motor
tone and upper airway patency has not been established. For
example, the more positive closing pressures after intravenous
ritanserin in anesthetized rats (32) is often cited as evidence of
a significant role for 5-HT in maintaining upper airway patency
in these animals. However, the increases in closing pressures
after 5-HT receptor antagonism are small in magnitude in both
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 GENIOGLOSSUS ACTIVITY IN OBESE ZUCKER RATS
lean and obese anesthetized Zucker rats, with increases of only
0.8 and 0.7 cmH2O, respectively (32). Similarly, in conscious
Zucker rats, systemic administration of ritanserin decreased
ventilation (as measured by noninvasive plethysmography) and
increased work of breathing (as reflected by oxygen consump-
tion) but only in older (8 mo) obese animals and not in younger
lean or obese rats (32). No measurements of the potential
effects on upper airway muscle activity were performed (32).
Our laboratory has developed an animal model for manipula-
tion of neurotransmission at the HMN in freely behaving rats
awake and asleep (1, 6, 47). Given the potential importance of
obese Zucker rats as a clinically relevant model of both
anatomic and neural control of the upper airway, with the latter
not yet determined (5, 24, 32, 35), the second aim of the
present study is to determine whether 5-HT receptor antago-
nism at the HMN reduces GG activity in obese and lean Zucker
rats consistent with a role of a significant 5-HT drive contrib-
uting to the maintenance of GG activity in this animal model of
obesity.
METHODS
Experiments were performed on seven obese [mean 645.1 35.0
(SE) g] and seven age-matched lean (393.1 20.0 g) male Zucker
rats. The obese phenotype (fa/fa) is due to a recessive trait, and lean
littermates (fa/?) were used as the controls (32, 35). Importantly, the
source (Vassar College, Poughkeepsie, NY), weights (see above) and
ages (9.2 0.7 and 8.9 0.3 mo, respectively) of rats were the same
as a previous study demonstrating increased upper airway collapsibil-
ity in anesthetized obese compared with lean Zucker rats (35), and the
study implicating a role for 5-HT modulation of ventilation and upper
airway stability in obese conscious rats via indirect measurements of
work of breathing and O2 consumption (32). All procedures con-
formed to the recommendations of the Canadian Council on Animal
Care, and the University of Toronto Animal Care Committee ap-
proved the experimental protocols.
Anesthesia and Surgical Procedures
Sterile surgery was performed under general anesthesia induced
with intraperitoneal ketamine (85 mg/kg) and xylazine (15 mg/kg) as
described previously ( 1 , 47, 48). In addition, rats were given saline
(3 ml, 0.9% ip) for fluid loading, atropine sulfate (1 mg/kg ip) to
reduce airway secretions, and buprenorphine (0.03 mg/kg ip) to
reduce potential postoperative pain. Following the onset of surgical
levels of anesthesia, as judged by abolition of the pedal withdrawal
and corneal blink reflexes, the abdomen, neck, and head regions were
shaved and cleaned with 70% alcohol and the antispeptic-germide
solution Triadine (10% povidone-iodine, Triad Disposables, Brook-
field, WI). Sterile 1% chloramphenicol ointment (Vetcom, Upton, PQ,
Canada) was applied to the cornea to prevent drying. An anesthesia
mask (12) was placed over the snout, and the rats spontaneously
breathed a 50:50 mixture of air and oxygen for the remainder of the
surgery. Any additional anesthesia was given by inhalation (isoflu-
rane, typically 0.22%).
With the rats supine the ventral surface of the GG muscle was
exposed via a submental incision and dissection of the overlying
geniohyoid and mylohyoid muscles. Two insulated, multistranded
stainless steel wires (AS631, Cooner Wire, Chatsworth, CA) were
implanted into GG muscle and secured with sutures and tissue glue.
Section of the medial branches of the hypoglossal nerves has shown
that GG activity is recorded with such electrode placements (30). To
record diaphragm activity, two insulated, multistranded stainless steel
wires (AS636, Cooner Wire) were sutured onto the costal diaphragm
via an abdominal approach. The size, configuration, and placement of
the GG and diaphragm electrodes were consistent across experiments.
J Appl Physiol VOL 102 JUNE 2007
2241
To assist in electrode placements during surgery, the GG and dia-
phragm signals were monitored for respiratory-related activity (AM8
Audio Amplifier, Grass). To ensure that the GG electrodes were
positioned correctly, incrementing test voltages were applied (Isolated
Pulse Stimulator, model 2100, A-M Systems) to confirm tongue
movements that were identified visually. The GG and diaphragm
wires were tunneled subcutaneously to an incision in the neck, and the
submental and abdominal incisions were closed with absorbable
sutures.
The rats were then placed in a stereotaxic frame (model 962, Kopf,
Tujunga, CA) with blunt ear bars. To ensure consistent positioning
between animals the flat skull position was achieved with an align-
ment tool (model 944, Kopf). Two multistranded stainless steel wires
were sutured onto the dorsal neck muscles to record the neck elec-
tromyogram (EMG). To record the cortical electroencephalogram
(EEG), two stainless steel screws attached to insulated wire (30
gauge) were implanted in the skull. One of the screws was implanted
2 mm anterior and 2 mm to the right of bregma, whereas the other was
implanted 3 mm posterior and 2 mm left of bregma (1, 47, 48). A
similar screw that served as a ground electrode was implanted adja-
cent to the sagittal suture 8 mm anterior to bregma on the nasal
bone. Finally, a small hole was drilled at the junction of interparietal
and occipital bones for placement of the microdialysis guides (CMA/
11, Acton, MA). The guides were lowered 13.96 0.04 mm posterior
to bregma, 7.37 0.19 mm ventral to bregma, and 0.30 mm lateral to
the midline for the obese rats, and they were implanted 14.00 0.00
mm, 7.14 0.32 mm, and 0.30 mm, respectively, for the lean rats. At
these coordinates the guides were targeted 3 mm above the HMN (1,
47). A dummy cannula was placed inside the microdialysis guide to
keep it patent until the day of the experiment. At the end of surgery,
all the electrodes were connected to pins and inserted into a miniature
plug (STC-89PI-220ABS, Carleton University, Ottawa, ON, Canada).
The plug and microdialysis guides are affixed to the skull with dental
acrylic and anchor screws.
After surgery, the rats were transferred to a clean cage and kept
warm under a heating lamp until full recovery as judged by normal
locomotor activity, grooming, drinking, and eating. The rats were
given soft food for the first day after surgery. The rats recovered for
an average of 9.3 0.7 days before the experiments.
Recording Procedures
For purposes of habituation the rats were placed in the recording
chamber (MD-1500, BAS, West Lafayette, IN) with fresh bedding,
food, and water on the evening before the experiments. The recording
chamber was placed inside an electrically insulated, sound-attenuated
cubicle (EPC-010, BRS/LVE, Laurel, MD). For recordings, a light-
weight electrically shielded cable was connected to the plug on the
rats head and was attached to a counterbalanced swivel that permitted
free movement. A video camera allowed continuous visual monitoring
without disrupting the rat. The chamber was covered with a custom-
ized lid with a center hole that allowed passage of the cable carrying
the electrical signals and microdialysis tubing. The volume inside the
rat chamber was 20.5 liters.
Air or CO2 mixtures (see below) were delivered to the chamber at
a flow rate of 5 l/min after humidification over a water reservoir (16,
47, 48). To disperse air as it flowed into the chamber, the lid contained
three inlet ports equipped with small computer fans (model FP-108G/DC,
12 V, Commonwealth). Measurements of sleep-wake states and re-
spiratory muscle activities were made during both room air and
CO2-stimulated breathing; 7.5% inspired CO2 was chosen because
this normally provides robust GG stimulation in awake and sleeping
rats (16, 47, 48) and is sufficient to overcome the strong vagal
inhibition of GG muscle in the intact rat (2, 16). Some evidence also
suggests that 5-HT raphe neurons are activated by CO2 (52, 54) and
so may also contribute to GG activation in hypercapnia, at least in
reduced preparations (7, 46), although this notion has recently been
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2242 GENIOGLOSSUS ACTIVITY IN OBESE ZUCKER RATS
questioned from studies in intact animals in vivo (48). The CO2
concentration inside the rat chamber was measured continuously
(Beckman LB-2) at a sample flow rate of 500 ml/min with the
sampled air returned to the chamber.
The electrical signals were amplified and filtered (Super-Z head-
stage amplifiers and BMA-400 amplifiers/filters, CWE, Ardmore,
PA). The GG and diaphragm signals were recorded at the same
amplification across all experiments. The EEG signal was filtered
between 1 and 100 Hz while the GG, neck, and diaphragm EMGs
were filtered between 100 and 1,000 Hz. The electrocardiogram was
removed from the diaphragm EMG using an oscilloscope and elec-
tronic blanker (model SB-1, CWE). The moving-time averages (time
constant 200 ms) of the EMGs were also obtained (model MA-821,
CWE). All signals were recorded on computer (Spike 2 software,
1401 interface, CED, Cambridge, UK).
Microdialysis
The rats were gently restrained while the internal cannula was
removed from the guide and the microdialysis probe (CMA/11 14/01)
was inserted. The probes were 240 m in diameter with a 1-mm
cuprophane membrane and a 6,000-Da cutoff. The probes projected 3
mm from the tip of the guide and were targeted to the HMN. In each
rat a transient burst of GG activity was recorded when the probe was
initially inserted and penetrated the HMN, and this was useful as a
preliminary confirmation of probe placement, and did not occur in the
neck or diaphragm signals (1, 47).
The probes were connected to Teflon tubing (inside diameter
0.12 mm) and 1.0 ml syringes via a zero-dead-space switch (Unis-
witch, BAS, West Lafayette, IN). During the experiments the probes
were continually flushed with artificial cerebrospinal fluid (ACSF) at
2.1 l/min using a syringe pump and controller (MD-1001 and
MD-1020, BAS). The composition of ACSF (mM) was 125 NaCl, 3
KCl, 1 KH2PO4, 2 CaCl2, 1 MgSO4, 25, NaHCO3, and 30 D-glucose
(30), with the ACSF made fresh on the day of each experiment. The
ACSF was bubbled with CO2 to a pH of 7.39 0.01 for both the lean
and obese rats. The experiments began the morning after insertion of
the microdialysis probes and were performed during the day when the
rats normally sleep.
Protocol
During microdialysis perfusion of ACSF into the HMN, recordings
were made across sleep-wake states during both room air breathing
and hypercapnia (see above). These measurements were made during
a 2.5-h period before the perfusion medium was switched to mianserin
dissolved in ACSF (mianserin HCl, formula weight 300.8, Sigma).
In the presence of mianserin, recordings were also performed during
room air breathing and hypercapnia, again for a 2.5-h period. Mian-
serin is a broad-spectrum 5-HT receptor antagonist with affinity for
5-HT2 receptors (56), i.e., those implicated in mediating the excitatory
effects at the HMN (9, 10, 23, 36). Mianserin was applied at a dose of
100 M. Importantly, our laboratory has shown using the exact same
methodology that 100 M mianserin at the HMN in anesthetized rats
produces robust decreases in GG activity during both room air and
hypercapnia (46), i.e., showing the efficacy of the interventions. Our
laboratory has also shown that similar effects to mianserin are ob-
served with the more specific 5-HT2 receptor antagonist MDL-100907
at the HMN in intact conscious rats (47). However, MDL-100907 is
less cost effective than mianserin for studies requiring continual
perfusion in intact rats. Mianserin was also chosen because we had
difficulty in dissolving other more specific 5-HT receptor antagonists
such as ritanserin in ACSF (53). The mianserin solution was applied
at a pH of 7.41 0.01 and 7.40 0.01 for the obese and lean rats,
respectively, i.e., similar to that of the ACSF controls (see above).
J Appl Physiol VOL
Data Analysis
The data were analyzed in consecutive 5-s time bins. The GG,
diaphragm, and neck EMG signals were analyzed from the respective
moving-time average signals (above electrical zero) and were quan-
tified both in arbitrary units and the percentage of maximum activity
recorded during the experiments for each individual rat. Electrical
zero was measured as the voltage recorded with the amplifier inputs
grounded. The GG and diaphragm signals were analyzed breath by
breath, which corresponded to 712 breaths for each 5-s epoch. For
each breath, the analysis of the GG EMG was time locked to breathing
as defined by the peak and trough of the diaphragm signal. For each
breath, GG activity was quantified as mean tonic activity (i.e., basal
activity in expiration), peak activity and phasic respiratory activity
(i.e., peak inspiratory activity tonic activity), and average values for
these measures of GG activity were calculated. Average diaphragm
amplitudes (i.e., phasic respiratory diaphragm activity) and respiratory
rates were also calculated for the breaths in each 5-s time bin. Neck
muscle activity was quantified as the mean signal over each 5-s
period. These average values of GG, diaphragm, and neck EMGs for
each 5-s period were then taken to a spreadsheet for later correspon-
dence with the prevailing sleep-wake state, level of respiratory stim-
ulation, and drug at the HMN. Each rat served as its own control with
all interventions performed in one experiment, therefore allowing for
consistent effects of experimental condition (e.g., ACSF and mian-
serin) to be observed across sleep-wake states within and between
rats. The EMG signals were recorded at the same amplifications
across all experiments. Data were analyzed at least 60 min after a
switch between drugs.
Sleep-wake states were identified by visual inspection and were
classified into wakefulness, non-rapid eye movement (REM) and
REM sleep using standard criteria (16, 28, 29, 47, 48). Periods of quiet
wakefulness were characterized by the lack of overt body movements,
identified from the EMG signals and video recordings, in contrast to
active wakefulness that included overt behaviors such as grooming
and exploring. Such periods of wakefulness were differentiated be-
cause 5-HT caudal raphe neurons are thought to be activated more
during motor tasks associated with such behaviors (18, 39), and hence
may be more responsive to the effects of 5-HT receptor antagonism at
those times, whereas the 5-HT raphe inputs may have a lesser role in
modulating the respiratory component of GG activity during basal
breathing (44). Only those epochs comprising at least 30 s of unin-
terrupted sleep without arousal, or wakefulness without drowsiness,
were included in the analysis. The data are presented as means of all
the 5-s epochs of a particular sleep or awake state. To minimize bias
in selecting periods, the EEG and neck EMG were scored for sleep-
wake states without reference to the GG or diaphragm signals.
Function of GG Electrodes and Histology
At the end of the experiments the rats were reanesthetized with
ketamine and xylazine as described above, and the GG electrodes
were again stimulated to confirm tongue protrusion. In all rats, the
electrodes were in place from the beginning to the end of the study. To
mark the sites of microdialysis, a microdialysis probe with the
membrane cut at the tip was inserted into the guide cannula and a 1%
aqueous solution of potassium permanganate was microinjected at a
flow rate of 2.1 l/min for 10 min (33, 49). The rats were then
euthanized with an overdose of pentobarbital sodium (325 mg), and
25 ml of 0.9% saline were perfused through the ascending aorta
followed by 40 ml of 10% formalin. The brains were then removed
and fixed in 10% formalin. The medullary regions were blocked,
transferred to 30% sucrose, and cut in 50-m coronal sections with a
cryostat (CM 1850, Leica, Nussloch, Germany). Each section around
the lesion site was mounted and stained with neutral red. Microdialy-
sis sites were localized from the stained sections and marked on
standard brain maps (38).
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 GENIOGLOSSUS ACTIVITY IN OBESE ZUCKER RATS
Statistical Analysis
For all comparisons, differences were considered significant if the
null hypothesis was rejected at P 0.05 using a two-tailed test.
Statistics were performed using analysis of variance with repeated-
measures (ANOVA-RM) and post hoc t-tests performed using Bon-
ferroni-corrected P values. All data are expressed as means SE.
Analyses were performed using Sigmastat (Jandel Scientific, San
Rafael, CA).
RESULTS
In the group of obese rats, a total of 31,854 5-s epochs (i.e.,
a total of 44.2 h of data) were included in the analysis, of which
25,939 epochs (81.4%) were from periods of wakefulness,
5,402 epochs (17.0%) were from non-REM sleep, but only 513
epochs (1.6%) were from REM sleep. Similarly, in the group
2243
of lean rats, a total of 24,697 5-s epochs (i.e., 34.3 h of data)
were included in the analysis, of which 19,154 epochs (77.6%)
were from wakefulness, 5,039 (20.4%) were from non-REM
sleep, and only 504 (2.0%) were from REM sleep. Because of
this low amount of data obtained in REM sleep in both the lean
and obese rats, data analysis was restricted to wakefulness and
non-REM sleep.
Function of GG Electrodes and Histology
The GG electrodes were in place and functional throughout
the study. Stimulation of the GG electrodes at the end of the
experiment showed that the voltages required to cause tongue
movements were not different from at the time of surgery
(0.92 0.14 vs. 0.93 0.13 V for the lean rats, and 0.90
0.14 vs. 0.87 0.13 V for the obese rats; both P 0.610,

Fig. 1. Example and group data showing location of the microdialysis probes. A: histological section showing an example of a lesion site made by the
microdialysis probe immediately adjacent to both hypoglossal motor nuclei (HMN). Also shown are the distribution of individual microdialysis sites from all
lean (B) and obese rats (C). The sizes of the bars represent the apparent size of the lesions from the histological sections. AP, area postrema; Cer, cerebellum;
Sol, nucleus of the tractus solitarius; 4V, fourth ventricle.

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2244 GENIOGLOSSUS ACTIVITY IN OBESE ZUCKER RATS

paired t-tests). Similarly, the voltages required to cause tongue
movements were similar for lean compared with obese rats
(P 0.740, unpaired t-test).
Figure 1A shows an example of a lesion site made by the
microdialysis probe in the HMN. The locations of all the
microdialysis sites from each of the lean and obese rats are
shown in Fig. 1, B and C, respectively. In all the experiments,
the microdialysis sites were within the HMN or in the midline
immediately adjacent to both the nuclei.
Breathing Pattern in Obese and Lean Rats
There was no evidence for any sleep-disordered breathing in
the obese rats (e.g., no recurring diaphragm recruitment or
periodic disturbances in sleep associated with abnormal breath-
ing episodes). Figure 2 shows examples of normal breathing
during sleep in the lean and obese Zucker rats.
Figure 3 shows values of respiratory variables recorded in
the lean and obese Zucker rats in active wakefulness, quiet
wakefulness, and non-REM sleep. There was a statistically
significant difference in respiratory rate between the lean and
obese Zucker rats (P 0.040, 2-way ANOVA-RM) that did
not depend on sleep-wake state (P 0.417), i.e., occurred
across all states. Post hoc analysis confirmed that respiratory
rate was increased in the obese vs. lean Zucker rats in both
wakefulness and sleep (P 0.048, post hoc unpaired t-test;
Fig. 3A). As shown in Fig. 3, B and C, this increased respira-
tory rate was due to reduced expiratory time in the obese
compared with the lean rats (P 0.007, post hoc unpaired
t-test after 2-way ANOVA-RM showed differences between
groups, P 0.005). Inspiratory time was not different between
the lean and obese rats (P 0.588, 2-way ANOVA-RM).
The amplitude of the diaphragm EMG signal was not sta-
tistically different between the lean and obese Zucker rats
whether analyzed as arbitrary units (P 0.632, 2-way
ANOVA-RM, Fig. 3D) or the percentage of maximum of
diaphragm activity (Fig. 3F; P 0.782, 2-way ANOVA-RM).
This lack of effect persisted even if diaphragm amplitude was
normalized for body weight (P 0.111, 2-way ANOVA-RM),
a normalization procedure routinely applied to the tidal volume
signal in studies with noninvasive plethysmography (32). The
maximum level of diaphragm activity recorded during the exper-
iments was also similar between the lean and obese Zucker rats
(P 0.696, unpaired t-test). Diaphragm minute activity (i.e.,
diaphragm amplituderespiratory rate, the surrogate for neural
ventilation) was not significantly different between groups
when analyzed as arbitrary units or the percent of maximum
diaphragm activity (P 0.168 and 0.661, respectively, 2-way
ANOVA-RM). Similarly, this lack of effect persisted even if
the diaphragm minute activity was also normalized for body
weight (P 0.128, 2-way ANOVA-RM).
GG Muscle Activity During Wakefulness and Non-REM
Sleep in Obese and Lean Zucker Rats
Figure 4 shows examples of GG muscle activities recorded
in an obese and lean Zucker rat during room air and CO2-
stimulated breathing in quiet wakefulness, in the presence of

Fig. 2. Examples showing respiratory muscle activities during non-rapid eye movement (REM) sleep in lean and obese Zucker rats. There was no evidence for
any sleep-disordered breathing, e.g., no recurring diaphragm recruitment or periodic disturbances in sleep associated with abnormal breathing episodes. Traces
show the electroencephalogram (EEG), neck electromyogram (EMG), diaphragm (DIA), and genioglossus (GG) signals. The GG and DIA are also displayed
as their moving-time averages (MTA) in arbitrary units (AU). The baseline of the integrator (i.e., electrical zero) is shown for the MTA signals and the arrow
denotes the direction of inspiration.

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 GENIOGLOSSUS ACTIVITY IN OBESE ZUCKER RATS 2245

Fig. 3. Group mean data showing breathing pattern in lean and obese Zucker rats (open and black bars, respectively). Data are shown for respiratory rate
(A; where min1 is breaths/min), expiratory time (B), inspiratory time (C), diaphragm amplitude (D), and diaphragm minute activity (E), as well as diaphragm
amplitude and diaphragm minute activity normalized to the percentage of maximum (%max; F and G respectively). AW, active wakefulness, QW, quiet
wakefulness; NREM, non-REM sleep. Values are means SE. *P 0.05 for obese compared with lean rats.

ACSF and mianserin at the HMN. Note the presence of
prominent tonic GG activity during room air breathing in
wakefulness as well as modest respiratory-related GG activity,
which is increased by hypercapnia, i.e., patterns typical of
previous studies (16, 28, 29, 48). Also note that GG activity is
not decreased by 5-HT receptor antagonism at the HMN.
Figure 5 shows group mean GG muscle activity recorded in
the lean and obese Zucker rats in active wakefulness, quiet
wakefulness and non-REM sleep. The data in Fig. 5 confirm
that GG muscle activity across sleep-wake states was not
increased in the obese rats compared with lean rats, and it even
had a tendency to be decreased; i.e., there was no evidence for
increased GG activity as stated in the hypothesis. Statistical
analysis showed that respiratory-related, tonic and peak GG
activities, whether expressed as arbitrary units (Fig., 5, AC) or
the percent of maximum (Fig. 5, DF), showed robust changes
across sleep-wake states (all P 0.001, 2-way ANOVA-RM).
However, although there was a tendency for GG activity to be
decreased in the obese compared with the lean rats this differ-
ence was not statistically significant whether analyzed as arbi-
trary units or the percent of maximum (all P 0.139, 2-way
ANOVA-RM). Importantly, any potential difference in GG
activity between the lean and obese rats did not depend on the
sleep-wake states being analyzed; i.e., the interaction term
J Appl Physiol VOL
from the analysis of variance was also not statistically signif-
icant whether analyzed as arbitrary units or the percentage of
maximum (all P 0.227, 2-way ANOVA-RM). The maxi-
mum level of GG activity recorded during the experiments was
also similar between the lean and obese Zucker rats (P
0.641, unpaired t-test).
5-HT Receptor Antagonism at the HMN on GG Activity in
Obese and Lean Zucker Rats
Figure 6 shows GG muscle activity recorded in the lean and
obese Zucker rats with 5-HT receptor antagonism at the HMN
during both room air and CO2-stimulated breathing. Data are
shown for respiratory-related, tonic, and peak GG activities,
both from the raw signal (Fig. 6, AC, respectively), as well as
the percentage of the maximum (Fig. 6, DF, respectively).
These data illustrate that there was no evidence for suppression
of GG activity with 5-HT receptor antagonism at the HMN in
either the lean or obese rats. Statistical analysis confirmed that
there was no significant difference in GG activity between
ACSF and mianserin at the HMN for any comparison (i.e.,
when analyzed as arbitrary units or the percentage of maxi-
mum) except for a significant effect on respiratory-related GG
activity in non-REM sleep (asterisk in Fig. 6, A and D), and
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2246 GENIOGLOSSUS ACTIVITY IN OBESE ZUCKER RATS

Fig. 4. Example in lean (A) and obese (B) rats

of the effects of 5-HT receptor antagonism at
the hypoglossal motor nucleus on GG activity
during room air and CO2-stimulated breathing.
Recordings are shown for periods of wakeful-
ness with both artificial cerebrospinal fluid
(ACSF) at the HMN (control) and mianserin.
Shown are the EEG, neck EMG, DIA, and GG
signals (abbreviations as for Fig. 2). The GG
and DIA are displayed as their MTA in AU.
The baseline of the integrator (i.e., electrical
zero) is shown for the MTA, and the arrow
denotes the direction of inspiration. Note the
tonic GG activity in wakefulness and the dis-
cernable respiratory-modulated GG activity
during room air breathing that is then increased
by CO2 stimulation. There is no major effect of
5-hydroxytryptamine receptor antagonism at
the HMN on GG activity in either the lean or
obese rats during room air or CO2-stimulated
breathing.

tonic and peak GG activities in quiet wakefulness in the obese
rats (asterisk in Fig. 6, E and F) (all P 0.05 from 2-way
ANOVA-RM). For these latter specific comparisons, however,
GG activity was increased in the presence of 5-HT receptor
antagonism (all P 0.05, post hoc paired t-test), i.e., in the
opposite direction predicted by the hypothesis, and a direction
of change also observed in a previous study (47).
DISCUSSION
The results of the present study show that GG activity across
sleep-wake states was similar between the lean and obese
Zucker rats, and there was no evidence for an augmented 5-HT
drive to the HMN. Similarly, there was no evidence of sleep-
disordered breathing in these obese rats. Overall, these results
suggest that despite the upper airway narrowing in obese
Zucker rats (5, 24, 32, 35) these animals have a sufficiently
stable upper airway such that pharyngeal muscle activity is
normal across sleep-wake states. Accordingly, the obese
Zucker rat appears to be a model most suited to investigations
of the impact of obesity on upper airway mechanics (5, 32,
35) and ventilation (8), rather than OSA per se or neuro-
muscular compensation for a narrow airway as occurs in
humans (26, 50).
J Appl Physiol VOL
Methodological Considerations
Data across sleep-wake states. Data analysis was restricted
to periods of wakefulness and non-REM sleep because of the
practical constraint of low amounts of data obtained in REM
sleep. This relative lack of REM sleep, unlike our laboratorys
previous studies (1, 6, 16, 47, 48), was probably because the
protocol was limited to measurements in a 2.5-h period in each
rat to accommodate each drug with both room air and hyper-
capnia. We did not wait for multiple REM episodes to occur in
a longer protocol because REM sleep is also limited by
hypercapnia (16). The percentage of data obtained in wakeful-
ness was also higher than in our laboratorys previous studies,
again likely because of the use of CO2 (16) or because these
animals were older, to be in the same age range as a relevant
previous study in obese Zucker rats (32). Nevertheless, suffi-
cient data were obtained in wakefulness and non-REM sleep
for the purposes of this study, especially to address the poten-
tial role of 5-HT receptor mechanisms in modulating GG
activity.
Quantification of EMG activity. Because comparisons of
EMG activity between groups of lean and obese animals using
the raw signal may theoretically be complicated by the possi-
bility of fat acting as an electrical insulator, potentially reduc-
ing EMG activity, the GG and diaphragm signals were ana-
102 JUNE 2007 www.jap.org
 GENIOGLOSSUS ACTIVITY IN OBESE ZUCKER RATS
Fig. 5. Group mean data showing levels of GG activity in lean and obese Zucker rats (open and black bars, respectively) during room air breathing in AW, QW,
and NREM sleep. Data are shown for respiratory-related, tonic, and peak GG activity quantified as the raw signal (A, B, and C, respectively) and for the percent
(%) of maximum activity (D, E, and F, respectively). Values are means SE.
lyzed both in arbitrary units and normalized to the percentage
of maximum activity recorded during the experiments. We did
not rely solely on the normalized signal for the analyses,
however, because unlike humans in whom behavioral tongue
activity can be standardized for comparisons between groups,
e.g., by maximum voluntary protrusion (26), maximum activ-
ity in such a freely behaving animal model cannot be controlled
and so data were analyzed also in arbitrary units. Analysis
showed that regardless of the method used to quantify muscle
activity, GG and diaphragm activity was similar between the
obese and lean Zucker rats across sleep-wake states, and there
was no evidence for a decrease in GG activity with 5-HT
receptor antagonism at the HMN. This lack of difference in GG
activity between lean and obese rats, with or without 5-HT
receptor antagonism, was not an artifact due to a lesser ability
to generate muscle tone in either group because maximum GG
activity was also similar between lean and obese rats, as was
maximum diaphragm activity. In addition, although tongue
movements in response to electrical stimulation of the GG
wires were only identified visually, it was noted that the
voltages required to cause such movements were similar for the
lean and obese rats.
Respiratory Motor Activity in Lean Obese Rats
The obese Zucker rats showed increased respiratory rates,
compared with lean animals, because of decreased expiratory
times (Fig. 3). The increased respiratory rate is in agreement
with a previous study using the same age and weights of rats
purchased from the same source (32). Interestingly, both tidal
volume and overall lung ventilation were reduced in the obese
Zucker rats when quantified by noninvasive plethysmography
(32), whereas measures of diaphragm muscle activity and
diaphragm minute activity (a surrogate for neural ventilation)
were similar to lean controls when measured by EMG, either
J Appl Physiol VOL
2247
quantified as the raw activity or the percent of maximum. This
result suggests that obesity imposes limitations to the mechan-
ical consequences of respiratory muscle activation in these
animals that leads to reduced tidal volumes for a given dia-
phragm activation (8, 32), an effect that is offset (at least in
part) by increased respiratory rates (32). Similar changes in the
pattern of breathing pattern are also observed in genetically
obese mice (34, 51).
Although the accuracy of tidal volume measurements by
whole body plethysmography in rats has been questioned,
measurements of respiratory rate are accurate with this tech-
nique (31). Of note, therefore, the average respiratory rates of
140 160 breaths/min reported for lean and obese Zucker rats
in the previous study investigating the role of systemically
administered 5-HT receptor antagonists in modulating breath-
ing (32) are high compared with the average respiratory rates
of 101120 breaths/min observed in quiet or active wakeful-
ness in the present study (Fig. 3). In the former study, the rats
were monitored in a small (4 liters) plethysmography chamber
and within a restrainer that did not permit backward rotation
(32). This restricted environment may have contributed to the
elevated respiratory rates in that study. In comparison, the rats
in the present study were placed in a larger (20.5 liters)
chamber in which they were able to move around and sleep
freely, habituated overnight and were free from interruption.
The potential for a more aroused animal in the former study is
relevant because it is under those conditions that systemic
administration of ritanserin increased the work of breathing (as
indicated by oxygen consumption) and decreased ventilation
measured by noninvasive plethysmography (32). Although
muscle activity was not measured in those animals (32), the
data were taken to suggest that the increase in work of
breathing was due to an effect at the upper airway. However,
the principal effect of ritanserin in reducing ventilation in those
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2248 GENIOGLOSSUS ACTIVITY IN OBESE ZUCKER RATS

Fig. 6. Group mean data showing levels of GG

activity in lean and obese Zucker rats with ACSF
or mianserin at the HMN (open and gray bars,
respectively). Data are shown for AW, quiet QW,
and NREM sleep during both room air and CO2-
stimulated breathing. GG is quantified as respi-
ratory-related, tonic, and peak GG activity, and
shown both for the raw signal (A, B, and C,
respectively) and the percent of activity maxi-
mum (%max; D, E, and F respectively). See text
for further details. Values are means SE. Data
were lacking for NREM sleep in the presence of
hypercapnia in lean rats, so these data are not
included. *P 0.05 for mianserin compared
with ACSF controls.

awake rats was via reduced respiratory rate (32), suggesting an
effect independent of the upper airway. In addition, the in-
creases in upper airway closing pressures in anesthetized rats
after 5-HT receptor antagonism occurred in both lean and
obese rats and were small in magnitude (1 cmH2O), even in
the supine position (32). A reinterpretation of the previous
observations in conscious rats (32), especially in the context of
the present experiments, would suggest that the reduced respi-
ratory rate in those animals by systemically administered
ritanserin could have been due to a sedating or anxiety-
reducing effect (27, 43) rather than an effect at upper airway
dilator muscles.
GG Activity and 5-HT Receptor Antagonism
GG activity was similar between the lean and obese Zucker
rats regardless of whether GG activity was quantified as the
raw signal or percentage of maximum activity. Importantly,
there was no evidence of any augmented GG activity in the
obese rats (Fig. 5) to indicate a neuromuscular compensation
for a narrow airway as occurs in humans (26). Magnetic
J Appl Physiol VOL
resonance imaging has shown that upper airway size increases
during inspiration in obese and lean anesthetized Zucker rats,
but it was less so in the obese animals (5). Simultaneous
measurements of pharyngeal wall tissue strain with noninva-
sive tissue tagging led to the suggestion that obesity imposes a
mechanical load that prevents a normal airway response to a
given change in pharyngeal wall tissue strain (5). The obser-
vation of similar levels of GG activity in obese and lean
animals in the present study is consistent with this suggestion.
From studies in reduced preparations, withdrawal of 5-HT
has been hypothesized to be a principal mechanism underlying
decreased GG activity in sleep (21, 23). However, in vivo
studies in conscious rats show that despite robust GG activa-
tion with delivery of 5-HT to the HMN (19), endogenous 5-HT
plays a minimal role in the normal modulation of GG activity
(47). Inhibition of serotonergic medullary raphe neurons, the
source of 5-HT inputs to the HMN, confirmed this result (48).
The results of the present study support this concept of a
minimal role for endogenous 5-HT receptor mechanisms at the
HMN in the normal modulation of GG activity across sleep-
102 JUNE 2007 www.jap.org
 GENIOGLOSSUS ACTIVITY IN OBESE ZUCKER RATS
wake states (47, 48), even in obese rats with an anatomically
narrow airway (5, 24). At first glance, these recent results may
seem to contradict the several previous studies in anesthetized
or decerebrate animals in which an endogenous 5-HT drive to
the HMN was first demonstrated (7, 10, 22, 23, 55), including
our laboratorys studies in anesthetized rats (46, 48) that used
the exact same methodology as the studies in conscious rats
(47, 48). However, those previous studies demonstrating an
endogenous 5-HT drive to the HMN were all performed in the
presence of vagotomy (7, 10, 22, 23, 46, 55), which was
subsequently shown to augment the role of 5-HT at the HMN
(47, 48). In contrast to the lack of influence of endogenous
5-HT mechanisms modulating GG activity, recent studies in
reduced (11) and conscious (6) rats suggest an important role
for noradrenergic mechanisms operating at the HMN across
sleep-wake states.
It was also noted, however, that GG activity could be
increased after mianserin (i.e., in contrast to the hypothesized
decrease), and this was statistically significant for some of the
comparisons shown in Fig. 6. Although studies in a larger
number of animals may have strengthened the validity of this
observation, this direction of change is also consistent with
previous experiments using both mianserin and the more spe-
cific 5-HT2 receptor antagonist MDL-100907 at the HMN (47).
These observations strengthen the case that there is a minimal
endogenous excitatory 5-HT drive operating at the HMN to
increase GG activity, and that the control by 5-HT of hypo-
glossal motoneurons may not be as straightforward as first
presumed. For example, in vitro evidence suggests that a major
component of the raphe inputs to hypoglossal motoneurons are
glutamatergic with 5-HT inhibiting the release of this excita-
tory neurotransmitter via presynaptic effects (4).
If relevant to humans, the lack of a significant role of
endogenous 5-HT in modulating pharyngeal muscle activity
across sleep-wake states may also help explain the lack of
clinically relevant responses to selective 5-HT reuptake inhib-
itors in OSA (3, 13, 20). Nevertheless, there is one animal
model where 5-HT appears relevant to augmenting pharyngeal
muscle tone and increasing upper airway stability. Pharyngeal
dilator muscle activity is diminished by systemic administra-
tion of the 5-HT receptor antagonist ritanserin in awake bull-
dogs, and this effect causes significant upper airway narrowing
(53). It remains to be determined whether this latter effect is
mediated by antagonism of an augmented 5-HT drive to
pharyngeal motoneurons that these dogs require to compensate
for a narrow air space (53). However, the airway narrowing
may also be due to antagonism of the afferent relay mecha-
nisms that mediate the neural compensatory reflex response for
pharyngeal muscle activation, e.g., via an influence of ritan-
serin on the 5-HT inputs to the nucleus of the solitary tract,
inputs which are also state dependent (55).
GRANTS
This work was supported by funds from the Canadian Institutes of Health
Research (Grant MT-15563).
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