J Appl Physiol 98: 1387–1395, 2005.
First published November 19, 2004; doi:10.1152/japplphysiol.00914.2004.
Orexin stimulates breathing via medullary and spinal pathways
John K. Young,1 Mingfei Wu,1 Kebreten F. Manaye,1 Prabha Kc,1
Joanne S. Allard,1 Serdia O. Mack,1 and Musa A. Haxhiu1,2
1
Howard University Specialized Neuroscience Research Program, Departments
of Physiology and Anatomy, Washington, District of Columbia, and 2Departments
of Pediatrics and Anatomy, Case Western University, Cleveland, Ohio
Submitted 20 August 2004; accepted in final form 10 November 2004
these associations remains to be determined because wakefulness is often accompanied by behavioral activation. Suppression of rapid eye movement sleep occurs through an inhibition
of the cholinergic neurons in the laterodorsal tegmental and
pedunculopontine nuclei (49).
In the central nervous system, orexin-containing neurons
innervate multiple sites, including cell groups in the brain stem
and spinal cord that are involved in the regulation of cardiovascular functions (10, 19, 25, 43, 45). However, there is no
information on projections of orexin-containing neurons to
respiratory-related brain stem regions or spinal cord motoneurons that innervate respiratory muscles such as the diaphragm.
Furthermore, it is not known whether the brain stem region
involved in the generation of inspiratory rhythmic activity
(pre-Bötzinger complex of the ventrolateral medulla) and/or
inspiratory motoneurons such as phrenic motor neurons express orexin receptors (42). We hypothesized that orexincontaining neurons that are involved in arousal also participate
in respiratory control via both direct projections to the preBötzinger complex and phrenic motor neurons.
hypothalamus; pre-Bötzinger region; phrenic motor neurons; orexin-1
receptors; sleep apnea
Animals
BREATHING IS AN ACTIVE NEURALLY controlled process that is
regulated by neural mechanisms that adjust respiratory-related
drive to the behavioral state and to the metabolic demands of
an organism. Hypothalamic neurons are part of this controlling
system and play an important role in the regulation of breathing
rate and depth (14, 26).
Neurons in the lateral hypothalamus synthesize orexin A and
orexin B, also called hypocretin-1 (hcrt-1) and hypocretin-2
(hcrt-2). These peptide neurotransmitters are processed from a
common precursor, prepro-orexin, encoded by a gene localized
to human chromosome 17q2. Orexin-containing neurons affect
autonomic, neuroendocrine, and sleep-wakefulness neuroregulatory systems that in turn could potentially influence breathing
(9, 13, 16, 19).
The orexins stimulate target cells via two orexin G proteincoupled receptors, orexin R1 and orexin R2 (40). It has been
proposed that orexin-containing neurons promote wakefulness
by excitation of cholinergic neurons in the basal forebrain,
which release acetylcholine and thereby contribute to the
cortical activation of wakefulness. However, the causality of
Address for reprint requests and other correspondence: J. K. Young, Dept.
of Anatomy, Howard Univ. College of Medicine, 520 W St., NW, Washington,
DC 20059 (E-mail: [email protected]).
http://www. jap.org
MATERIALS AND METHODS
Experimental protocols were approved by Howard University Institutional Animal Care and Use Committee. All experiments were
performed in male Sprague-Dawley rats (Harlan, Indianapolis, IN;
300 –350 g) according to the National Institutes of Health Guide for
the Care and Use of Laboratory Animals. Male rats were chosen to
reduce potential effects of hormonal cycling in females.
Neuroanatomic Experiments
In the present study, the distribution of neurons that express orexin-1
receptors within the rostral ventrolateral medulla and the cervical spinal
cord was determined. To define whether phrenic motor neurons express
orexin receptors, animals (n ⫽ 3 rats) were anesthetized with pentobarbital sodium (40 mg/kg) and a retrograde tracer, cholera toxin B subunit
(0.1% CTB; List Biological Laboratories, Campbell, CA), was injected
into the costal region of the diaphragm at 10 sites with 300 nl/site. This
procedure specifically identifies phrenic motoneurons that project to the
diaphragm. Seven days after CTB injections, the animals were deeply
anesthetized (50 mg/kg ip pentobarbital) and rapidly perfused transcardially with 0.9% buffered saline followed by 4% paraformaldehyde in 0.1
M PBS (pH 7.4). The perfused brains were removed from the animals
and postfixed with 4% paraformaldehyde in 0.1 M PBS (pH 7.4) for 24 h
at 4°C. After cryoprotection by immersion in 30% sucrose in 0.1 M PBS
at 4°C for 48 h, 40-␮m-thick frozen sections of brains were prepared by
using a Leitz microtome.
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
8750-7587/05 $8.00 Copyright © 2005 the American Physiological Society
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Young, John K., Mingfei Wu, Kebreten F. Manaye, Prabha Kc,
Joanne S. Allard, Serdia O. Mack, and Musa A. Haxhiu. Orexin
stimulates breathing via medullary and spinal pathways. J Appl
Physiol 98: 1387–1395, 2005. First published November 19, 2004;
doi:10.1152/japplphysiol.00914.2004.—A central neuronal network
that regulates respiration may include hypothalamic neurons that
produce orexin, a peptide that influences sleep and arousal. In these
experiments, we investigated 1) projections of orexin-containing neurons to the pre-Bötzinger region of the rostral ventrolateral medulla
that regulates rhythmic breathing and to phrenic motoneurons that
innervate the diaphragm; 2) the presence of orexin A receptors in the
pre-Bötzinger region and in phrenic motoneurons; and 3) physiological effects of orexin administered into the pre-Bötzinger region and
phrenic nuclei at the C3–C4 levels. We found orexin-containing fibers
within the pre-Bötzinger complex. However, only 0.5% of orexincontaining neurons projected to the pre-Bötzinger region, whereas
2.9% of orexin-containing neurons innervated the phrenic nucleus.
Neurons of the pre-Bötzinger region and phrenic nucleus stained for
orexin receptors, and activation of orexin receptors by microperfusion
of orexin in either site produced a dose-dependent, significant (P ⬍
0.05) increase in diaphragm electromyographic activity. These data
indicate that orexin regulates respiratory activity and may have a role
in the pathophysiology of sleep-related respiratory disorders.
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OREXIN AND RESPIRATORY CONTROL
J Appl Physiol • VOL
hypoglossal nuclei, and within the anatomic borders of the preBötzinger complex (see also Fig. 2). Seven days after injection,
animals were deeply anesthetized and perfused as described above,
and the brains were removed and processed immunohistochemically
for CTB and orexin labeling. Transverse sections of the whole brain
of each animal were cut at 40 ␮m. The immunohistochemical procedures used were previously described (26) for staining tissue sections
of the hypothalamus. Briefly, free-floating sections were washed in
PBS containing 0.3% Triton X-100, and then a 1-in-5 series of tissue
sections was exposed for 30 min to a PBS-Triton solution containing
1% BSA to block nonspecific binding sites. After a further wash, the
tissue was placed overnight at 4°C in a primary polyclonal antibody
solution (1:2,000 dilution of rabbit anti-orexin A in 1% normal goat
serum in PBS-Triton; Oncogene Research Products) for 48 h at 4°C.
The sections were washed twice in PBS, and they were incubated for
2 h in a solution of biotinylated goat anti-rabbit secondary antibody
(Vector Laboratories). The sections then were washed with PBS and
incubated for 60 min in avidin-biotin-peroxidase complex, as recommended by the supplier.
To identify retrogradely labeled neurons within the lateral hypothalamus that produce orexin, after being labeled for orexin sections
were washed in PBS and placed in a primary polyclonal antibody
solution (1:20,000 dilution of goat anti-CTB in PBS-Triton) for 48 h
at 4°C. The sections were washed twice in PBS, and they were
incubated for 2 h in a 1:200 solution of donkey anti-goat IgG
conjugated with Alexa Fluro 594 (Texas red; Molecular Probes). The
sections were washed and mounted on gelatin/alum-coated glass
slides. A drop of Vecta Shield (Vector Laboratories) was applied to
air-dried sections, and the slides were coverslipped. Sections were
viewed with either bright-field microscopy for orexin immunoreactivity (brown diaminobenzidine precipitate) or with fluorescence microscopy for CTB immunoreactivity (red fluorescence). Digitized
bright-field images were merged with fluorescence images, using
imaging software, to detect double-labeled cells (Sigma Scan Pro,
SPSS, Chicago, IL)
Physiological Experiments
Rats were anesthetized with urethane (1.2 g/kg), and the carotid
artery and external jugular vein were catheterized for recording blood
pressure and heart rate and for administering fluid and anesthetic
(one-tenth of initial concentration per hour). After bilateral vagotomy
and tracheotomy, the rats were placed in a stereotaxic apparatus in a
prone position. Diaphragm electromyographic activity (DEMG) was
recorded via bipolar electrodes placed in the costal diaphragm. Animals were ventilated with O2 at a pump rate that provided 30% of total
ventilatory activity obtained with 7% CO2 in O2, as previously
described (14, 26).
To investigate the effects of orexin on phrenic motoneurons, the
cervical spinal cord segment (C3–C5) was exposed after laminectomy and the dura was opened and retracted (n ⫽ 5 rats). After
control values were recorded, the spinal cord segment was microperfused with 5 ␮l of saline or orexin A dissolved in saline
applied to the surface of the cord, and respiratory variables were
recorded for 5 min for each solution. Respiratory and cardiovascular responses to three doses of orexin (4, 40, and 200 ␮g/ml in
sterile saline) were studied. These doses corresponded to doses that
have proven effective in stimulating neuronal firing and/or arousal
in other studies (e.g., Ref. 19).
To examine the medullary effects of orexin, 200 nl of either saline
or of the three doses of orexin were microinjected into the preBötzinger complex of an additional five rats via a Hamilton syringe
(same coordinates as described above). Respiratory variables were
recorded as described above. Finally, at the end of the physiological
experiments, three of the five rats were microinjected at the same
medullary stereotaxic coordinates with 200 nl of 1% toluidine blue to
mark the anatomic location of the perfusion site. These rats were
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In these experiments, immunostaining procedures for orexin receptors were performed sequentially rather than simultaneously, because
previous studies in our laboratory demonstrated that this approach
provides better specificity and lower background staining (26).
Briefly, to enhance immunoreactivity, the free-floating sections of the
brain stem and cervical spinal cord (C3–C5 region) were washed in
PBS and then incubated in 10 mM citric acid at 37°C for 1 h, followed
by three washes in PBS and incubation for 30 min in PBS-Triton
solution containing 1% normal serum to block nonspecific binding
sites. After a further wash, the sections were incubated for 24 h at 4°C
in a PBS-Triton solution containing rabbit anti-orexin-1 receptor
antibody (Chemicon International; 1:400 dilution). The sections were
rinsed and incubated for 2 h with a 1:200 dilution of goat anti-rabbit
IgG conjugated with Alexa Fluro 488 fluorescein isothiocyanate
(FITC) (Molecular Probes, Eugene, OR).
Briefly, in the second step, the sections were rinsed in PBS and
stained for CTB. Sections were placed in a primary polyclonal
antibody solution (1:20, 000 dilution of goat anti-CTB in PBS-Triton;
List Biological Laboratories) for 48 h at 4°C. The sections were
washed twice in PBS, and they were incubated for 2 h in a 1:200
solution of donkey anti-goat IgG conjugated with Alexa Fluro 594
(Texas red; Molecular Probes). The sections were washed and
mounted on gelatin/alum-coated glass slides. A drop of Vecta Shield
(Vector Laboratories, Burlingame, CA) was applied to air-dried sections, and the slides were coverslipped.
Selected sections of the medulla oblongata were also stained for
orexin-1 receptors with peroxidase-labeled antibody and diaminobenzidine chromogen (ABC technique, as described below). Control
experiments for each experiment were done to determine whether the
primary or the secondary antibodies produced false-positive results. In
these experiments, sections were stained with all possible combinations of primary and secondary antibodies in which a single immunoprobe was omitted. Omission of primary or secondary antibodies
resulted in the absence of labeling, demonstrating that no falsepositive results were obtained with these reagents. Slides were viewed
with a bright-field and fluorescence microscope (Olympus AX70,
Olympus America) equipped with the adequate filter systems to
observe the Texas red and green FITC fluorescence. Colocalization of
CTB and orexin-1 receptors was identified by alternating between
filters to view Texas red and FITC fluorescence and by analyzing
merged images of the exact same sites. In this qualitative approach to
the problem, we did not attempt to quantitatively estimate total
numbers of identifiable phrenic motor neurons between C3 and C5 or
the percentage of phrenic motor neurons that stained positively for
orexin receptors.
In a second set of experiments, orexin-containing, lateral hypothalamic cells that project to the phrenic nucleus were identified by
neuroanatomic tracing. In anesthetized animals (n ⫽ 3 rats), CTB was
unilaterally injected into the ventral horn of the cervical spinal cord,
extending from C3 to C5 (total of ⬃300 nl of 0.1% solution) as
previously described (26). Injections were made with a glass micropipette (40- to 60-␮m diameter), 0.6 mm from the midline and 1.4 mm
from the dorsal surface of the spinal cord. Seven days after CTB
injections, animals were perfused and brains were prepared as outlined above.
In a third set of experiments, in three experimental subjects,
projections from orexin-containing neurons to the pre-Bötzinger complex were determined by unilateral microinjection of CTB, 12.30 mm
caudal to bregma, 2.2 mm lateral to midline, and 0.8 mm dorsal to the
ventral surface of the ventrolateral medulla oblongata (right site; 100
nl of 0.1% CTB). These coordinates were used in previous studies in
our laboratory to examine respiration-regulating neurons possessing
neurokinin-1 receptors within the so-called pre-Bötzinger complex
region (26, 42). These same coordinates were later utilized for
microinjection of either orexin or toluidine blue marker dye (see
below). Histological inspection of sections confirmed that this injection site was caudal to the facial nuclei, rostral to the level of the
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perfused as above, brains were sectioned, and medullary sections were
dried down onto slides, counterstained with 0.1% basic fuchsin, and
mounted in Vecta-Shield without exposure of the slides to alcohol.
This procedure avoided the elution of the marker dye, toluidine blue,
from the sections by alcohol.
Data Analysis
Moving average DEMG was used to determine the amplitude as well
as the frequency of inspiratory bursts. Respiratory responses were
quantified by averaging these parameters for the control period for 10
consecutive breaths and 10 breaths at the peak response after microperfusion of the saline or increasing concentrations of orexin A.
Blood pressure and heart rate were measured in the control period and
when peak changes occurred after orexin A administration. Average
values (means ⫾ SD) for the analyzed variables were compared by
analysis of variance for repeated measures and Tukey’s protected
t-tests. Statistical significance was considered achieved when P ⬍ 0.05.
RESULTS
Connectivity of orexin-ir neurons with the phrenic motor
nucleus of the spinal cord. After CTB injection into the spinal
phrenic nucleus, retrogradely labeled cells were most abundant
in the lateral hypothalamus, dorsal to the fornix. This distribution showed considerable overlap with the distribution of
orexin-immunoreactive (ir) neurons in the lateral hypothalamus. In these rats, 386 orexin-ir cells and 173 CTB labeled
cells were examined; a total of 11 double-labeled cells, mainly
concentrated in the portion of orexin-ir neurons dorsolateral to
the fornix, were found (Fig. 1).
Connectivity of orexin-ir neurons with the pre-Bötzinger
complex. After CTB injection into the pre-Bötzinger complex,
labeled cells tended to be located somewhat closer to the third
ventricle than cells projecting to the phrenic motor nucleus. In
this hypothalamic area, 227 orexin-ir cells and 362 CTB-
Physiological Experiments
Physiological effect of orexin A receptor activation on
respiratory drive. To analyze the effects of microinjection of
orexin into the pre-Bötzinger complex, changes induced by
orexin were expressed as percentage of baseline discharge and
compared (Fig. 4). This analysis showed that the two higher
doses of orexin elicited statistically significant changes in peak
DEMG compared with the response to saline (P ⬍ 0.05 for 40
␮g/ml and for 200 ␮g/ml; Tukey’s protected t-test for analysis
of variance). Toluidine blue dye infusion confirmed that the
anatomic position of the infusion site was within the preBötzinger complex (Fig. 2). A close inspection of the infusion
site under the microscope showed that the intense color of the
dye was confined to an area with a diameter of ⬃200 ␮m; no
dye could be detected farther than 500 ␮m away from the
center of the infusion site.
Heart rates and blood pressure values for rats in this study
were ⬃5% higher than in our previous experiences and were
slightly elevated from typical values reported by others (e.g.,
Fig. 1. A: Schematic diagram of the area of
the hypothalamus investigated (2.2 mm posterior to bregma) (37). Boxed region shows
the location of orexin-immunoreactive neurons. B: bright-field microscopy of the lateral
hypothalamus, showing orexin-immunoreactive neurons (brown) labeled with diaminobenzidine. C: same field as in B, viewed with
fluorescence microscopy to show neurons
retrogradely labeled with CTB (rhodaminelabeled antibody) that had been infused into
the phrenic motor nucleus. D: A and B digitally combined. Neurons labeled only with
CTB appear white, whereas an orexin-immunoreactive neuron also labeled with CTB
appears red (arrow). Calibration bar ⫽ 100
␮m; F, fornix.
J Appl Physiol • VOL
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Neuroanatomic Experiments
labeled cells were examined; only one cell was double labeled.
However, orexin-containing fibers were observed in the preBötzinger complex region of the rostral ventrolateral medulla
(Fig. 2).
Detection of orexin-1 receptors. Immunoreactivity was
present in neurons of the nucleus ambiguus and nearby areas in
the ventrolateral medulla roughly corresponding to the preBötzinger complex (Fig. 2C). This area precisely corresponds
to the region recently reported to contain an unusually dense
accumulation of orexin-ir fibers (12).
Immunocytochemistry for orexin receptor protein showed
positive staining in neurons of the phrenic motor nucleus that
were identified by retrograde transport of CTB from the diaphragm (Fig. 3).
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OREXIN AND RESPIRATORY CONTROL
Ref. 10). We have no exact explanation for this, although these
values may be related to the specific experimental conditions
that we employed (i.e., multiple vascular cannulae, vagotomy,
tracheal cannula, etc.). No statistically significant effects of
orexin on heart rate or blood pressure were detected (Table 1).
A recording from a single rat that is representative of these
group differences between treatment conditions is shown in
Fig. 5.
Similarly, microperfusion of the two higher doses of orexin
onto the spinal cord elicited statistically significant changes in
peak DEMG compared with the response to saline (P ⬍ 0.05 for 40
␮g/ml and P ⬍ 0.01 for 200 ␮g/ml). No statistically significant
changes (P ⬎ 0.05) in breathing rate between treatment conditions
were detected (Fig. 6). Orexin produced a slight but statistically
significant increase in heart rate (P ⬍ 0.05 for 4 and 40 ␮g/ml,
P ⬍ 0.01 for 200 ␮g/ml of orexin) (Table 2). A recording from a
single rat that is representative of these mean results is shown in
Fig. 7.
J Appl Physiol • VOL
DISCUSSION
Hypothalamic Orexin-Producing Neurons and the Brain
Stem-Spinal Cord Respiratory Related Network
The results of this study showed for the first time that a
discrete population of orexin-ir neurons in the lateral hypothalamus projects to the ventrolateral medulla and phrenic nuclei.
These findings extend previous reports demonstrating that long
descending orexin-containing axonal projections innervate the
spinal cord (13, 24). However, our results suggest that direct
synaptic contact between pre-Bötzinger neurons and orexin-ir
neurons are infrequent, despite the presence of numerous
orexin-containing fibers in this region.
Conceivably, the apparent projection of a subset of lateral
hypothalamic orexin-containing neurons to pre-Bötzinger neurons and to phrenic nuclei could be partly due to nonspecific
labeling produced by some diffusion of retrograde tracer into
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Fig. 2. A: schematic diagram of the area of the medulla investigated (12.3 mm posterior to bregma) (37) paired with a section showing the distribution of toluidine
blue dye microperfused by using the same coordinates and volume as those used to infuse orexin. Boxed region shows the area examined at higher magnifications
in B, C, and D. Calibration bar ⫽ 0.5 mm. Amb, ambiguus; PrBo, pre-Bötzinger complex; sol, solitary tract; ml, medial lemniscus; py, pyramid; sp5, spinal tract
of 5. B: orexin-immunoreactive, beaded axons present within the region of the pre-Botzinger complex. Calibration bar ⫽ 50 ␮m. C: low-magnification view of
medulla, showing orexin-A receptor immunoreactivity (brown) in the nucleus ambiguus and subjacent neurons of the pre-Botzinger complex in a section
counterstained with thionin. Calibration bar ⫽ 250 ␮m. D: higher magnification view of neurons of the nucleus ambiguus that are immunoreactive for orexin
receptors. Calibration bar ⫽ 50 ␮m.
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the regions that contain nonrespiratory-related cells, and/or the
uptake of tracer by descending fibers of orexin neurons that
innervate the intermediate cell column or the thoracolumbar
portion of the spinal cord (24). In addition, CTB may also be
taken up by fibers of passage. However, CTB continues to be
regarded as a useful retrograde tracer in even the most recent
neuroanatomic studies (11). Furthermore, our results are in
accord with other reports of transsynaptic transport of pseudorabies virus from the diaphragm to the lateral hypothalamus
(18, 21, 27).
Considerable numbers of orexin containing fibers were
present within the rostral ventrolateral medulla of the rat. This
finding is in accordance with recent data that orexin-containing
neurons innervate vagal preganglionic cells within the nucleus
ambiguus (12). However, we found little retrograde transport
of CTB infused into this area to hypothalamic orexin-ir neurons, and CTB-labeled cells were mostly located medial to
orexin-ir neurons. Because CTB is taken up by synaptic vesicles, the possibility exists that synapses between pre-Bötzinger
complex neurons and orexinergic fibers of passage may be
sparse. It may be that orexin released from passing fibers
nonsynaptically activates receptors or that orexin circulating
within cerebrospinal fluid may activate neurons that contain
orexin receptors (volume transmission), in analogy with the
effects of endogenously released catecholamines in this region
(2, 23). Medullary neurons ventral to the compact portion of
the nucleus ambiguus, which in our study also were immunoreactive for orexin A receptors, may belong to the external
(loose) portion of the nucleus ambiguus. Such vagal preganJ Appl Physiol • VOL
glionic neurons may contribute to the control of respiration by
the pre-Bötzinger complex or else may not be directly involved
in the control of respiratory timing. A diversity of function of
orexin-sensitive neurons in the medulla may explain the stimulatory effects of orexin on respiratory amplitude and the
apparent lack of an effect on respiratory timing.
Orexin Receptor Expression on Brain Stem-Spinal Cord
Respiratory Related Neurons
The results of the present study showed that neurons within
a rostral ventrolateral medullary region corresponding to the
pre-Bötzinger complex did stain positively for orexin-A receptors. This finding is in accord with numerous other studies of
orexin receptors found throughout the brain stem and in more
rostral brain regions (6, 7, 15, 19, 28, 46). Furthermore, these
findings for the first time indicate that identified phrenic motor
neurons express orexin receptors.
Respiratory Effects of Orexin Activation
Orexin microinjected into the pre-Bötzinger region or administered topically to the cervical spinal cord increased
DEMG, without affecting discharge frequency in bilaterally
vagotomized rats. An increase in peak integrated moving
average activity of the diaphragm without changes in respiratory timing indicates that these neurons are involved in the
regulation of tidal volume activity of inspiratory pumping
muscles, independent of frequency discharge. However, these
results do not exclude a critical role of inspiratory rhythm-
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Fig. 3. A: diagram of the location of phrenic motor
neurons (boxed region) at segment C4 of the spinal cord
(32). B: phrenic motor neurons in the ventral horn of the
spinal cord, retrogradely labeled with CTB infused into
the diaphragm (green FITC-labeled secondary antibody). Calibration bar ⫽ 50 ␮m. C: same view as in B,
displaying the same section showing red fluorescence
(rhodamine-labeled secondary antibody) at sites of
orexin-A receptor immunoreactivity. D: B and C digitally combined. Double-labeled cells demonstrate that
phrenic motor neurons possess orexin receptors.
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Fig. 4. A: mean peak integrated respiratory diaphragmatic voltages expressed
as percent deviations from electromyographic values observed during baseline
recording from the diaphragm. The respiratory responses to microperfusion of
40 and 200 ␮g of orexin into the ventrolateral medulla were significantly
different from the responses to saline or to 4 ␮g/ml of orexin (*P ⬍ 0.05,
protected t-test). B: breathing rates during the 5 treatment conditions.
generating cells of the pre-Bötzinger complex for the control of
respiratory timing (17).
Orexin may also affect respiration via projections to other
sites, e.g., it may cause an increase in respiratory drive via
activation of hypothalamic neurons that project to the preBötzinger complex and to phrenic nuclei (14, 26, 29, 41,
50). Furthermore, the activity of upper airway dilating
muscles such as the genioglossus muscle could be affected
via direct projections of orexin-containing neurons to hypoglossal motoneurons and activation of orexin-1 receptors
Table 1. Cardiovascular variables following microperfusion
of test solutions into the pre-Bötzinger complex of the rostral
ventrolateral medulla
Orexin
Baseline
Saline
4 ␮g/ml
40 ␮g/ml
200 ␮g/ml
Heart rate, beats/min 414⫹6
Systolic pressure,
mmHg
154⫹12
Diastolic pressure,
mmHg
131⫹6
413⫹3
414⫹6
418⫹8
418⫹11
154⫹10
167⫹14
156⫹13
166⫹16
136⫹7
146⫹9
143⫹11
144⫹13
Values are means ⫹ SE.
J Appl Physiol • VOL
expressed by hypoglossal motor cells that innervate that
muscle (48). In addition, dense orexinergic projections innervate the intermediolateral cell column of the spinal cord
where the majority of sympathetic preganglionic neuronal
cell bodies reside (13, 46). This indicates that lateral hypothalamic orexinergic neurons may act directly on different
brain stem and spinal cord cell groups, providing parallel
control to cardiovascular and respiratory related output
neurons during behavioral state control, defensive responses, and energy homeostasis (22, 42).
Orexin, when topically applied to the spinal cord, had slight
but significant effects on heart rate. These findings are in
accord with other data showing that intracisternal or intrathecal
injections of orexin A or B provoke a dose-related increase in
heart rate and sympathetic neuronal activity in urethane anesthetized rats. Orexin given by these routes could activate
multiple central nervous system and spinal orexin-sensitive
sites, including excitatory brain stem neurons and spinal cord
sympathetic preganglionic neurons (3, 10). We observed no
effects of medullary infusions of orexin on heart rate, possibly
because our rats were vagotomized (12).
The physiological effects observed in this study showed a
reliable dose-response curve but were obtained by using relatively high concentrations of orexin. Possibly, some of these
responses could in part be pharmacological. A blockade of
these results by pretreatment with an orexin antagonist (e.g.,
SB-334867) would be required to completely eliminate the
possibility that they could partly be non-orexin receptor mediated (5).
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Fig. 5. Respiratory activity of rat 13 after microperfusion of saline into the
ventrolateral medulla (A) and after application of 40 ␮g/ml of orexin into the
medulla (B). 兰DEMG, integrated electromyographic activity of the diaphragm.
OREXIN AND RESPIRATORY CONTROL
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Fig. 6. A: mean peak integrated respiratory diaphragmatic voltages expressed
as percent changes from electromyographic values observed during baseline
recording from the diaphragm. Respiratory responses to application of 40 and
200 ␮g/ml of orexin onto the spinal cord were significantly different from the
responses to saline or to 4 ␮g/ml of orexin (*P ⬍ 0.05, **P ⬍ 0.01, protected
t-test). B: breathing rates during the 5 treatment conditions.
Physiological Relevance of These Findings
Orexin neurons provide a link between central nervous
system mechanisms that coordinate sleep-wakefulness states
and central nervous system control of autonomic functions, via
multiple projections (38). These neurons also, as shown by the
present study, are involved in regulation of the breathing
activity, via projections to the brain stem-spinal cord respiratory related network, presumably through the orexin R1 sigTable 2. Cardiovascular variables following microperfusion
of test solutions into the phrenic motor nucleus
of the spinal cord
Orexin
Baseline
Saline
4 ␮g/ml
40 ␮g/ml
200 ␮g/ml
Heart rate, beats/min 373⫹27 380⫹26 384⫹25* 387⫹22* 389⫹23†
Systolic pressure,
mmHg
160⫹18 154⫹13 147⫹9
144⫹10 147⫹9
Diastolic pressure,
mmHg
118⫹13 117⫹12 115⫹10 122⫹6
120⫹8
Values are means ⫹ SE. *P ⬍ 0.05, different from baseline, ANOVA for
repeated measures, Tukey’s protected t-test; †P ⬍ 0.01.
J Appl Physiol • VOL
naling pathway. This is in accord with a recent report that
stimulation of the perifornical (lateral) hypothalamus elicits
increases in respiratory activity, a response diminished in mice
genetically engineered to lack orexin (25). If, in humans,
orexin proves to stimulate respiration, then a deficit in orexin
action may contribute to a decrease in respiratory drive during
sleeping (for review, see Ref. 23). This assumption is consistent with recent findings indicating that orexin plays an important role in the maintenance of wakefulness (for reviews, see
Refs. 28, 49).
A decreased influence of orexin in brain regions that regulate
sleep and breathing could cause simultaneous decreases in both
arousal and respiratory drive. Recently, it was reported that
patients with sleep apnea had decreased blood levels of orexin,
relative to controls (34). The mechanisms that could produce a
decreased production of orexin are unknown but may be
related to the apparent link between obesity and sleep apnea. It
is well known that obesity is the most prominent risk factor for
sleep apnea (30, 56). Diabetes mellitus and hyperglycemia also
appear to provoke sleep apnea (47). It seems likely that some
metabolic alterations provoked by obesity, possibly elevations
in circulating nutrient molecules, could partly explain the
association between obesity and sleep apnea.
In genetic and diet-induced obesity in rodents, blood concentrations of a number of circulating molecules are elevated.
An increased effect of glucose, free-fatty acids, or leptin on
hypothalamic function could provoke an altered activity of
orexin-containing neurons (1, 4, 8, 20, 35, 36, 39, 51, 52, 54,
55). Thus orexin-containing neurons may be a key component
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Fig. 7. Respiratory activity of rat 5 after application of saline to the spinal cord
(A) and after application of 200 ␮g/ml of orexin to the spinal cord (B).
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OREXIN AND RESPIRATORY CONTROL
in the connection between obesity and sleep apnea. Further
study will be required to investigate this hypothesis.
In summary, hypothalamic orexin-containing neurons are
part of a brain stem-spinal cord respiratory-related network.
Orexin, acting via orexin R1 expressed by pre-Bötzinger neurons and the phrenic nuclei, increases respiratory drive. This
indicates that the orexin-orexin R1 pathway may play an
important role in linking central nervous system nuclei involved in regulation of sleep-wakefulness states with the central nervous system control of respiration. Alterations in this
regulatory system could lead to autonomic dysfunctions and
sleep-related respiratory disorders.
GRANTS
A preliminary account of these findings has appeared in abstract form (53).
This study was supported by National Institutes of Health Grants NINDS 1
U54 NS39407 and NCRR HL-5027 to M. A. Haxhiu and RO1 NSO45859 to
S. O. Mack, and a Howard University Mordecai W. Johnson Research Support
Grant to J. K. Young.
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