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J Appl Physiol 98: 1387-1395, 2005. First published November 19, 2004; doi:10.1152/japplphysiol.00914.2004
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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

1Howard 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


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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 orexin-containing 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.

hypothalamus; pre-Bötzinger region; phrenic motor neurons; orexin-1 receptors; sleep apnea

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 protein-coupled 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 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 orexin-containing neurons that are involved in arousal also participate in respiratory control via both direct projections to the pre-Bötzinger complex and phrenic motor neurons.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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Animals

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-µm-thick frozen sections of brains were prepared by using a Leitz microtome.

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 false-positive 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-µm 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 hypoglossal nuclei, and within the anatomic borders of the pre-Bötzinger 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 µm. 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.



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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 µm. 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 µm. D: higher magnification view of neurons of the nucleus ambiguus that are immunoreactive for orexin receptors. Calibration bar = 50 µm.

 
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 µl 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 µg/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 pre-Bötzinger 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 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
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Neuroanatomic Experiments

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).



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  		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 (rhodamine-labeled 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 µm; F, fornix.

 
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-labeled cells were examined; only one cell was double labeled. However, orexin-containing fibers were observed in the pre-Bötzinger 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 pre-Bötzinger 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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  		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 µm. 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.

 
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 µg/ml and for 200 µg/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 pre-Bötzinger 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 µm; no dye could be detected farther than 500 µm away from the center of the infusion site.



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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 µg of orexin into the ventrolateral medulla were significantly different from the responses to saline or to 4 µg/ml of orexin (*P < 0.05, protected t-test). B: breathing rates during the 5 treatment conditions.

 
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., 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.


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  		Table 1. Cardiovascular variables following microperfusion of test solutions into the pre-Bötzinger complex of the rostral ventrolateral medulla

 


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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 µg/ml of orexin into the medulla (B). {int}DEMG, integrated electromyographic activity of the diaphragm.

 
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 µg/ml and P < 0.01 for 200 µg/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 µg/ml, P < 0.01 for 200 µg/ml of orexin) (Table 2). A recording from a single rat that is representative of these mean results is shown in Fig. 7.



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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 µg/ml of orexin onto the spinal cord were significantly different from the responses to saline or to 4 µg/ml of orexin (*P < 0.05, **P < 0.01, protected t-test). B: breathing rates during the 5 treatment conditions.

 

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  		Table 2. Cardiovascular variables following microperfusion of test solutions into the phrenic motor nucleus of the spinal cord

 


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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 µg/ml of orexin to the spinal cord (B).

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
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 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 preganglionic 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-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 pre-Bötzinger 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 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).

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 signaling 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 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
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
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 NSO45859to S. O. Mack, and a Howard University Mordecai W. Johnson Research Support Grant to J. K. Young.


   FOOTNOTES
 

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: jyoung{at}howard.edu)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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