1 Department of Pharmacology and Center of Excellence for Neuroscience, Louisiana State University Health Sciences Center, New Orleans 70112; and 2 Neurobiology of Nutrition, Pennington Biomedical Research Center, Baton Rouge, Louisiana 70808
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ABSTRACT |
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Orexins regulate food intake, arousal, and the sleep-wake cycle. They are synthesized by neurons in the lateral hypothalamus and project to autonomic areas in the hindbrain. Orexin A applied to the dorsal surface of the medulla stimulates gastric acid secretion via a vagally mediated pathway. We tested the hypothesis that orexins in the dorsal motor nucleus (DMN) of the vagus regulate gastric motor function. Multibarelled micropipette assemblies were used to administer vehicle, L-glutamate, orexins A (1 and 10 pmol) and B (10 pmol), and a dye marker into this site in anesthetized rats. When the pipette was positioned in the DMN rostral to the obex (where excitation of neurons by L-glutamate evoked an increase in contractility), orexins A and B increased intragastric pressure and antral motility. In contrast, 10 pmol orexin A microinjected into the DMN caudal to the obex (where L-glutamate evokes gastric relaxation through a vagal inhibitory pathway) did not significantly alter gastric motor function. In separate immunocytochemical studies, orexin receptor 1 was highly expressed in neurons in the DMN. Specifically, it was present in retrogradely labeled preganglionic neurons in the DMN that innervate the stomach. These data are consistent with the idea that orexin A stimulates vagal excitatory motor neurons. These are the first data to suggest that orexins in the DMN have potent and long-lasting effects to increase gastric contractility.
brain-gut, hindbrain, gastrointestinal, feeding, motility
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INTRODUCTION |
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OREXINS ARE NOVEL PEPTIDES that are synthesized exclusively by neurons in the lateral hypothalamic area (17). This single population of neurons supplies a widespread distribution of orexins in the brain (12, 13). There are two forms, orexins A and B (also known as hypocretin1 and hypocretin 2), which have affinity for orexin receptors 1 (OR1) and 2 (OR2), respectively.
Orexins are thought to be involved in diverse functions, such as the regulation of the sleep-wake cycle, pain, and arousal; however, many studies have emphasized their role in energy homeostasis. For example, orexin A stimulates feeding when applied to regions of the hypothalamus (5). Orexins of hypothalamic origin have been linked to the dorsal vagal complex (see below). This is where they could integrate energy balance homeostasis with sensory information from the viscera and bloodstream. The vagus nerve conveys both descending influences that prepare and coordinate the gut to receive food and sensory feedback from the gastrointestinal tract about meal size and composition. Integration of vagal afferent-efferent pathways from the gut occurs at the level of the dorsal vagal complex of the hindbrain medulla. This complex comprises the dorsal motor nucleus (DMN) of the vagus, where preganglionic motor neurons innervating the gastrointestinal tract are located, and the nucleus of the solitary tract (NTS), where primary visceral afferents terminate.
The emerging data linking orexins of hypothalamic origin to the dorsal vagal complex are as follows. The terminal field innervation of these orexin-containing cell bodies are nuclei frequently associated with autonomic function (4). Specifically, 15% of orexin A-positive neurons in the lateral hypothalamic area project to the dorsal vagal complex (6). Intracerebroventricular injections of orexins also induce c-Fos expression in the NTS and DMN of the vagus (4). In the latter study, hypoglycemic rats that were fasted showed significantly more c-Fos-positive neurons in the lateral hypothalamus and dorsal vagal complex than fed controls. Interestingly, many of the c-Fos-positive neurons in the lateral hypothalamus contained orexins. These investigators concluded that neurons in the NTS detect the decreasing glucose signal and activate lateral hypothalamic neurons that express orexin.
Although orexins A and B do not stimulate feeding by acting at the level of the dorsal vagal complex (5), there is evidence that orexin A may stimulate feeding-related functions of the gut. For example, orexin A applied to the dorsal surface of the medulla stimulates gastric acid secretion through vagal pathways (20). Although the site of action of this effect is unknown, it is likely to be via an action in the dorsal vagal complex. In addition, acid secretion is temporally and possibly causally linked to gastric contractile activity; however, little is known about the effects of centrally administered orexin A on gastric contractile activity. Therefore, we microinjected orexins A and B into the dorsal vagal complex in anesthetized rats while monitoring indexes of gastric contractile activity. In addition, we used antibodies to the peptides and receptors and immunocytochemistry to determine the location of the endogenous receptor in the dorsal vagal complex that could account for the observed gastric motor effects.
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METHODS |
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General. Male Sprague-Dawley rats (200-390 g) obtained from Charles River Laboratories (Wilmington, MA) were used in all experiments after an overnight fast with ad libitum access to water. The study was approved by the Louisiana State University Health Sciences Center Institutional Animal Care and Use Committee.
Microinjection experiments.
The animals were initially anesthetized with ketamine and xylazine
mixture (36 and 3.6 mg/kg im, respectively), and separate indwelling
cannulas were placed in the left femoral artery and vein. Afterwards,
-chloralose (60-80 mg/kg) was administered intravenously, and a
tracheotomy was performed to connect the animal to a small animal
respirator (Kent Scientific, Litchfield, CT). A laparotomy was
performed, and an intraluminal latex balloon was inserted into the
stomach through an incision in the fundus for recording intragastric
pressure (IGP). The imparting pressure within the intragastric balloon
was maintained at ~5 cmH20 before starting microinjection
experiments in all animals. A small strain gauge (Warren Research
Products, Charleston, SC) was sutured onto the surface of the distal
antral region of the stomach for continuous recording of circular
smooth muscle. Rectal temperature was kept between 37.0 and
37.5°C by radiant heat.
Immunocytochemistry.
Animals were deeply anesthetized by overdose of pentobarbital before
cardiac perfusion with saline followed by 4% paraformaldehyde. The
hindbrains were postfixed and then placed in 20% sucrose at 4°C for
24-48 h. Coronal sections at 40 µm were cut through the medulla
by either vibratome or a cryostat from approximately 1.5 to +2.00 mm
relative to the obex, and the sections were sequentially collected in
four wells of Tris · HCl at pH 7.5. Sections were incubated in
antibodies raised against OR1, OR2, orexin A, or orexin B (all from
Alpha Diagnostic International, San Antonio, TX; 1:1,000-1:4,000)
and stained using a tyramide system amplification kit tagged with
flourescein (New England Nuclear, Boston, MA), a protocol that
amplifies the signal by repeated exposure to biotin-tyramide (3). These antibodies have been well characterized for use in immunocytochemistry. For example, preadsorption of antisera with the
respective immunizing peptides completely abolished staining in rat
central nervous system (antisera to orexin A and OR1; see Ref. 2) and guinea pig enteric neurons (all four
antisera; see Ref. 8). Brains analyzed after staining for
each antiserum were as follows: eight for orexin A, four for orexin B,
eight for OR1, and two for OR2. In some brains, adjacent sections were stained for more than one antiserum, so that 16 brains were used for
final analysis.
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RESULTS |
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Figure 1A is a sample
chart recording demonstrating the gastric motor responses elicited on
microinjection of L-glutamate and 10 pmol orexin A into the
DMN rostral to the obex. Within 20 s after L-glutamate
microinjection, there was a marked but brief increase in antral
contractility and phasic changes in IGP. This is a typical response on
excitation of vagal neurons that increase contractility via cholinergic
postganglionic/enteric neurons. Within 1 min after microinjection of
orexin A at a dose of 10 pmol into the same site, there was a marked
and prolonged increase in antral contractility and phasic increases in
IGP. The location of the microinjection site in this example is within the DMN at the level where the area postrema is prominent (Fig. 2A).
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Whereas a majority of microinjections of orexin A into the DMN evoked
increases in gastric contractility, some were ineffective. Therefore,
we mapped the location of the microinjection sites in terms of the
responses to L-glutamate and orexin A (Fig.
3). Only those sites within the DMN at
the level of the area postrema resulted in increases in response to
orexin A (10 pmol). Indeed, at more rostral sites, there was absolutely
no response to the highest dose of orexin A tested (100 pmol). This
suggests that the response to orexin A is quite site specific and that
the responses obtained are unlikely to be due to the diffusion of
orexin A to adjacent structures. In one case, an ineffective
microinjection of orexin A (10 pmol) was located at the border of the
DMN and NTS. In addition, microinjection of orexin A (100 pmol) into
the midline nucleus raphe obscurus did not result in significant
changes in gastric contractility (change in peak IGP +0.8 ± 0.4 cmH20; antral MMI +1.7 ± 0.8, n = 5).
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The compiled data from the microinjections of orexin A (1 and 10 pmol)
in the DMN at the level of the area postrema are shown in Fig.
4. This figure demonstrates that, similar
to L-glutamate microinjection, there is a significant
increase in peak IGP (orexin A: 1 and 10 pmol) and antral motility
index (orexin A: 10 pmol) into this region of the DMN. In two
additional animals, a dose of 100 pmol microinjected into this region
of the DMN evoked a similar increase in peak IGP (4.3 ± 0.3 cmH2O; P < 0.05 compared with
vehicle). Thus 10 pmol probably evoke a maximal response at
this site. With the use of this dose, the time to peak response is
5.5 ± 1.5 min, and there is a long duration of the gastric effects evoked by these microinjections of 27.2 ± 5.7 min. In three cases, 10 pmol orexin B were microinjected into the DMN rostral
to the obex. This resulted in a peak IGP increase of 3.8 ± 0.7 cmH2O, a motility index increase of 10.3 ± 1.8 MMI,
and the effects lasted for 33-47 min.
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Excitation of gastric motor function evoked by microinjection of orexin A (10 pmol) into the DMN was completely abolished by ipsilateral vagotomy (peak IGP was 3.4 ± 0.7 cmH2O before and 0.2 ± 0 cmH2O after vagotomy, P < 0.05; MMI was 5.8 ± 1.2 before vagotomy and 0.5 ± 0.2 after vagotomy P < 0.05). In a few cases, two doses of orexin A were microinjected into the same sites in some of the animals, after allowing 15-30 min after recovery to baseline before the second injection. We were able to evoke a gastric excitatory response to the second microinjection, and, therefore, it is unlikely that the inability to evoke responses in vagotomized animals was due to tachyphylaxis.
Immunostaining of orexins and their receptors was very consistent in
all brains analyzed, and examples are shown for orexin A, OR1, and
orexin B in the dorsal vagal complex at the level of the area postrema
(Fig. 5, A-C).
Orexins A (Fig. 5A) and B (Fig. 5C)
immunoreactive varicosities were noted throughout the dorsal
vagal complex, with no noticeable subnuclear organization. Immunocytochemical staining of OR1 consistently indicated that the
receptor is highly expressed in the majority of neurons in the DMN
(Fig. 5B). Neuronal cell bodies in the hypoglossal nucleus also express the receptor (not shown). In general, OR2 immunostaining was weaker than OR1, but it was observed within cell bodies within the
DMN of the vagus (not shown).
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Because OR1 was highly expressed in the DMN, we performed
double-labeling experiments to combined OR1 and retrograde tracer (CTB)
injected into the stomach. Figure 6
illustrates these results and shows that OR1 is expressed in the
majority of preganglionic neurons innervating the stomach.
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To further investigate the regional responses to orexin A within the
DMN, we performed additional microinjections into the DMN at the level
of, and caudal to, the obex. Direct excitation of cell bodies by
L-glutamate microinjection in this region decreased IGP (Fig. 7), a response that would be
expected after activation of vagal output to inhibitory
(nonadrenergic-noncholinergic) postganglionic/enteric neurons. However,
microinjections of 10 pmol orexin A into the same sites resulted in
either no response (1 case) or a modest increase in gastric motor
function (Fig. 7). The compiled data, using only those cases in which
microinjection of L-glutamate evoked decreases in IGP,
demonstrated that there was no significant effect of orexin A
microinjected into the caudal DMN (Fig. 4B). A sample chart
recording (Fig. 1B) shows that L-glutamate
microinjection evokes a brief decrease in IGP and slight inhibition of
antral contractility. Orexin A (10 pmol) microinjected into the same site did not decrease intragastric pressure but rather evoked a modest,
though prolonged, increase in phasic IGP oscillations and antral
motility. The site of microinjection in this case is clearly in the DMN
caudal to the obex (Fig. 2B).
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DISCUSSION |
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The major emphasis of the present study was on orexin A, and our results indicated that this peptide evokes prolonged and marked increases in gastric contractility when microinjected into the DMN rostral to the obex. Supportive of this, there is a high level of expression of OR1 in vagal preganglionic motor neurons that project to the stomach. Therefore, one mechanism of action for the excitatory gastric motor effect of orexin A could be via OR1 on vagal preganglionic neurons innervating the stomach. We were not able to visualize OR2 in neurons in the DMN to the same extent as OR1, but it is highly likely that both peptides act on their respective receptors to evoke the observed gastric motor effects. We base this conclusion on the following findings: 1) mRNA for both OR1 and OR2 is present in the DMN and NTS (11); 2) orexin B microinjected into the DMN evoked a marked gastric excitatory effect; and 3) orexin B-immunostained fibers are visualized in this region.
The only other report of the direct effects of orexin A on central vagal circuitry controlling gastric function suggests that dorsal application of the peptide increases gastric acid output (20). The time course of orexin A on the dorsal vagal complex to increase gastric acid is very comparable with the increase in gastric motor function when microinjected directly into the DMN. Our mapping study demonstrates that microinjection of orexin A into the DMN rostral to the obex at the level of the area postrema is the most effective site for the stimulatory gastric effects of this peptide. In the DMN caudal to the obex, where L-glutamate evokes inhibition of gastric motor function, orexin A did not significantly increase motility. However, OR1 expression is evident in DMN neurons throughout the rostrocaudal extent of the nucleus. We cannot explain why activation of postsynaptic OR1 does not seem to be effective in the caudal DMN. Possibly, it is related to the dose of orexin A microinjected; however, increasing the dose microinjected into the caudal DMN would increase the likelihood of diffusion of high concentrations of the peptide into the adjacent commissural NTS and compromise the selectivity of the stimulus.
Preganglionic neurons in the DMN may be functionally divided into a group rostral to the obex, where stimulation results in upper gastrointestinal excitation, and a more caudal area located behind the obex, where stimulation results in upper gastrointestinal muscle (lower esophageal sphincter) relaxation (1, 18). Gastric motor excitation evoked by stimulation of the DMN is abolished by atropine and involves cholinergic preganglionic neurons that synapse onto postganglionic cholinergic neurons. The data presented here illustrate that L-glutamate microinjected into the caudal DMN evokes gastric relaxation. This response probably mediated cholinergic preganglionic neurons that synapse onto postganglionic/enteric NANC motor inhibitory neurons (21). If orexin A were activating postsynaptic OR1 on vagal preganglionic motor neurons in the caudal DMN, then inhibition of gastric motor function would be predicted. In contrast, gastric relaxation was never observed after microinjection of orexin A into this site. Thus our data in the caudal DMN do not support a postsynaptic site of action of OR1. Preliminary data in whole cell patch clamp in a brain stem slice preparation show that OR1 activates inhibitory inputs onto vagal neurons (19). Therefore, a presynaptic site of action of OR1 to influence vagal output is likely, but we cannot determine this based on the techniques used in the present study. However, we can conclude that orexin A is similar to thyrotropin-releasing hormone, a peptide that potently increases contractility in DMN rostral to the obex but has no effect in the caudal DMN, where vagal-NANC inhibitory neurons are located (14).
These data suggest that orexins act on sites in the DMN to promote gastric contractility. This is consistent with the observation that orexin A given centrally increases c-Fos expression in neurons of the DMN (4). The action of orexin A on vagal pathways may contribute to the gastrointestinal changes associated with feeding. For example, similar to hypoglycemia-induced feeding (which is associated with c-Fos expression in the DMN and increased gastric motility), orexins could act on vagal motor neurons to increase both motility and gastric emptying. The result is that the stomach empties faster, and this could contribute to the orexigenic effects of the peptide.
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ACKNOWLEDGEMENTS |
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The investigators acknowledge the support of National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-42714 (to P. J. Hornby) and DK-57542 (to H.-R. Berthoud) for these studies.
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FOOTNOTES |
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* Z. K. Krowicki and M. A. Burmeister contributed equally to this work.
Preliminary reports of this study have been published elsewhere (7, 9).
Address for reprint requests and other correspondence: P. Hornby, Enterology, Johnson & Johnson Research Development L.L.C., Welsh and McKean Roads, Spring House, PA 19477-0776 (E-mail: phornby{at}prdus.jnj.com).
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.
October 10, 2001;10.1152/ajpgi.00264.2001
Received 18 June 2001; accepted in final form 4 October 2001.
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