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NEUROHUMORAL CONTROL OF CARDIOVASCULAR FUNCTION
Departments of 1Pharmacology and 2Medicine (Endocrinology), Georgetown University Medical Center, Washington, District of Columbia
Submitted 6 January 2005 ; accepted in final form 19 July 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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2-adrenergic blockers microinjected into the DMV (n = 7), but not into the NTS, blocked the vagovagal reflex. These data, in combination with our earlier pharmacological microinjection data with nicotine, indicate that both esophageal distension and nicotine produce nitric oxide in the NTS, which then activates noradrenergic neurons that terminate on and inhibit DMV neurons.
vagus nerves; dorsal motor nucleus of the vagus; medial subnucleus of the tractus solitarii; 2-adrenoreceptor; esophageal distension; c-Fos; nitric oxide synthase; central nervous system
The principal second-order neurons of the NTS that engage the third-order DMV neurons have been thought to be GABAergic and glutamatergic (7, 9, 18, 52, 53, 58). In these reports, noradrenergic neurons are never considered as candidates for second-order neurons in the vagovagal reflex pathways. However, this is surprising for several reasons. First, Rea et al. (34) have shown that the DMV exhibits more noradrenergic terminals than any other major nucleus in the medulla oblongata. The origin of these terminals presumably arises from several noradrenergic cell groups, namely, A1, A2, A5, and A6 (23, 41, 46, 48, 56). Second, Sumal et al. (44), report that A2 noradrenergic neurons within the NTS are innervated by terminals from first-order vagal neurons in the nodose ganglion. Third, vagal afferent nerve stimulation has been described as affecting the firing rate of A2 neurons (26). Fourth, electrical stimulation of the A2 noradrenergic cell group evokes inhibitory postsynaptic potentials (IPSPs) in the DMV, which are blocked by the 2-adrenoreceptor antagonist yohimbine (14). This also has been shown to be the case with some spontaneously recorded IPSPs at the DMV (14).
Our interest in the role of noradrenergic neurons in a vagovagal reflex was prompted by results obtained with intravenously administered nicotine showing that this substance also activates a vagovagal reflex, resulting in decreases in intragastric pressure (IGP) and fundus tone in the rat (12). Once we realized that nicotine exerted this effect, we employed this alkaloid as a tool to learn what central nervous system (CNS) neurotransmitters mediated the reflex-induced decreases in IGP and fundus tone. We assumed that nicotine was acting to excite a second-order inhibitory neuron and that the inhibitory neurotransmitter released was either GABA (18, 51, 53) or norepinephrine (14, 26, 34, 44). Blockade of the GABAA receptor at the DMV did not alter nicotine-induced decreases in IGP. However, blockade of the 2-adrenoreceptor at the DMV did abolish most of the response (12). Our study also revealed that for this vagovagal reflex to operate, nitric oxide was required to act as a signaling molecule in the NTS. Our data indicated that the efferent projecting pathway out of the DMV affected by nicotine appeared to be only the cholinergic excitatory pathway. Evidence for this was a lack of any direct excitation by nicotine of peripherally projecting DMV neurons, based on c-Fos expression data (12).
Subsequent to our study showing that norepinephrine and nitric oxide are involved in synaptic signaling in gastrointestinal vagovagal reflex pathway and that NANC neurons are not involved, Rogers et al. (39) published results at odds with our findings. These investigators used esophageal distension to activate a vagovagal reflex and produce fundic relaxation in the rat. Although esophageal distension increased c-Fos expression in NTS neurons immunoreactive for tyrosine hydroxylase (TH), blockade of 2-adrenoreceptors in the hindbrain only partially antagonized the reflex-induced fundic relaxation. To produce a blockade equivalent to that which we observed using yohimbine, they required a combination of yohimbine and the 1-adrenoreceptor antagonist prazosin. Furthermore, suppression of nitric oxide formation in the hindbrain failed to influence this vagovagal reflex. Finally, in contrast to our findings, the NANC pathway was concluded to play a major role in mediating their vagovagal reflex.
The specific purpose of the present study was to employ the same reflex-stimulating technique as Rogers et al. (39), namely, esophageal distension, and to test whether previous results obtained with nicotine also hold true for esophageal distension.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Experiments were performed on male Sprague-Dawley rats (n = 77) weighing 275–400 g (Taconic, Germantown, NY) in accordance with the National Institutes of Health guidelines for use of animals in research and with the approval of the Georgetown University Animal Care and Use Committee.
Before all experiments, with the exception of the c-Fos immunohistochemistry experiments, food was withheld overnight, whereas water was provided ad libitum. Animals were anesthetized with an intraperitoneal injection of a mixture (3 ml/kg) containing urethane (800 mg/kg) and -chloralose (60 mg/kg) dissolved in 3 ml of 0.9% saline. Body temperature was monitored with a rectal thermometer and maintained at 37 ± 1°C with an infrared heating lamp. To minimize brain swelling, we pretreated all animals that underwent neurosurgery with dexamethasone (0.8 mg sc).
Rats were intubated via the trachea to maintain an open airway and to institute artificial respiration when necessary. The carotid artery and the jugular vein were also cannulated with polyethylene tubing (PE-50) for monitoring blood pressure and for systemic infusion of drugs, respectively. Blood pressure was monitored with a pressure transducer that was coupled to a PowerLab data acquisition system (AD Instruments, Colorado Springs, CO).
To monitor intraluminal gastric pressure, an intragastric balloon (made from the little finger of a small latex glove, connected to polyethylene tubing, PE-160) was inserted into the stomach via the fundus and positioned around the corpus/antrum areas. The balloon was inflated (with warm saline, 2–3 ml) to produce a baseline pressure of 6–15 mmHg. This tubing was also connected to a pressure transducer, whose signal was channeled via a quad-bridge amplifier (AD Instruments) onto the PowerLab data acquisition system connected to a G4 Macintosh computer (Apple). Data obtained were analyzed as the decrease in IGP (mmHg) from the baseline value obtained over a 3-min period of time before drug or vehicle microinjection.
For brain microinjection experiments, to gain access to the dorsal medulla, the rats were positioned in a stereotaxic apparatus (David Kopf, Tujunga, CA). A partial dorsal craniotomy was performed to expose the medulla. After retraction of the cerebellum, the underlying dura and pia were cut and reflected. The caudal tip of the area postrema, the calamus scriptorius (CS), was viewed as a reference point for determining the microinjection coordinates.
Brain Microinjection Technique and Histological Verification of Microinjection Sites
Drugs were infused via a double-barrel pipette with an overall tip diameter of 30–60 µm. All microinjections were given bilaterally into either the medial subnucleus of the tractus solitarii (mNTS) or the DMV. Microinjections were administered within 5–10 s in a volume of 60 nl via hand-controlled pressure. Stereotaxic coordinates for injection (pipette was angled at 30° from the perpendicular) into the mNTS were 0.3–0.5 mm rostral to CS, 0.5–0.7 mm lateral to the midline, and 0.4–0.6 mm ventral to the dorsal surface of the medulla. Coordinates for the DMV were 0.3–0.5 mm rostral to CS, 0.3–0.5 mm lateral to the midline, and 0.5–0.7 mm ventral to the dorsal surface of the medulla. These coordinates are similar to those reported in an earlier study (12). In these earlier studies in our laboratory (12, 13), we demonstrated our ability to place microinjected drug into either the NTS or the DMV.
At the end of each experiment, the rat was euthanized with an overdose of pentobarbital. The brain was removed and fixed in a mixture of 4% paraformaldehyde and 20% sucrose for at least 24 h. It was then cut into 50-µm-thick coronal sections and stained with neutral red. The location of the microinjection sites was studied in relation to nuclear groups by using the atlas of Paxinos and Watson (32). Initially, the gross micropipette track was located. The track was then followed under both dark-field and light-field optics to its deepest point and plotted. In most cases, our 
50-µm pipette tip produced a distinct track that could be clearly identified. In rare cases, when the pipette tip could not be located with confidence, data from those experiments were discarded. Camera lucida drawings were performed for each experiment to document all microinjection sites. It must be noted that although our micropipette produced tissue damage along its track, stable responses to L-glutamate microinjection could be elicited that were reproducible.
Esophageal Distension Technique
The description of the following technique applies to all experiments except those involving c-Fos immunohistochemistry. A Fogarty 5-F balloon catheter (Edwards LifeSciences, Irvine, CA) was used to produce stretch of the esophagus. The catheter was placed orally in the thoracic esophagus 1 cm above the esophageal hiatus, as described by Rogers et al. (39). Its location was confirmed by direct visualization at the termination of the experiment. The effects of esophageal distension on gastric tone were tested by distending the balloon with fluid for a duration ranging from 15 to 60 s. In any given experiment, the duration of stretch was kept constant throughout the entire study. For most of the pharmacological microinjection studies, the volume used ranged from 0.6 to 0.8 ml.
c-Fos Immunohistochemistry
For c-Fos studies, male Sprague-Dawley rats, 275–400 g, were housed three to a cage in a temperature-controlled room with a regular light cycle. All rats were allowed to acclimate to the facility for at least 5–7 days on standard rat chow and tap water before experimentation. On the day of study, rats were denied access to food and water from 8:00 AM. Included in this study are brains from rats of a previous study in which intravenous doses of 56.5, 113, and 226 nmol/kg nicotine were administered (12). The purpose for including these brain tissues was for comparing data obtained by activating vagovagal reflexes with nicotine with data obtained by activating vagovagal reflexes with esophageal distension.
For c-Fos studies to be optimal, studies need to be performed in conscious animals, because most anesthetics that have been tested induce c-Fos expression (47). This was possible for our intravenous nicotine experiments (12), but anesthesia was required for investigations of the effects of esophageal stretch, because the stress involved with stretching the thoracic esophagus in conscious animals was assumed to be too great. In preliminary studies, we sought an anesthetic that would not cause a high baseline level of c-Fos activity. This was achieved by administering pentobarbital (70 mg/kg ip) to three rats and perfusing their brains 1 h later for c-Fos immunoreactivity. Three conscious animals were given intraperitoneal vehicle injection and euthanized 1 h later with an overdose of pentobarbital. On visual inspection of c-Fos immunoreactivity in brain stem sections, no apparent difference between the pentobarbital-anesthetized group and the conscious group was noted. On the basis of these data from our preliminary studies, we used pentobarbital-anesthetized rats for testing esophageal distension on c-Fos expression in the hindbrain.
After rats were anesthetized with 70 mg/kg ip pentobarbital, the balloon catheter was inserted orally and placed in the esophagus so that the tip was located 1 cm above the esophageal hiatus in the thoracic esophagus. The balloon was then distended with fluid to produce a catheter diameter of 1.0 cm (0.7-ml volume). Distension duration was for 1 min.
In these experiments, two different protocols were used to distend the thoracic esophagus. One protocol involved distending the thoracic esophagus for 1 min and removing the brain for c-Fos analysis 60 min later. A control consisted of placing the catheter into the thoracic esophagus for 1 min but not inflating the balloon to distend the esophagus, followed by removal of the brain 60 min later for c-Fos analysis. The other protocol involved distending the thoracic esophagus for three 1-min periods. Each 1-min period of distension was separated by a 3-min rest period. Sixty minutes after this 12-min "test" protocol, brains were harvested and examined for the presence of c-Fos. A control consisted of placing the catheter into the thoracic esophagus for 12 min but not inflating the balloon to distend the esophagus. The brain of each animal was removed 60 min later for c-Fos analysis. Data for both protocols were similar and thus were pooled. Visual inspection of the dissected esophagus of control and test animals at the end of the experiment did not reveal any observable injury to the esophagus from either a 1-min-duration balloon distension or a 1-min-duration balloon distension applied three times separated by a 3-min rest period. By using a similar version of the first protocol for drug microinjection studies, relaxation of the fundus could be reproduced by repeated esophageal distension consistent with no injury to the esophagus.
In all of the above studies, 5 days before euthanasia, animals were given intraperitoneal injections of 0.8 mg of Fluoro-Gold (FG; Fluorochrome, Denver, CO) to retrogradely label DMV neurons projecting to the periphery. Of the 12 animals receiving FG, the DMV was analyzed for c-Fos expression in five rats undergoing esophageal distension and three rats serving as controls.
To remove the brain for c-Fos analysis (and for phenotyping neurons, see below), the thoracic cavity was opened, the inferior vena cava was clamped, and an 18-gauge gavage needle was inserted into the apex of the heart and routed to the entrance of the ascending aorta. Five hundred units of heparin were injected into the heart, and the right atrium was punctured to allow drainage. The animal was then perfused transcardially using methods described previously (42). The brains were post-fixed overnight in phosphate-buffered 4% paraformaldehyde and then stored in 25% sucrose until sectioned. Brain stems were cut into sequential 25-µm coronal sections with a freezing stage microtome (Jung Histoslide 2000; Deerfield, IL). The sections were collected into serially ordered sets through the rostrocaudal extent of the DMV so that each set contained 1:6 series of hindbrain sections spaced 150 µm apart. The sections were stored at –20°C in tissue culture dishes containing cryoprotectant (55) until they were processed.
To ensure that the immunohistochemical analyses were representative of the entire extent of the sectioned brain area, each analysis consisted of sections that were cut 150 µm apart (every 6th section). The tissue was processed using methods described previously (42). To identify FG-containing neurons, the same sections were double-stained with an antibody directed against FG (Chemicon, Temecula, CA), diluted 1:70,000 in PBS-Triton X-100. Peroxidase was attached to the antibody as described previously (42), and the presence of peroxidase was detected by incubating with diaminobenzidine and hydrogen peroxide in 0.05 M Tris-buffered NaCl (pH 7.2, 0.15 M). This reaction product was light brown. Throughout the staining procedure, the tissue was rinsed with PBS multiple times after each incubation step. The tissue was mounted on Superfrost Plus slides (Fisher Scientific), air-dried overnight, serially dehydrated in alcohol, cleared in Histoclear, and coverslipped with Histomount (National Diagnostics, Atlanta, GA).
Tissue slices were visualized using a Nikon Eclipse E600 microscope fitted with a linear encoder (type MSA 001-6; RSF Electronics, Rancho Cordova, CA) connected to a digital readout device (Microcode II; Boeckler Instruments, Tucson, AZ), a video camera (DEI-750; Optronics Engineering, Goleta, CA), and a microcomputer running the Bioquant software package (Bioquant Image Analysis, Nashville, TN). Using x10 and x20 objective lenses, the brain regions of interest (NTS and DMV) were outlined by using the brain atlas of Paxinos and Watson (32) as a guide. The numbers of total c-Fos-positive (+) cells in the NTS and the numbers of c-Fos(+) and FG(+) immunoreactive cells in the DMV were counted separately on each section. With the Bioquant software package, each individual immunoreactive cell was marked during the counting process, eliminating the possibility of double counting identified cells. For each animal, all single- and double-labeled neurons were counted from sections that were 1.05 mm rostral and 0.60 and/or 0.90 mm caudal to CS. The total number of positive cells in the area counted was then divided by the number of sections counted, and the result was expressed as c-Fos(+) (NTS) or c-Fos(+) and FG(+) (DMV) neurons per section.
TH and Nitric Oxide Synthase Immunocytochemistry
Alternate sets of c-Fos-labeled sections were further processed for the immunocytochemical localization of TH or nitric oxide synthase (NOS). Sections were rinsed thoroughly in PBS and then incubated for 48 h at 4°C in one of the following antisera: monoclonal mouse anti-TH (Chemicon; 1:50,000) or polyclonal rabbit anti-NOS antibody (Chemicon; 1:50,000). After incubation, sections were rinsed and incubated in the appropriate biotinylated secondary antiserum (anti-rabbit or anti-mouse IgG; Vector) and rinsed in PBS. Sections were then processed as described above, using Vectastain reagents. Cytoplasmic TH or NOS immunoreactivity was detected with unintensified diaminobenzidine. Tissue sections were then rinsed thoroughly in PBS, mounted on glass slides, air-dried, dehydrated through graded alcohols, cleared in Histoclear, and coverslipped with Histomount (National Diagnostics).
Cytoarchitectural and Quantitative Analysis
The Bioquant Tracing System was used to map the locations of immunolabeled cells in tissue sections. First, the distribution of c-Fos(+) cells was plotted in 12 coronal sections (spaced 150 µm apart) through the medulla, beginning at the caudal NTS and extending through the rostral DMV. With a x10 objective, the locations of all visible blue-black c-Fos immunoreactivity profiles were plotted, regardless of labeling intensity. For these plots and for quantitative analysis (see below), TH-immunoreactive (TH-IR) neurons were considered c-Fos(+) if they displayed a blue-black nucleus. Neurons were considered c-Fos negative (–) if they displayed either no nucleus or a nucleus free of blue-black immunoreactivity. The criteria used for plotting (and counting) a neuron as TH-IR or NOS-IR included the presence of brown cytoplasmic immunolabeling, even if the neuronal nucleus was not visible.
The TH(+) and NOS(+) neurons in the NTS were counted, and the percentage of these neurons expressing nuclear c-Fos immunoreactivity was determined. In each case, immunoreactive cells were counted in 12–14 sections from each set of double-labeled material, corresponding to the rostrocaudal levels used for mapping (see above).
Experimental Protocols
Before testing our hypothesis, namely, that CNS NO and CNS noradrenergic neurons are important components of a gastrointestinal vagovagal reflex, namely, the esophageal reflex, it was necessary to establish that esophageal distension-evoked reflex-induced changes involved only efferent vagal excitatory pathways to the stomach (with no sympathetic nervous system component) and also involved the hindbrain. To assess whether the esophageal distension reflex involved the hindbrain, we performed bilateral microinjection of tetrodotoxin (TTX) into the mNTS. Details about these protocols are listed below.
Bilateral cervical vagotomy (n = 6 animals). First, two control responses to esophageal distension were obtained. To ensure reproducible reflex-induced responses, we used a 25- to 30-min interval between reflex tests for this and the following protocols. Acute denervation of the parasympathetic nervous system was performed by exposing and isolating the vagus nerves in the cervical region and sectioning these nerves bilaterally. At least 10 min were allowed after vagotomy before esophageal distension was tested.
Spinal cord transection (n = 6 animals). Acute denervation of the sympathetic nervous system was performed by first placing animals on a ventilator and then making two complete incisions through the exposed spinal cord at the level of the first cervical vertebra. These incisions were made laterally from opposite sides to ensure complete interruption of all descending fibers. An interval of at least 45 min was allowed after transection before the esophageal distension was tested. This interval was needed to ensure that the preparation had stabilized after spinal cord transection.
Intravenous NG-nitro-L-arginine methyl ester (n = 5 animals). In this study, two control responses were obtained for esophageal distension. Twenty-five minutes after the second control response, NG-nitro-L-arginine methyl ester (L-NAME) was administered in a bolus intravenous dose of 10 mg/kg. At the 5- and 30-min time points following L-NAME administration, reflex testing was performed.
Intravenous atropine methylbromide (n = 4 animals). This drug was administered as an intravenous bolus injection of 0.5 mg/kg in four of the five animals that had been evaluated for the effects of L-NAME on esophageal distension-induced gastric relaxation. Ten minutes after atropine methylbromide was administered, esophageal distension was repeated.
TTX microinjection in mNTS (n = 6 animals). For these studies, esophageal distension was performed and the decrease in IGP noted. Distension was repeated using a 10-min interval. Once two stable control responses were obtained, TTX (10 pmol) was microinjected bilaterally into the mNTS. Five minutes later, the esophagus was distended and the decrease in IGP noted.
Once the general neural circuit involved in the esophageal distension-induced reflex was established, we proceeded to the studies of c-Fos expression and phenotyping of c-Fos expression (n = 7 experiments). Parallel studies were performed with intravenous nicotine (n = 5 experiments).
Next, microinjection studies of kynurenic acid (NTS, 6 experiments; DMV, 3 experiments), L-NAME (mNTS, 5 experiments; DMV, 3 experiments); 2-adrenoreceptor blocking agents (DMV, 7 experiments; mNTS, 6 experiments), and bicuculline (DMV, 3 experiments) were performed. Before performing these drug studies, we would locate the target site (either NTS or DMV) by microinjecting 500 pmol of L-glutamate. The NTS location would be reflected by noting a decrease in IGP with L-glutamate, whereas the DMV location would be reflected by noting the opposite response. After, at least two microinjections of 500 pmol of L-glutamate separated by at least a 5- to 10-min interval were tested to show reproducibility of the response.
Drugs
All of the following drugs were purchased from Sigma (St. Louis, MO): -chloralose, atropine methylbromide, nicotine hydrogen tartrate, bicuculline methiodide, kynurenic acid, L-NAME, TTX, urethane, yohimbine hydrochloride, and L-arginine hydrochloride. Dexamethasone was purchased from Elkins-Sinn (Cherry Hill, NJ). SKF 86466 was a gift from Dr. Paul Heible (Glaxo-SmithKline Beecham Pharmaceuticals, King of Prussia, PA). Sodium nitroprusside was purchased from Abbott Labs (North Chicago, IL). All drugs were dissolved in 0.9% saline except yohimbine, which was dissolved in double-distilled water and sonicated for 2–3 min. The pH of drug solutions in microinjection and intravenous studies was 7.0–7.2 and 7.0–7.4, respectively.
Rationale for Drug Doses Used
Information about drug doses and the rationale for selecting specific doses is provided in a previous report (12). This includes information on nicotine hydrogen tartrate, kynurenic acid, L-NAME, L-arginine, yohimbine, SKF 86466, and bicuculline. The dose of TTX used for microinjection into the NTS was the same dose that was effective in an earlier study published by our laboratory (54). The dose of sodium nitroprusside used was the same as used in the study of Ishiguchi et al. (19). The dose of atropine methyl bromide used (0.5 mg/kg iv) was assumed to selectively block muscarinic receptors in the periphery.
Data Analysis
In all cases, statistical analysis was performed on both raw data and percentage of change. Paired sample t-test was performed when animals served as their own controls. Independent-sample t-test was performed on data from separate control and experimental test groups. Comparison between more than two means from different groups of rats was made by two-way ANOVA followed by post hoc analysis using the Newman-Keuls test. Differences were considered significant at P < 0.05. All values are expressed as means ± SE.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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In six neurally intact rats, distension of the thoracic esophagus produced significant decreases in IGP, and a representative experimental tracing from one of these animals is shown as Fig. 1A. The magnitude of the decrease in IGP was linearly related to the volume of distension of the thoracic esophagus (Fig. 1B). A decrease in IGP was noted in some animals when the volume of distension was 0.6 ml and decreased further when the volume of distension was increased in roughly 0.1-ml increments up to 1.0 ml (Fig. 1B). Bilateral cervical vagotomy was performed in six animals, and IGP was monitored during distension of the thoracic esophagus. Data are presented in Fig. 1B and indicate that vagotomy completely prevented esophageal distension from decreasing IGP. Experimental tracings of one representative animal showing the blocking effect of bilateral cervical vagotomy on esophageal distension-induced decrease in IGP are shown in Fig. 1A. Spinal cord transection was performed in six animals, and IGP was again monitored during distension of the thoracic esophagus. Data are summarized in Fig. 1B and indicate that spinal cord transection had no effect on the relationship between esophageal distension and decreases in IGP.
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In a previous study in our laboratory, c-Fos expression was used as a marker of neuronal activation to identify hindbrain sites where intravenous administered doses of nicotine that selectively reduced gastric tone acted (12). These nicotine doses produced significant increases in c-Fos expression in the NTS area compared with the effect of saline vehicle.
Seven animals underwent esophageal distension as described in METHODS, and five animals served as controls (i.e., a catheter was placed in the thoracic esophagus, but the balloon was not inflated). Data from a representative animal of each group are presented in Fig. 4. As shown, esophageal distension produced significant increases in c-Fos expression in the NTS (Fig. 4, distension) relative to the effect of no esophageal distension (Fig. 4, sham). c-Fos expression occurred throughout the rostrocaudal extent of the NTS and generally dorsal to the DMV. The c-Fos-labeled cells were too diffusely distributed throughout the NTS for us to conclude that any particular subdivision was selectively excited. c-Fos data from all animals tested are summarized in Fig. 5 and provide quantitative values for brain sections taken at 150-µm intervals. The profile of esophageal distension-induced activation of neurons indicates that significant excitation occurred from +1.05 mm rostral to CS to –0.6 mm caudal to CS, with the maximal number of NTS neurons activated at the CS.
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We did not observe a significant activation of NOS-IR neurons after nicotine administration (data not shown). This analysis was performed in brain stems of four saline control animals and five nicotine-treated animals. Finally, in regard to the effects of intravenously administered nicotine in doses of 56.5, 113, and 226 nmol/kg on DMV neurons, investigators in our group already reported in a previous study that these doses did not activate FG retrogradely labeled DMV neurons projecting to the stomach (12).
Effect of Bilateral Microinjection of Kynurenic Acid and L-NAME Into mNTS on Esophageal Distension-Induced Decrease in IGP
The protocol used for the TTX experiments was employed for studies of kynurenic acid. Kynurenic acid (100 pmol) was microinjected bilaterally into the mNTS in six experiments and was found to significantly counteract the decreases in IGP produced by esophageal distension (Fig. 9A). Bilateral microinjection of kynurenic acid into the DMV of three animals exerted no significant antagonism of the reflex-induced response (Fig. 9A).
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Effect of Bilateral Microinjection of 2-Adrenoreceptor Blocking Agents and Bicuculline Into DMV on Esophageal Distension-Induced Decrease in IGP
In the first series of experiments, two drugs known to block 2-adrenergic receptors, namely, yohimbine and SKF 86466, were tested at the DMV to determine whether they would counteract the decrease in IGP produced by distension of the thoracic esophagus. In the second series of experiments, a drug known to block the GABAA receptor, namely, bicuculline, was tested at this site. As shown in Fig. 9C, bilateral microinjection of either yohimbine (500 pmol; n = 4) or SKF 86466 (100 pmol; n = 3) into the DMV significantly counteracted esophageal distension-induced decreases in IGP. In contrast, the same dose of each of these agents bilaterally microinjected into the mNTS (n = 3 for each drug) had no effect (data not shown). A representative experiment showing the ability of yohimbine microinjected into the DMV to antagonize the reflex-induced response is shown in Fig. 10. The microinjection sites for yohimbine and SKF 86466 into the DMV and mNTS are depicted in Fig. 10. Data obtained with bicuculline (50 pmol) microinjected into the DMV in three experiments did not counteract esophageal distension-induced decreases in IGP (data not shown).
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DISCUSSION |
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Additional evidence that esophageal distension activates noradrenergic neurons in the NTS that project to the DMV is that microinjection of 2-adrenoreceptor antagonists, namely, yohimbine and SKF 86466, into the DMV counteracted esophageal distension-induced decreases in IGP. Fukuda et al. (14), using a rat brain slice preparation, reported that norepinephrine activated 2-adrenoreceptors located on DMV neurons, which then resulted in inhibition of these neurons. Later confirmation of this result was made by Martinez-Peña et al. (24). This finding, together with our evidence, suggests that esophageal distension activates NTS noradrenergic neurons, causing release of norepinephrine, which then activates 2-adrenoreceptors and inhibits the cholinergic excitatory pathway to the stomach.
Consistent with our findings are data of other investigators providing anatomical and electrophysiological evidence that noradrenergic neurons connect the NTS to the DMV (5, 14, 33, 43). In fact, the DMV has been described as receiving more noradrenergic terminals than any other medullary nucleus (34). We assume that the A2 norepinephrine neurons are the important source of the terminals in our study, but there also is evidence that A6 norepinephrine neurons are another source of these terminals (48). We have singled out the A6 noradrenergic cell group from the noradrenergic cell groups, i.e., A1 and A5, because of the report that excitation of A6 neurons produces vagus nerve-mediated decreases in gastric motility (30). Rogers et al. (39) were the first to demonstrate that noradrenergic neurons in the rat NTS were activated by esophageal distension, and they provided data indicating that these neurons participated in the esophageal-gastric relaxation reflex. Immunohistochemical techniques were used and showed that esophageal distension excited 53% of TH-IR neurons in the central division of the NTS. Excitation was reflected by colocalization of the TH immunoreactivity with c-Fos. This activation of a high percentage of the total number of noradrenergic neurons in the NTS, combined with data showing that pharmacological blockade of 1- and 2-receptors in the dorsomedial medulla counteracted the esophageal-gastric relaxation, provided strong evidence that noradrenergic neurons in the nucleus tractus solitarii are a necessary component of this vagovagal reflex. In our study, we observed far fewer TH-IR neurons that were activated by esophageal distension. The maximal percent of the total noradrenergic neurons excited at a specific distance from the CS was 19.7 ± 2.3%. The reason for this finding probably relates to the differences in the intensity of the esophageal distension stimulus utilized. Rogers et al. (39) used repetitive distension for 90 min, whereas we used a protocol consisting mainly of three 1-min periods of distension. Because of the lower number of TH-IR neurons excited by our method of esophageal distension, we sought further confirmatory evidence for the participation of NTS noradrenergic neurons in the operation of a vagovagal reflex. Our earlier studies indicated that intravenous nicotine activated this reflex, resulting in gastric relaxation (12), and associated with gastric relaxation was an increase in c-Fos in the mNTS neurons. In addition, 2-receptor antagonists microinjected into the DMV blocked nicotine-induced gastric relaxation. This raised the question as to the phenotype of the NTS neurons that exhibited c-Fos after nicotine administration, and particularly whether a significant portion of those neurons were also TH immunoreactive. We therefore felt it was important for the interpretation of these studies to compare the percentage of TH-IR neurons activated by nicotine to our esophageal distension data. We found that nicotine activated the same general percentage of TH-IR neurons (19.7 ± 4.2%) as were activated by esophageal distension and, furthermore, that these activated neurons were localized to a similar rostrocaudal location in the NTS. Together, these data therefore suggest that activation of as little as one-fifth of the noradrenergic neurons in a specific rostrocaudal location of the mNTS is associated with vagovagal-mediated reflex-induced inhibition of the stomach.
Rogers et al. (39) reported that blockade of 2-adrenoreceptors in the hindbrain area with yohimbine (which was applied to the floor of the 4th ventricle) reduced esophageal distension-induced fundic relaxation to 56% of control. Blockade of 1-adrenoreceptors in the hindbrain with prazosin (also applied to the floor of the 4th ventricle) reduced esophageal distension-induced fundic relaxation to 55% of the control, and a combination of the two blockers reduced the distension-induced response to 28% of control. We found that 2-adrenoreceptor blockade elicited a response in our study equivalent to the combination of combined 2-receptor and 1-receptor blockade in the study by Rogers et al. (39). On the basis of our results with 2-receptor blockade produced at the DMV, we conclude that activation of an NTS noradrenergic pathway projecting to the DMV results in synaptic release of norepinephrine that, in turn, activates predominantly 2-adrenoreceptors on DMV neurons. In agreement are the findings of Rosin et al. (40), who described 2C-adrenergic receptor-like immunoreactivity located on the perikarya of DMV neurons of the rat. These 2-adrenoreceptors (2C subtype?) are located on cholinergic excitatory neurons, and activation results in inhibition of vagal outflow. Decreased vagal outflow then leads to relaxation of gastric smooth muscle tone and decreases in IGP. Consistent with our view are the findings of Fukuda et al. (14), demonstrating that excitation of the NTS noradrenergic pathway to the DMV only affects 2-adrenoreceptors on DMV neurons and not 1-adrenoreceptors on the same neurons (presumably, the 1-adrenoreceptor is extrasynaptic). Also in support of sole inhibition of DMV neurons by the NTS noradrenergic projection is the finding that c-Fos expression was not activated in DMV neurons after esophageal distension, similar to previous findings after nicotine stimulation (12).
Our data obtained with bilateral microinjection of kynurenic acid into the NTS demonstrated antagonism of esophageal distension-induced decreases in IGP. These data indicate that L-glutamate acting on ionotropic receptors is involved in mediating the reflex-induced response. It is known from studies of McCann and Rogers (25) that glutamate is released from vagal afferent nerves, which could directly excite second-order NTS noradrenergic neurons.
Because some responses to glutamate are mediated by nitric oxide (15, 50), we evaluated whether this signaling molecule might act as a link between released glutamate and NTS noradrenergic neurons. First, we assessed whether esophageal distension would activate NTS neurons that express NOS. Our c-Fos expression studies combined with NOS immunoreactivity indicated that only a small number of neurons were double labeled with c-Fos and NOS immunoreactivity, which is unlikely to be of physiological significance. Second, we assessed whether microinjection of L-NAME, an inhibitor of NOS, into the mNTS area would counteract the esophageal distension-induced decrease in IGP. The magnitude of the antagonism was equal to the antagonism observed with kynurenic acid and TTX microinjected into the same site. Microinjection of the same dose of L-NAME into the nearby DMV had no effect on reflex-induced decrease in IGP. Microinjection of L-arginine into the mNTS at the peak of the block with L-NAME reversed the L-NAME-induced antagonism. The lack of effect of L-NAME in the DMV and the reversal of the blockade with L-arginine microinjections into the mNTS strongly suggests that L-NAME was acting at the NTS and its blocking effect was due to inhibition of synthesis of nitric oxide.
Results obtained using nicotine to activate a vagovagal reflex were similar to those obtained with esophageal distension. In the present study, intravenous nicotine did not excite NTS neurons that were immunoreactive for NOS. On the other hand, microinjection studies reported previously by our group (12) indicated that L-NAME microinjected into the NTS blocked nicotine-induced effects on IGP and that L-arginine microinjected into the same site reversed the blockade.
To reconcile the findings of a lack of activation of NOS-containing NTS neurons during esophageal distension, as assessed by c-Fos expression, with the ability of L-NAME microinjected into the mNTS to counteract the reflex-induced decreases in IGP, we suggest that glutamate, instead of exciting the cell bodies of NOS neurons, may instead excite NOS-containing nerve terminals in the NTS neuropil. The site where vagal afferent nerves link the esophagus to the NTS is the subnucleus centralis, which is defined by the high density of NADPH-diaphorase-stained terminals and cell bodies within it (22, 31). It is known that presynaptic nerve terminals can serve as a source of nitric oxide and that stimulation of glutamate receptors on terminals can lead to nitric oxide production (28). We speculate that glutamate released from vagal afferent terminals synapsing in the subnucleus centralis activates glutamate ionotropic receptors on NOS-containing terminals. This causes nitric oxide to be formed at the terminals and then released, where it then acts intercellularly to excite nearby noradrenergic projection neurons that synapse in the DMV (Fig. 11). Nitric oxide could also act as a retrograde messenger (27) to evoke additional release of glutamate from nearby vagal afferent terminals (Fig. 11).
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2-adrenoreceptor at the DMV largely abolished the vagovagal reflex, whereas only a partial blockade was observed in the other study; and 2) inhibition of nitric oxide synthesis in the NTS in our study also abolished the reflex, whereas inhibition of NOS in the hindbrain in the other study was ineffective in altering the reflex. These differences may be related to the different methods for applying drugs to the NTS and DMV neurons. In our study using antagonists to the 2-adrenoreceptor and an inhibitor of NOS, these agents were microinjected using a volume of 60 nl into the specific areas of interest (i.e., NTS and DMV). Rogers et al. (39) delivered their antagonist via the floor of the fourth ventricle. Drug applied (microdropped in 2-µl volume) to the surface of the fourth ventricle would have ready access to neurons of the area postrema (AP) and NTS, as well as the DMV. Because 2-adrenoreceptors are present in the AP (4) and both 2-adrenoreceptors and nitric oxide are present in the NTS (10, 11, 49), it is difficult to interpret the data obtained with microdropping the drug in a 2-µl volume onto the dorsal surface of the medulla. The reason given for microdropping the drug onto the brain surface was that microinjection of the antagonist in the DMV would only cover a small portion of the total area of the DMV. Because of their earlier anatomical findings showing that NTS neurons in the ventral commissural nucleus of the tractus solitarii (TS) (36) and the central subnucleus of the TS (35) project throughout the entire anterior-posterior extent of the DMV, it seems reasonable to be concerned that an uninterpretable "negative result" would occur with drug microinjected into locally into DMV tissue. We did not have this concern for several reasons. First, in a previous nicotine study, investigators in our group were successful in obtaining "positive results" with microinjections of antagonist drugs (12). Second, the second-order neuron we appear to be dealing with is noradrenergic, and no data exist that describe the length of DMV that is innervated by noradrenergic terminals. Third, retrograde tracing studies show that the density of DMV neurons that project specifically to the fundus in the rat is greatest at the site of our microinjection and drops off precipitously in rostral and caudal areas of the DMV (29).
The other major difference between our findings and those of Rogers et al. (39) is that our data led us to conclude that the NANC vagal pathway plays no role in mediating esophageal distension-induced gastric relaxation. The data that supports this position are 1) intravenously administered L-NAME had no effect on distension-induced decrease in IGP (Fig. 2); and 2) an absence of c-Fos expression in DMV neurons that project to peripheral structures. Rogers et al. did not evaluate L-NAME against esophageal distension-induced gastric relaxation. In addition, they did not address whether gastric projecting DMV neurons exhibit c-Fos after distension of the esophagus. Instead, their evidence for involvement of a NANC pathway is indirect, consisting of data obtained by microdropping prazosin onto the floor of the fourth ventricle. They suggest that 1 antagonism blocks the direct postsynaptic effects of norepinephrine to activate the NANC component of DMV projections to the stomach. This suggestion is at odds with the findings of Fukuda et al. (14), who reported that excitation of the NTS noradrenergic pathway to the DMV only affects 2-adrenoreceptors on DMV neurons and not 1-adrenoreceptors. Other differences between the two studies were that of anesthesia and species of rats. In the study by Rogers et al., experiments were performed on thiobutabarbital-anesthetized Long-Evans (Charles River) rats, whereas our studies were carried out on urethane-chloralose- and/or pentobarbital-anesthetized Sprague-Dawley (Taconic) rats.
It should be noted that our study has several potential limitations, but these limitations, in our opinion, do not impact adversely on our findings. One limitation is that our experimental studies were conducted under three different experimental conditions. The functional studies were carried out using a mixture of urethane and -chloralose. The c-Fos response to esophageal distention studies was carried out using pentobarbital. The reason for this is that anesthesia had to be used because the stress involved with stretching the thoracic esophagus in conscious animals was assumed to be great (see METHODS). Pentobarbital was used because urethane induces c-Fos expression (47). The nicotine studies were carried out in conscious rats because the minor experimental intervention did not put the animals under duress. Even though the experimental interventions differed, the c-Fos expression evoked by esophageal distension and intravenous nicotine were of similar magnitude and pattern in the NTS, indicative of roughly equivalent reflex-induced activation of this part of the hindbrain. Furthermore, Rogers et al. carried out functional studies in barbiturate-anesthetized animals, specifically, thiobutabarbital-anesthetized rats, and observed both c-Fos in the NTS and esophageal distension-induced gastric relaxation (39). In their earlier study (35), Rogers et al. used rats anesthetized with urethane in a dose that was described as producing "a deep plane of anesthesia," and these animals exhibited gastric relaxation with esophageal distension. Thus deep anesthesia does not abolish this vagovagal reflex. Another potential limitation is the weight we have placed on the c-Fos data obtained in the DMV neurons that project to the periphery. We have argued that the lack of c-Fos expression in these neurons during esophageal distension and nicotine administration indicates that DMV neurons are not excited by these two interventions. However, as pointed out in an earlier review (17), "absence of c-Fos expression does not necessarily mean that stimulation did not occur." It should be pointed out, however, that our c-Fos methodology clearly is able to detect excitation of DMV neurons that project to the stomach. That is, we recently published results showing excitation of DMV neurons based on c-Fos expression after insulin-induced hypoglycemia (42). Furthermore, we also reported that cholecystokinin given intravenously also activates DMV neurons as determined by c-Fos expression (42). Interestingly, DMV neurons expressing c-Fos after cholecystokinin did not project to the stomach.
A final potential limitation and/or concern is that although intravenous L-NAME had no effect on esophageal induced gastric relaxation, microinjection of L-NAME into the mNTS did counteract this reflex-evoked response. Presumably, when L-NAME is administered intravenously in a dose of 10 mg/kg, the concentration of drug within the mNTS never rises to that attained with local microinjection of the drug.
In summary, on the basis of our present findings, we speculate that the scheme depicted in Fig. 11 represents the pathways and CNS signaling molecules by which esophageal distension and systemically administered nicotine evoke their effects on gastric motility. Both stimuli release glutamate in the NTS, with events initiated at sites 1 (distension) and 2 (nicotine activating a 4
2-nicotinic acetylcholinergic receptor; Ref. 12). Glutamate, in turn, activates an ionotropic receptor on NOS-containing terminals in the NTS, and nitric oxide acts to excite second-order noradrenergic neurons projecting to the DMV. Norepinephrine is released and activates 2-adrenoreceptor on cholinergic excitatory neurons, causing them to hyperpolarize. This reduces ACh release at the ganglion and at the postganglionic parasympathetic nerve terminal. The subsequent reduction in stimulation of muscarinic postsynaptic receptors on gastric smooth muscle decreases IGP. Evidence that the esophageal distension reflex is a vagovagal mediated reflex was obtained by demonstrating that vagotomy but not spinal cord transection abolishes the decrease in IGP. Evidence that esophageal distension-induced decreases in IGP involves only inhibition of the cholinergic excitatory pathway and that the NANC inhibitory pathway likely plays no role was obtained from three types of experiments. First, atropine methylbromide completely blocked the end-organ response. Second, L-NAME given intravenously in a dose that others have shown blocks the NANC pathway (19, 45) had no effect on the end-organ response. Third, esophageal distension, while exciting neurons in the NTS area based on c-Fos expression, had no effect on c-Fos expression in DMV neurons that project to the periphery.
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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.
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), or saline (
) in the left and right NTS. At right are injection sites where 
) or DMV (
). Note that, for the sake of clarity in presentation, right injections sites for TTX, Kyn, L-NAME, L-ARG, or saline are shown together with left injection sites in the left NTS and DMV. Similarly, left microinjection sites for 
