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1Department of Pediatrics, Kosair Children's Hospital Research Institute, Departments of 2Physiology and Biophysics, 3Medicine, and 4Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, Kentucky 40202; and 5Department of Physiology, Loyola University Chicago, Stritch School of Medicine, Maywood, Illinois 60153
Submitted 21 April 2003 ; accepted in final form 30 October 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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68% (P < 0.01) and attenuated baroreflex sensitivity by 83% (P < 0.01) at 15 days. Similarly, electrical stimulation of the vagal trunk of DA-lesioned animals led to attenuated decreases in HR responses. NA neuronal counts were reduced by 81% (P < 0.01) and mean HR responses to L-glutamate injection into the lesioned NA were attenuated by 65% (P < 0.01) at 180 days. Therefore, the NA plays a major role in baroreflex control of HR, and the integrity of the NA is critically important for the normal baroreflex control. In addition, NA lesions produce long-term anatomic and functional dysfunction of the nucleus, and thus it may provide an useful model for functional assessment of respective roles of the NA and DmnX. excitotoxin; brain stem; parasympathetic; vagal efferent; cardiac ganglia
Because antidromic stimulation of intrathoracic cardiac nerve branches activated more NA neurons than DmnX neurons (21, 22) and because injection of the retrograde tracers horseradish peroxidase, cholera toxin-horseradish peroxidase, or 1,1-dioleyl-3,3,3',3'-tetramethylindocarbocyanine (DiI) into cardiac tissues labeled much more NA neurons than DmnX neurons (9, 14, 23), the classic model assigned the major role to the NA in controlling the heart. In contrast, the DmnX was considered as the nucleus that primarily mediated gastrointestinal functions and only played a minor role in cardiac functions (16, 19). However, using anterograde tracing and confocal microscopic techniques, we recently demonstrated that the DmnX and NA both project strongly to all cardiac ganglionic plexuses and generate extensive, but no overlapping, basket endings around ganglionic principal neurons, suggesting that both may play important roles in cardiac functions (5, 6, 8). Indeed, several lines of physiological evidence support this latter concept: 1) selective stimulation of vagal efferent B fibers (presumably from the NA) and vagal C fibers (presumably from the DmnX) evokes bradycardia, atrioventricular block, and reduction of cardiac contractility (10, 17, 18, 31); 2) stimulation of the DmnX elicits bradycardia and reduces myocardial contractility (3, 11, 26); 3) activation of the NA induces negative chronotropic, dromotropic, and inotropic effects (1, 2, 20, 29); and 4) both DmnX and NA neurons are barosensitive (32). However, these anatomic and physiological data still do not necessarily imply that the integrity of the NA or DmnX is necessary or sufficient for baroreflex control, nor do they support that the two nuclei play an equal role in baroreflex.
To study the roles of DmnX, we recently made bilateral lesions of the dorsal vagal complex with domoic acid (DA) and found that baroreflex sensitivity, i.e., HR responses to blood pressure changes induced by intravenous administration of the vasoactive drugs phenylephrine (PE) and sodium nitroprusside (SN) was not significantly affected at 15 days, despite a reduction of 90% in the DmnX neuronal population (4). On the basis of these findings and the markedly distinct anatomic projections of the NA and DmnX to cardiac ganglia (5, 6, 8), we hypothesized that the NA was the critical nucleus underlying baroreflex control of the HR.
To test this hypothesis, we lesioned the NA by appling DA, a potent neurotoxin that acts via glutamate receptors. We assessed the loss of the NA motoneurons and tested the importance of the integrity of this nucleus in baroreflex control of the HR during PE and SN infusion. Because a permanent damage of NA may provide a new strategy to differentiate the roles of the NA and DmnX in controlling the heart, we further examined functional reduction and anatomic loss of NA cardiac motoneurons at 180 days post-DA lesions.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Injections of DA into the NA. Animals were anesthetized with pentobarbital sodium (60 mg/kg ip) and artificially ventilated with oxygen-enriched room air. The surgical procedure to expose the brain stem was identical to that previously described (5, 8). Briefly, animals were placed in a stereotaxic instrument equipped with a head holder adapted to permit the neck to be sharply flexed. A dorsal incision was made over neck muscles, which were retracted to expose the atlanto-occipital membrane. The membrane was opened with an incision, exposing the cisterna magna and the dorsal medulla. The occipital bone was trimmed with the bit of a dental drill until the caudal cerebellum was visible. The caudal end of the area postrema was used as a rostrocaudal reference for stereotaxic coordinates. A glass micropipette (inner diameter: 10–20 µm) was filled with DA and then advanced into the NA by using a micromanipulator. DA (0.75 mM) was instilled unilaterally in small aliquots (2.5–12.5 nl each) at nine different sites (left; rostrocaudal: -1,600–1,600 µm; lateral: 1,300–2,100 µm; ventral: -1,800 to -2,200 µm; total volume: 22.5–112.5 nl), separated by 400 µm longitudinally along the NA. The amount of DA injected into the NA was limited to 0.75 mM, because higher doses were lethal. Initial experiments were conducted, whereby bilateral injections to the NA were performed. However, this approach produced seizures followed by death in all animals. Therefore, it was abandoned in favor of the unilateral NA injection approach. After injections, the surgical wounds were closed. Animals were then returned to their cages, injected intraperitoneally with 2.5 ml of normal saline, and provided with free access to water and rat chow. Control animals received vehicle saline injections within NA.
Labeling of vagal motoneurons with tetramethylrhodamine dextran: examination of DA lesions in the brain stem. Retrograde labeling technique was used to label NA and DmnX motoneurons, and anterograde tracing technique was used to label peripheral afferent terminals in the nucleus of the solitary tract (NTS). Tetramethylrhodamine dextran (TMR-D; 7%, molecular weight of 3,000, catalog number D-3308, Molecular Probes) is an anterograde as well as a retrograde tracer. Because the nodose ganglion and petrosal ganglion are very close to each other and form the vagal-petrosal complex in rats, we injected TMR-D into the nodose ganglion to label both vagal motoneurons in the NA and DmnX, and vagal and glossopharyngeal afferent terminals in the NTS.
Animals (vehicle and DA treated) were anesthetized with pentobarbital sodium (50 mg/kg ip) and injected with atropine sulfate (1 mg sc). A midline incision was made along the neck, and ventral neck muscles were gently separated by blunt dissection to expose the nodose ganglion medial to the internal carotid artery (7). Multiple injections of TMR-D (total dose of 500 nl) were made into the left nodose ganglion through a micropipette under continuous visual inspection with a surgical microscope and a picospritzer (54 lbs. of pressure, 4 ms, 20- to 40-µm inner diameter micropipette). After each injection, the micropipette was left in place for 1 min before being withdrawn to reduce dye leakage of the micropipette track. After completion of all injections, the surgical wound was closed with sutures and the animal was returned to its cage.
After a survival period of 7 days to allow for tracer transport to the brain stem, each animal was anesthetized with an overdose of pentobarbital sodium (100 mg/kg) and perfused through the heart with 0.9% saline (300 ml) and phosphate-buffered (pH 7.4, 600 ml) 10% formalin. The brain stem and the nodose-petrosal ganglion complex were removed. Each brain stem containing the entire NTS, DmnX, and NA was stored in 15% sucrose formalin overnight and sectioned transversely at 100 µm by using a cryostat on the second day. All tissues were then dehydrated through a graded series of ethanol rinses (70%, 2 min; 90%, 2 min; and 2 x 100%, 1.5 min/each). Finally, the tissue was mounted and coverslipped in Cytoseal XYL.
Serial brain stem sections containing the NA, DmnX, and NTS were examined systematically and completely by using epifluorescence and confocal microscopes to determine the loss of NA neurons and the effects of DA treatment on the DmnX and NTS. Brain stem sections of vehicle and DA-treated animals were scanned, the total number of the TMR-D-labeled NA neurons were counted, and confocal projection images were made to display neural degeneration.
Baroreflex measurement. DA-injected and vehicle-treated rats were reanesthetized at day 15 after the lesion, and indwelling catheters (PE-50; 0.56-mm inner diameter, 0.88-mm outer diameter) were introduced into the femoral artery and vein. Animals were initially anesthetized (50 mg/kg), and additional pentobarbital sodium (10 mg/kg) was administrated as needed. Recordings were conducted while animals were under anesthesia, as evidenced by the absence of paw withdrawal reflex to a pinch of the paw. Rectal temperature was monitored and maintained at 37.5°C with a Harvard homeothermic blanket. Animals were artificially ventilated with oxygen-enriched room air. To examine whether DA microinjections into the left NA were associated with a baroreceptor reflex sensitivity reduction, the right vagus nerve just distal to the nodose ganglion was transected to eliminate the baroreflex effect via the contralateral vagus. Thus any baroreflex was due to the left vagus plus sympathetic innervation.
For determination of baroreceptor function, the vasoactive drugs PE and SN were administered by using a microinfusion pump (solution concentration of 100 µg/ml; infusion rate: 10, 20, and 30 µl/min iv). Each infusion lasted 2 min. Mean steady-state values of the arterial blood pressure (ABP) and HR during the 20 s preceding each dose administration were considered as baseline values for ABP and HR. At least 15 min were allowed after PE infusion and 30 min after SN infusion for the hemodynamic variables to have time to return to the baseline values. In all animals, ABP was measured from the arterial line connected to a calibrated pressure transducer. The blood pressure was visually monitored and displayed in the first channel of the Powerlab Data Acquisition System. HR was calculated in beat-by-beat mode and displayed in the second channel that was synchronized with the blood pressure in the first channel. The analog signals were digitized and processed by using Chart 4.4 software.
First, the baseline values of ABP and HR were measured as the average of the ABP (mABP) and HR during the 20-s period before drug administration. At the end of the 2-min drug infusions, both blood pressure and HR changes had already reached a stable plateau. All responses returned to the previous baselines after the termination of drug infusion. The average changes in mABP and mean HR during the last 20 s of the stimulation period, relative to the baseline values of ABP and HR, were measured. The ratios of change in HR over change in mABP were then calculated and averaged for all doses of each drug. The average ratios of HR change to mABP change for PE and SN were used as the indicators of baroreflex sensitivity. The averaged baroreflex sensitivity across DA-treated animals was calculated and then compared with the sensitivity of controls. Values were reported as means ± SE, and t-tests were used to compare differences between groups.
Electrical stimulation of the vagal nerve. To determine the effect of the DA lesion on functional reduction of NA cardiac axons, the left vagal nerve was transected distal to the nodose ganglion. The vagal nerve distal to the cut was stimulated by a pair of bipolar electrodes with the conventional arrangement, i.e., the cathode was near to the heart. Square pulses (0.5 mA, 1 s) were generated with a stimulator (S48K, Grass Instruments) at frequencies of 0.1, 0.5, 1, 5, 10, 20, and 30 Hz for 30 s. A stimulus isolation unit (PSIU6, Grass Instruments) was used to provide the nerve with steady current during stimulation. Interstimulus interval was 5 min. HR and blood pressure baseline values were determined from the average values during the 20 s before stimulation. Mean responses of the HR and blood pressure were calculated from the 30-s stimulation period. HR and blood pressure returned to the steady baselines after stimulation. Response curves of the HR and blood pressure were plotted against corresponding stimulus frequency.
Pharmacological blockade. To determine the baroreflex component contributed by the sympathetic system and to test whether DA injection into the NA region also lesioned sympathetic neurons in the brain stem, we transected vagal trunks bilaterally distal to the nodose ganglion in another group of vehicle-treated animals to eliminate vagal efferent drives to the heart (n = 6). Baroreflex sensitivity was then measured as described above. An alternative approach to eliminate the vagal efferent effect consists of administration of atropine to block the parasympathetic pathway. However, because we had to transect the right vagus to study baroreflex sensitivity in the animals whose left NA had been lesioned, we considered bilateral transecting vagus as a preferred control compared with atropine treatment. Animals were then pretreated with propranolol [4 mg/kg iv at a volume of 0.1 ml/injection through the femoral vein (15)] to block sympathetic inputs to the heart. Ten to fifteen minutes after treatment with propranolol, the HR reflex elicited by PE was completely abolished, confirming that the response was due to sympathetic efferents.
To further examine whether HR and blood pressure responses to electrical stimulation were elicited by parasympathetic activation, methylatropine [3 mg/kg iv in 0.1 ml (15)] was administrated in these animals, and 10–15 min later the vagal nerve was stimulated at 20 Hz. HR and blood pressure responses were completely abolished, confirming the specificity of the parasympathetic stimulation protocol.
Microinjection of L-glutamate into the NA. To study the long-term effect of DA lesion on the functional loss of NA neurons, cardiovascular responses to microinjection of L-glutamate (L-glu) into the lesioned NA were performed at 180 days postlesion (n = 9). The physiological conditions were maintained as described above. Before each experiment, the right cervical vagus was cut to block the right vagus effect. Anesthetic and pharmacological drugs were delivered through different catheters in the left and right femoral veins. L-glu (10 mM/30 nl) was microinjected into the left NA at the level of the area postrema where, in preliminary experiments, repeated injections reliably induced maximal bradycardia and hypotension. The injection sites for the maximal responses were experimentally determined by injecting the different sites along and around the NA at the level of the area postrema. Microinjections were made in 1 s with a pneumatic microinfusion pump. The volume delivered was controlled by monitoring the displacement of the meniscus in the pipette through the microscope fitted with an eyepiece graticule. Saline was injected at the same site to provide a vehicle control. Repeated injections into the same site were possible because the small mark produced by the previous injection was identifiable by using a surgical microscope, and repeated penetration did not cause tissue depression. Across animals, injection sites were comparable because similar coordinates were used, and DiI was delivered into injection sites to leave markers for verification of L-glu injection sites. The left vagus was then injected with Fluoro-Gold to retrogradely label NA neurons. Five days later, animals were perfused, brain stems were removed and sliced, and injection sites were examined by using a confocal microscope as described in Refs. 5 and 8. Those animals whose injections were out of target boundaries were not included in the analysis. The average changes in HR and blood pressure relative to the prestimulus baselines were measured. Data were presented as means ± SE, and statistical differences were assessed by t-tests. Differences were considered significant at P < 0.05.
In another group of control animals (n = 6), the 
1-adrenoceptor antagonist atenolol (1 mg/kg iv in 0.3 ml) was first injected, and then the muscarinic receptor antagonist methylatropine (3 mg/kg iv, 0.1 ml) was administered to study sympathetic and parasympathetic components in HR responses to L-glu injection.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Attenuation of baroreflex sensitivity at 15 days post-DA injections into the left NA. Before recording, the right cervical vagus was transected. Mean blood pressure baselines [control (n = 9): 125.5 ± 5.3 mmHg; DA (n = 12): 125.7 ± 5.8 mmHg; P > 0.05] and mean baselines of HR [control (n = 9): 345.4 ± 18.8 beats/min; DA (n = 12): 333.4 ± 18.beats/min; P > 0.05] were comparable. However, baroreflex sensitivity of DA-treated animals was substantially reduced as shown in Fig. 2. Mean baroreflex sensitivities at 15 days post-DA lesions for PE and SN were -0.14 ± 0.05 and -0.11 ± 0.04 beats·min-1·mmHg-1 (n = 12), respectively, whereas the baroreflex sensitivities measured in saline-treated animals (n = 9) were -0.72 ± 0.07 and -0.73 ± 0.11 beats·min-1·mmHg-1, respectively (P < 0.001). Therefore, DA-induced lesions in the NA reduced baroreceptor-mediated control of the HR by 83%.
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It should be emphasized that the baroreflex sensitivity measured above reflected activity for the left vagus and the sympathetic system. To evaluate the sympathetic baroreflex, we also measured sympathetic baroreflex sensitivity in another six control animals after bilateral vagotomy. Figure 3 is an example from one such animals. Averaged sympathetic baroreflex sensitivities were -0.30 ± 0.06 beats·min-1·mmHg-1 in PE and -0.26 ± 0.08 beats·min-1·mmHg-1 in SN. Complete abolition of such responses occurred 15 min after treatment with the -blocker propranolol (PE: +0.01 ± 0.05 beats·min-1·mmHg-1; SN: -0.02 ± 0.04 beats·min-1·mmHg-1; P < 0.01, n = 6), thereby confirming that these responses were due to the sympathetic system. Assuming that both left and right sympathetic nerves equally mediate the baroreflex, the unilateral sympathetic baroreflex sensitivity would be -0.14 beats·min-1·mmHg-1. Therefore, the baroreflex mediated by the sympathetic nerve is 19%, whereas the vagal nerve contributes the majority of the reflex responses, i.e., 81%. Consistent with the literature, our data indicate that baroreflex control of the HR is essentially through vagal nerves. Of note, the baroreflex mediated by sympathetic nerves is much slower than vagally mediated baroreflex (Figs. 2A and 3A).
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It should be pointed out that the animals used for examination of degeneration of brain stem neurons were not used for the physiological testing because tracer injection into the no-dose ganglion significantly reduced baroreflex sensitivity (Z. Cheng, D. Zhang, and H. Gozal, unpublished observation).
Reduction of the HR and blood pressure responses to electrical stimulation of the vagal trunk at 15 days post-DA injections into the left NA. At baseline, blood pressure for saline-treated (n = 6) and DA-treated (n = 6) animals was similar (118.0 ± 7.4 mmHg in controls vs. 117.0 ± 10.1 mmHg in DA-treated rats; P > 0.05), as were their mean HR (361.8 ± 17.7 beats/min in controls vs. 335.3 ± 20.7 beats/min in DA-treated rats; P > 0.05). Of note, the right cervical vagus had been transected before physiological recordings were initiated.
In six DA-treated animals, in which mean baroreflex sensitivity was -0.10 ± 0.05 beats·min-1·mmHg-1, the distal end of the vagal trunk ipsilateral to the DA injection site was electrically stimulated for 30 s, and this induced a significant reduction of HR and blood pressure. However, HR and blood pressure responses of DA-treated animals were significantly attenuated compared with controls (Fig. 4). Because the baroreflex responses were very small in these animals (see Fig. 2B), responses to electrical stimulation were most likely due to the DmnX axonal innervation of the heart. Figure 4A is an example showing that electrical stimulation (0.5 mA, 1 ms, 20 Hz) of the distal end of the left cervical vagus induced large responses of HR and blood pressure in a control animal. In contrast, Fig. 4B shows that responses of HR and blood pressure to the electrical stimulation (0.5 mA, 1 ms, 20 Hz) of the vagus in the DA-treated animal were substantially attenuated. When HR and blood pressure response curves were plotted against stimulation frequency, significant shifts to the right emerged in DA-treated animals (Fig. 5). Therefore, consistent with the anatomic findings, DA effectively degenerated NA cardiac efferent axons and reduced the vagal control of the heart. In addition, the residual HR responses could be due to functional projections from DmnX.
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To further evaluate whether the HR and blood pressure responses to electrical stimulation were due to parasympathetic activity, saline-treated animals (n = 6) were pretreated with muscarinic blocker methylatropine. This led to the complete disappearance of HR and blood pressure responses (vehicle control: -215.5 ± 18.2 beats/min, -62.2 ± 7.7 mmHg; methylatropine: -4.4 ± 2.6 beats/min, 2.2 ± 2.4 mmHg). Figure 6 shows such an example. These findings indicate that responses evoked previously by electrical stimulation were indeed due to vagal cardiac efferents.
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Long-term NA dysfunction (180 days post-DA lesion). To determine whether DA lesions of the NA lead to a long-term dysfunction of the nucleus, we microinjected L-glu into the left NA and examined the HR and blood pressure response to L-glu 180 days after the DA injection. Again, the right vagus was transected before recording. Mean blood pressure baselines (vehicle control: 120.5 ± 4.3 mmHg; DA: 124.6 ± 6.1 mmHg; P > 0.05, n = 9) and mean baselines of the HR (vehicle control: 334.2 ± 17.1 beats/min; DA: 330.2 ± 19.3 beats/min; P > 0.05, n = 9) were comparable. However, the averaged HR and blood pressure responses to L-glu injections at 180 days were significantly reduced by 65 and 67%, respectively [HR: -155 ± 14.8 beats/min (control) vs. -54 ± 15.2 beats/min (DA); blood pressure: -40 ± 6.2 mmHg (control) vs. -13 ± 4.1 mmHg (DA); P < 0.01, n = 9]. Figure 7A shows responses in one rat, whereas Fig. 7B indicates that DA injection was restricted to the NA region.
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It should be pointed out that the HR and blood pressure responses to L-glu injections into the NA were due to activation of both parasympathetic and sympathetic neurons. However, the responses were primarily mediated by parasympathetic stimulation, because injection of 1-adrenoceptor antagonist atenolol (1 mg/kg) only mildly lowered HR and attenuated the HR response to the L-glu stimulation, whereas subsequent injection of the muscarinic receptor blocker methylatropine (3 mg/kg iv) completely eliminated responses. As shown in Fig. 8, the baselines of HR and blood pressure declined by 53 beats/min (from 309 to 256 beats/min) and by 12 mmHg (from 126 to 114 mmHg), respectively, after atenolol, and the magnitude of the HR and BP responses to L-glu injection was reduced by -53 beats/min (from -182 beats/min before atenolol to -129 beats/min after atenolol) and-7 mmHg (from -53 mmHg before atenolol to -46 mmHg after atenolol), respectively. Because the averaged HR response to L-glu injection after atenolol (-125 ± 19.2 beats/min; n = 6) was still significantly much larger than that of the DA-treated animals even without atenolol administration (-54 ± 15.2 beats/min; n = 9; P < 0.001), we conclude that the injection of DA into the NA leads to a long-term dysfunction of this region.
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Loss of NA motoneurons at 180 days after DA injections. The number of the NA motoneurons at 180 days post-DA injection was still significantly reduced compared with saline controls but was not significantly different from the number of NA neurons at 15 days. Therefore, DA injection appears to eliminate NA motoneurons permanently (Fig. 9).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Anesthesia. We have used pentobarbital sodium to anesthetize animals. Because baroreflex sensitivity is affected by anesthesia and different anesthetic drugs may impose different degrees of inhibition on baroreflex, the baroreflex sensitivity obtained from pentobarbital sodium-anesthetized animals was smaller than that obtained from unanesthetized animals. Because the pentobarbital sodium anesthesia was used to examine baroreflex sensitivities in vehicle- and DA-treated animals, the type of anesthetic used should not have affected our findings that DA lesion of the NA reduced the baroreflex.
Elimination of NA motoneurons: DA lesion approach. Two important issues should be addressed. The first issue is whether DA lesions effectively eliminated vagal cardiac motoneurons within the NA. The NA is a relatively large nucleus spanning longitudally in the brain stem, and even multiple injection sites may not completely cover the entirely targeted region. In addition, although NA cardiac neurons may contain glutamatergic receptors (30), whether DA lesions would completely eliminate such neurons is still uncertain. The second issue is whether DA lesions in the NA region also potentially damaged other cardiac neurons and axons in the baroreflex loop, such as sympathetic cardiac neurons in caudal ventrolateral medulla (CVLM), DmnX cardiac axons dorsal to the NA, or NTS cardiac axons that project to the NA and CVLM (28). Notwithstanding such concerns, our anatomic and physiological findings show that 68–81% of NA neurons disappeared after DA injections and that such lesions were accompanied by a comparable reduction in cardiovascular responses to vagal stimulation, indicating that the DA lesion had effectively destroyed a significant number of vagal cardiac motoneurons. However, whether all of the neurons eliminated by DA were indeed cardiac neurons was not tested. Regarding sympathetic cardiac neurons in the CVLM, they are indeed located very close to the NA and could be particularly susceptible to damage by DA injections. These neurons contain glutamate receptors, receive NTS glutamate inputs, and project to and inhibit the sympathetic cardiac neurons in the rostral ventrolateral medulla (RVLM) through GABAergic mechanisms. Increases in blood pressure would decrease HR through this sympathetic baroreflex pathway, such that damage to the neurons may have contributed to the reduced responses reported herein. Indeed, the sympathetic baroreflex sensitivity, measured after bilateral cervical vagotomy in control rats, was -0.28 ± 0.04 beats·min-1·mmHg-1, and it was completely abolished by pretreatment of -blocker propranolol. Compared with the sympathetic baroreflex sensitivity, the baroreflex sensitivity (-0.12 ± 0.03 beats·min-1·mmHg-1) derived from DA-treated animals (unilateral vagotomy) was significantly smaller (P < 0.05). Therefore, the DA injection might have also damaged the inhibitory cardiac neurons in the CVLM and RVLM (because these sympathetic cardiac neurons are located near the compact formation of the NA in the rostal medulla).
All of these considerations could also apply to DmnX and NTS cardiac neurons. Using epifluorescent microscopy, we have qualitatively examined TMR-D-labeled DmnX neurons and vagal afferent terminals in the NTS in brain stem sections in all vehicle and DA-lesioned animals in a rostrocaudal fashion. Compared with controls, we did not observe any significant changes in DmnX neurons and vagal afferent terminals within the NTS, such that this possibility is unlikely. In addition, our laboratory's previous study (4) showed that the baroreflex sensitivity at 15 days post-DA lesion of the bilateral DmnX and NTS was unchanged. Therefore, even if DA injections into the NA through the dorsal brain stem might damage some DmnX and NTS neurons, such damage should not have significantly altered the baroreflex sensitivity.
L-Glu injection into the NA. As addressed above, DA lesion eliminated NA cardiac motoneuron as well as damaged inhibitory cardiac neurons in the CVLM. Therefore, the reduced HR response to L-glu injection in DA-lesioned animals might be due to damage to vagal motoneurons and sympathetic cardiac neurons in the CVLM. However, three lines of evidence indicate that damage of the parasympathetic system may be the primary cause for such a reduction in NA-lesioned animals. First, the DA injection eliminated 81% of NA motoneurons at 180 days. Second, HR responses to L-glu injection after application of 1-adrenoceptor antagonist atenolol in vehicle control only mildly attenuated the HR response to L-glu stimulation, whereas subsequent injection of the muscarinic receptor blocker methylatropine completely eliminated responses. Third, the baroreflex control of the HR is primarily mediated by the vagus. Therefore, we conclude that the reduced HR response to L-glu injection into the NA of DA-treated rats should be mainly due to the disruption of the parasympathetic pathway in these animals.
NA and DmnX cardiac motoneurons: functional significance. We demonstrate for the first time that the integrity of the NA is critical for baroreflex control of the HR. Although it is known that NA neurons exert important cardiac functions, i.e., activation of the NA elicits negative chronotropic, dromotropic, and inotropic responses, and also that these neurons are barosensitive, i.e., they can be activated by baroreceptor inputs (25, 32), we now extend such observations and show that the integrity of the NA not only is necessary for the baroreflex but is also sufficient. These findings clearly contrast with the preservation of the baroreflex control of HR after chronic lesions of the DmnX (4), where barosensitive neurons are also found (32).
Selective lesion of vagal motor nucleus may provide a potential tool to dissect the differential functional roles of the DmnX and NA. Whether the DmnX has any significant functional role in cardiopulmonary reflexes remains intriguing. Several lines of evidence may support the idea that the DmnX controls the heart in a complementary role to the NA. First, the DmnX strongly innervates the principal neurons in all cardiac ganglionic plexuses (8). Second, DmnX and NA cardiac axons differ in caliber and projection fields (5). Third, the DmnX and NA project to completely different sets of cardiac neurons (6). Fourth, physiological studies indicate that NA cardiac axons are mainly B fibers, whereas DmnX fibers are C fibers (10, 17, 31). Finally, electrical stimulation of the vagal nerve could still significantly decrease the HR and blood pressure even in the animals whose NA neurons had been largely destroyed and whose baroreflex sensitivity was almost completely abolished. However, these responses were much smaller than those observed in saline controls, indicating that the DmnX axons may exert a smaller influence on the HR than the NA axons.
To further examine the role of the DmnX, we would need to selectively stimulate the DmnX. Conventionally, any attempts to stimulate the DmnX electrically or chemically may also activate the adjacent NTS, which might secondarily activate the cardiac neurons in the NA, CVLM, and RVLM. Therefore, it is very important to effectively disconnect the NA, CVLM, and RVLM from their reflex circuitry before the DmnX is activated. To this end, lesions of the NA region may provide a new tool to dissect the differential roles of the DmnX and NA in different cardiac reflexes.
In summary, selective lesions of vagal motor nuclei suggest that the integrity of the NA is critically important for baroreflex control of the HR. Along with our previous anatomic findings that the NA and DmnX may project to the heart very differently and that chronic lesion of the DmnX does not affect baroreflex sensitivity, we propose that the NA plays different roles from the DmnX in baroreflex control. The differential roles of the NA and DmnX in other cardiac reflexes should be further elucidated.
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ACKNOWLEDGMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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GRANTS
Z. J. Cheng is supported by National American Heart Association Grant 9930173N and National Institute of Health (NIH) Grant AG-021020-01A1. D. Gozal is supported by NIH Grants HL-63912, HL-69932, and HL-65270, and the Commonwealth of Kentucky Research Challenge Trust Fund.
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FOOTNOTES |
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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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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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 E. K. A. Corbett, D. A. S. G. Mary, P. N. McWilliam, and T. F. C. Batten Autonomic Neuroscience: Age-related loss of cardiac vagal preganglionic neurones in spontaneously hypertensive rats Exp Physiol, November 1, 2007; 92(6): 1005 - 1013. [Abstract] [Full Text] [PDF]
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 H. Gu, M. Lin, J. Liu, D. Gozal, K. E. Scrogin, R. Wurster, M. W. Chapleau, X. Ma, and Z. Cheng Selective impairment of central mediation of baroreflex in anesthetized young adult Fischer 344 rats after chronic intermittent hypoxia Am J Physiol Heart Circ Physiol, November 1, 2007; 293(5): H2809 - H2818. [Abstract] [Full Text] [PDF]
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M. Lin, R. Liu, D. Gozal, W. B. Wead, M. W. Chapleau, R. Wurster, and Z. Cheng Chronic intermittent hypoxia impairs baroreflex control of heart rate but enhances heart rate responses to vagal efferent stimulation in anesthetized mice Am J Physiol Heart Circ Physiol, August 1, 2007; 293(2): H997 - H1006. [Abstract] [Full Text] [PDF]
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G. K. Soukhova-O'Hare, Z. Cheng, A. M. Roberts, and D. Gozal Postnatal intermittent hypoxia alters baroreflex function in adult rats Am J Physiol Heart Circ Physiol, March 1, 2006; 290(3): H1157 - H1164. [Abstract] [Full Text] [PDF]
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A. J. M. Verberne Differential cardiac parasympathetic innervation--what is the functional significance? Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2004; 287(2): R485 - R486. [Full Text] [PDF]
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) and DA (
) microinjections to the left NA. Top: HR responses. Bottom: BP responses. Both responses were significantly reduced after DA lesions at all vagal stimulation frequencies that elicited significant responses. *Significant difference in DA vs. saline (P < 0.001; 2-way ANOVA).
