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NEUROHUMORAL CONTROL OF CARDIOVASCULAR FUNCTION
Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, Nebraska
Submitted 19 August 2006 ; accepted in final form 13 October 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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cardiovascular reflexes; losartan; renal sympathetic nerve activity; microinjection; extracellular recording; barosensitivity
The nucleus tractus solitarii (NTS) receives sensory inputs from a vast array of peripheral receptors located on visceral, somatic, and cardiorespiratory organs and would be an effective region for modulating cardiovascular reflexes (1, 29). The NTS is known to be the central termination site of baroreceptors and plays a critical role in integrating the baroreflex (29, 30). On the other hand, axonal tracing studies have shown cardiac sympathetic afferents project to the dorsal horn of the upper thoracic spinal cord through the stellate ganglia and the sympathetic chain (6, 13). The ascending fibers from the dorsal horn of the spinal cord have been demonstrated to terminate in the NTS (22). Electrophysiological evidence also suggests a subgroup of the NTS neurons can be excited by stimulation of cardiac sympathetic afferents (31). Therefore, the NTS has been implicated to be an important area for integrating the central transmission of the CSAR (15, 31).
Although at least two ANG II receptor subtypes, AT1 and AT2, are expressed in rat brain, competition binding studies suggest that only AT1 receptors are found in the NTS (28, 32). It has been suggested that an NTS-ANG II mechanism plays an important role in controlling cardiovascular activity (2, 37). Microinjection of ANG II into the NTS has been found to modify AP and depress baroreflex function, whereas injection of ANG II receptor antagonists facilitates the baroreflex (4, 21). However, no evidence is available to address the question of whether an ANG II mechanism contributes to the interaction between the baroreflex and the CSAR at the level of the NTS.
Hence, the present study was designed to test the role of an NTS-AT1 receptor mechanism in processing of the interaction between the CSAR and the baroreflex in anesthetized rats. We investigated the effects of bilateral microinjection of losartan into the NTS on the blunted baroreflex evoked by CSAR activation. We further determined the effects of CSAR activation on the activity of NTS barosensitive neurons before and after treatment with intravenous injection of losartan.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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General surgery.
Rats were anesthetized with urethane (800 mg/kg ip) and 
-chloralose (40 mg/kg ip). The trachea was cannulated, and the rat was paralyzed with pancuronium bromide (1 mg/kg iv, 0.1 mg/kg thereafter as needed) and ventilated artificially with room air supplemented 100% oxygen. The left common carotid artery was cannulated, and the AP was measured with a pressure transducer (model PT300; Grass Instruments, Quincy, MA) for measurement of mean arterial pressure (MAP). HR was derived from the AP pulse with a PowerLab model 16S (ADInstruments, Colorado Springs, CO). The femoral vein was cannulated for intravenous injections. Rats were placed in a stereotaxic frame (Stoelting, Chicago, IL), and the dorsal surface of the medulla was surgically exposed by incising the atlantooccipital membrane and removing part of the occipital bone and dura. Supplemental doses of -chloralose (20 mg/kg iv) were administered to maintain an appropriate level of anesthesia. Body temperature was maintained at 
37°C with an animal temperature controller (ATC1000; World Precision Instruments).
Recording of RSNA. The left renal sympathetic nerves were exposed, identified, dissected free of the surrounding connective tissue, and placed on a pair of platinum-iridium recording electrodes. Both the nerve and the electrodes were covered with a fast-setting silicone (Wacker Sil-Gel). The signal was amplified (band pass 100–1,000 Hz) with a preamplifier (model P 18D; Grass Instruments). The amplified discharge was monitored on a storage oscilloscope (model 121 N; Tektronix, Beaverton, OR), imported to a computer system with other parameters, and then stored on disk until analyzed. Respective noise levels were subtracted from the nerve recording data before percentage changes from baseline were calculated. Integrated RSNA was normalized as 100% baseline in the control period (15).
Activation of the CSAR and the baroreceptors. Epicardial application of capsaicin has been demonstrated to effectively stimulate the cardiac sympathetic afferents (9, 39). The chest was opened through the fourth intercostal space. The pericardium was removed to expose the left ventricle. A filter paper containing capsaicin (3 x 3 mm, 0.4 µg in 2 µl) was applied to the anterior surface of the left ventricle. The baroreflex was tested about 2 min after capsaicin was applied, and then the epicardium was rinsed three times with 10 ml of warm normal saline (38°C). The time interval of repeated capsaicin was at least 30 min to allow the AP, HR, RSNA, and discharge of NTS neurons to return to, and stabilize at, their control levels. To test and evaluate the barosensitivity of NTS neurons in neuronal recording experiments, we placed a vascular occluder (Harvard Apparatus) around the descending thoracic aorta above the diaphragm, to elevate AP by constricting the aorta.
Microinjections into the NTS. Microinjections were made from four-barrel micropipettes with total tip diameters of 20–30 µm and performed using a four-channel pressure injector (PM2000B; World Precision Instruments). The injections were made over a 10-s period, and a 50-nl injection volume was measured by observing the movement of the fluid meniscus along a reticule in a microscope. The dorsal medial NTS (coordinates in mm with respect to calamus scriptorius: 0.4–0.5 rostral, 0.5–0.6 lateral, and 0.4–0.5 deep) was identified by injecting L-glutamate (2 nmol) and observing a depressor response of at least 25 mmHg. Losartan and L-glutamate were dissolved in artificial cerebrospinal fluid (aCSF). The time interval between bilateral injections was within a 60-s period. At the end of the experiments, 50 nl of 2% Pontamine sky blue were injected for marking the injection sites.
Extracellular recording of the NTS neurons.
A single-unit extracellular recording was obtained using a single micropipette (tip diameter 1–2 µm; resistance 5–12 M
) filled with 0.5 M sodium acetate dissolved in 2% Pontamine sky blue. The electrode was advanced using a microdrive (HSE-HA 864/1) into the dorsal medial NTS at a speed of 1–2 µm/s until single-unit activity was recorded. The spontaneous action potentials were amplified using a high-impedance preamplifier (Dagan; band pass 100–3,000 Hz) and fed into a window discriminator (Mentor N-750 spike analyzer), which generates a standard pulse for each spike. Potentials were visualized on an oscilloscope (model 121N; Tektronix). The pulse output of the discriminator was then fed into a rate/interval monitor (HFC) whose analog output is proportional to the number of spikes per unit time (1 s). These signals were displayed online on the computer and recorded on the data acquisition system.
The barosensitivity of NTS neurons was identified by their excitatory response to transient AP elevation (20–50 mmHg) by aorta occlusion (5–10 s). Baroreceptor stimulation by aorta occlusion or balloon inflation has been performed to conveniently identify the barosensitivity of neurons in the brain stem (7, 16). Some specific NTS barosensitive neurons that were inhibited by aorta occlusion were not further observed in the present study. After a neuron of interest was recorded, a small amount of 2% Pontamine sky blue was iontophoresed (–15 µA, 10 min) to mark the recording location for histological analysis.
Histological analysis. At the end of the experiment, the rat was given a lethal injection of pentobarbital sodium (100 mg/kg iv) and perfused with 10% formaldehyde solution (100 ml) intracardially. The brain stem was then quickly removed and fixed in 10% buffered Formalin. Frozen 50-µm coronal sections were made on a freezing microtome and mounted on slides. The dye spot and spread area for injection or cell recording sites were identified and plotted on standardized sections according to the atlas of Paxinos and Watson (24). Data were excluded if the injection or recording sites were not located in the medial NTS area. Figure 1 shows the locations of the microinjection sites (A) and the recorded neurons (B) in the NTS. There was no clear difference in topographical distributions between barosensitive and nonbarosensitive neurons within the NTS.
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The barosensitivity of NTS neurons was evaluated as percent changes of baseline discharge during aorta occlusion. The magnitude of barosensitivity of NTS neurons in the control period was normalized as a barosensitivity of 100%. The barosensitivity was evaluated again after treatments at the same AP elevation used during the control test. Decreases or increases in barosensitivity were expressed as a percent reduction or elevation from the magnitude of barosensitivity in the control period. Importantly, we also examined the change in relationship between neuronal discharge and AP during aorta occlusion (spikes/mmHg) for further evaluation of the barosensitivity.
Statistical analysis. The data are presented as means ± SE. We constructed composite baroreflex curves by averaging the four parameters of the logistic equation for all curves and using the mean parameters to construct a single curve. The changes in integrated RSNA and neuronal activity after treatments were evaluated as percent changes from control because of the variability in baseline RSNA in each animal and discharge in each neuron. Comparisons between control and experimental interventions were made using either Student's t-test (paired and unpaired) or one-way analysis of variance. Differences were considered to be statistically significant when P < 0.05.
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RESULTS |
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Figure 6 shows original representative tracings of baroreflex activation by aorta occlusion and the responses of a barosensitive NTS neuron to epicardial application of capsaicin. The magnitude of AP elevation during aorta occlusion before and after treatments was similar. This magnitude of AP elevation (35.7 ± 3.2 mmHg, n = 8) in several rats was verified to effectively activate the baroreflex control of HR and RSNA (Fig. 6A). In 17 of 24 (70.8%) barosensitive NTS neurons (4.9 ± 0.7 spikes/s), the resting discharge was significantly (P < 0.05) inhibited by 35.6 ± 4.0% during epicardial capsaicin application. In these neurons, the barosensitivity was also markedly (P < 0.01) attenuated to 33.5 ± 6.8% (
discharge/AP: from 0.142 ± 0.022 to 0.032 ± 0.006 spikes/mmHg) (Fig. 7). The remaining seven (29.2%) barosensitive NTS neurons (5.4 ± 0.9 spikes/s) did not respond to capsaicin. In contrast, neither discharge (4.8 ± 1.3 vs. 5.0 ± 1.5 spikes/s, P > 0.05) nor barosensitivity (discharge/AP: 0.137 ± 0.038 vs. 0.126 ± 0.028 spikes/mmHg, P > 0.05) in seven capsaicin-sensitive neurons was changed during epicardial application of 0.9% normal saline (2 µl).
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Effects of intravenous losartan on the NTS neuronal barosensitivity in response to epicardial application of capsaicin.
Eleven barosensitive NTS neurons from 11 rats that were sensitive to capsaicin were tested in this experiment. Figure 8 shows the responses of neuronal barosensitivity to epicardial application of capsaicin 10 min after intravenous losartan (2 mg/kg in 0.3 ml normal saline). It was found that intravenous injection of losartan significantly decreased AP (from 96 ± 2 to 82 ± 3 mmHg, P < 0.01) and HR (from 368 ± 7 to 332 ± 7 beats/min, P < 0.01), whereas it increased ongoing activity of barosensitive neurons by 30.1% (from 6.0 ± 1.2 to 7.5 ± 1.4 spikes/s, P < 0.05). The neuronal barosensitivity was increased to 119 ± 9% (discharge/AP: from 0.124 ± 0.018 to 0.159 ± 0.011 spikes/mmHg, P < 0.05) 7 min after intravenous losartan. Ten minutes after losartan, capsaicin did not significantly decrease the baseline discharge (7.3 ± 1.3 vs. 6.4 ± 1.1 spikes/s, P > 0.05) or barosensitivity (discharge/AP: 0.159 ± 0.011 vs. 0.144 ± 0.014 spikes/mmHg, P > 0.05). We also noted that the baseline AP and neuronal discharge between 7 and 10 min after losartan were similar. The magnitudes of percent changes in discharge and barosensitivity of NTS neurons during capsaicin application before and after intravenously injected losartan are shown in Fig. 8B. In addition, intravenous normal saline (0.3 ml, n = 3) did not modify capsaicin-induced inhibition of NTS neurons (data not shown).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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In chronic heart failure, the CSAR has been reported to be augmented, and its enhancement may be one important factor that contributes to sympathetic hyperactivity and baroreflex impairment (8, 9, 33–35). The current data also show that in normal rats, chemical activation of the CSAR by application of capsaicin to the left ventricle produced a sympathoexcitation and a reduction in baroreflex control of RSNA, which was consistent with the previous studies (8, 9, 34). Consistent with our previous study (9), we noted that CSAR activation by capsaicin significantly attenuated the values of slope and maximum gain of baroreflex curve without affecting the values of range of RSNA response and minimum RSNA. Because increases in AP and RSNA after capsaicin usually recovered to control levels within 2 min, prebaseline levels of baroreflex testing before and after capsaicin were similar. This may explain why the baroreflex curves after capsaicin are not significantly shifted right and upward. The baroreflex test was conducted 2 min after capsaicin treatment, when the variables of AP and RSNA had returned to their control values, which indicates that at this time the effects of capsaicin had dissipated. Although we also have observed a significant attenuation of baroreflex sensitivity 2 min after capsaicin treatment, the observed effects on the baroreflex may be less than those that would have occurred at an earlier time. To address the question of the central mechanism responsible for interaction between the CSAR and the baroreflex, there are at least two essential factors to understand. These are to identify the integrative region(s) in the brain and the neurotransmitters/receptors that participate in this interaction. Because the NTS receives convergent inputs from baroreceptor afferents and cardiac sympathetic afferents (22, 31) and the NTS ANG II mechanism participates in integrating the baroreflex (4, 5), we chose the NTS as the target region where the interaction between the CSAR and the baroreflex may occur.
The present results from the microinjection experiments showed that AT1 receptor blockade in the NTS significantly increased the baroreflex function without modifying baseline AP, HR, and RSNA. This observation is consistent with previous studies from other laboratories (5, 21). These data suggest that endogenous ANG II acting on the NTS AT1 receptors modifies baroreflex function. Importantly, we also found that AT1 receptor blockade in the NTS significantly attenuated the increases in AP, HR, and RSNA evoked by capsaicin and also effectively reversed the attenuation of the baroreflex by capsaicin. We also observed that the capsaicin-induced barosensitivity after NTS losartan not only was reversed to the preinjection level but also was enhanced (Fig. 5B). Therefore, we believe that NTS losartan abolishes capsaicin-induced depression of baroreflex function, as well as increasing baroreflex sensitivity. These data strongly indicate that the functional states of the NTS AT1 receptors are very important in mediating both the central transmission of the CSAR and the interaction between the CSAR and the baroreflex. The present data suggest that during CSAR activation, endogenous ANG II release and action on NTS AT1 receptors results in an increase in sympathetic outflow and attenuation of the baroreflex. However, no direct evidence is available to demonstrate that, at the NTS level, release of ANG II is increased and the function of AT1 receptors is upregulated when the CSAR is activated. Importantly, previous data support this idea. For instance, 1) angiotensin-converting enzyme mRNA expression within the NTS is higher in heart failure rats (27); and 2) AT1 receptor density in the NTS is upregulated in a rabbit with heart failure in a radioautographic study (40). Although the CSAR is significantly augmented in heart failure (33, 34), contribution of the CSAR enhancement to increased ANG II or upregulated AT1 receptors in the NTS has not been confirmed and awaits further study. In addition to the NTS, other cardiovascular centers such as the rostral ventrolateral medulla (RVLM) and the paraventricular nucleus also may participate in this interaction (16, 38).
To further elucidate the role of NTS-AT1 mechanism in this interaction, we directly observed the effect of CSAR activation on NTS barosensitive neurons. In this work, we did not electrically stimulate the aortic or carotid sinus nerve to identify NTS neurons receiving baroreceptor afferents, and clearly, this may be considered a limitation. We believe that AP elevation by aorta occlusion may be similar to AP elevation by intravenous injection of phenylephrine, which has been shown to activate NTS neurons that are driven by single-pulse stimulation of the aortic nerve (26). In the present study, an average of 35 mmHg of AP elevation during aorta occlusion was verified to effectively stimulate the baroreceptors and activate the baroreflex (Fig. 6A). Moreover, we also found that in some tested NTS neurons, there existed a significantly positive correlation (r = 0.76; data not shown) between the change in AP by aorta occlusion and the firing rate of NTS neurons. In addition, a large change in AP (>50 mmHg) during recording of single units often resulted in the loss of the recording, because the distance between the stationary microelectrode and the neuron of interest increased.
At the level of NTS barosensitive neurons, CSAR activation inhibited the basal discharge rate and attenuated the barosensitivity, which could be prevented by intravenous losartan. Losartan has been demonstrated to cross the blood-brain barrier in rats (17). Because losartan was given intravenously in this work, its exact targets could not be identified. However, some important observations from the current data indicate that endogenous ANG II acted at AT1 receptors to mediate the CSAR-induced decreases in baseline discharge and barosensitivity of NTS neurons. We believe that the inhibitory effect of capsaicin on NTS neurons is not due to neuronal baroreflex activation, because AP elevation by CSAR activation will produce an excitation of NTS barosensitive neurons. It is well known that NTS barosensitive neurons, which receive input from the peripheral baroreceptors, excite the caudal ventrolateral medulla (CVLM) neurons via a glutamatergic projection, whereas the CVLM neurons inhibit the RVLM vasomotor neurons via a GABA-ergic projection (1, 29). The RVLM has been recognized as a critical site for generating sympathetic outflow and controlling baroreflex function (29). Therefore, it is logical to assume that the inhibitory effect of CSAR activation on NTS barosensitive neurons would increase sympathetic outflow and that attenuation of the barosensitivity of NTS neurons would decrease the baroreflex function. Therefore, these new findings contribute to our understanding of the mechanism responsible for CSAR-induced sympathetic hyperactivity and baroreflex impairment. Because there are several pathways between the NTS and RVLM, we guess that reduction of barosensitive neurons by CSAR activation may not be a unique pathway (NTS-CVLM-RVLM pathway) to cause sympathoexcitation. More recently, we furthermore found that CSAR activation significantly excites the chemosensitive neurons within the commissural NTS, where neurons directly send the excitatory projection to the RVLM (36). Therefore, in addition to inhibition of barosensitive neurons, excitation of chemosensitive neurons may be another important mechanism within the NTS to mediate the CSAR-induced sympathoexcitation. Taking these findings together, we think that several mechanisms may simultaneously and together mediate the processing of CSAR-induced sympathoexcitation.
It has been reported that a neuron group exists within the NTS that can be excited by stimulation of cardiac sympathetic afferents and that excitatory amino acids may mediate this central transmission (15, 31). Because these NTS neurons have not been further identified by barosensitivity, we assume these neurons directly receive cardiac sympathetic afferent input rather than baroreceptor input. Our results provide evidence for this possibility, because a majority (56%) of nonbarosensitive neurons were excited by epicardial capsaicin. Therefore, it is possible that the CSAR-excitatory neurons are interneurons that release inhibitory neurotransmitters. CSAR-induced excitation of this inhibitory interneuron may produce an inhibition of NTS barosensitive neurons, which is consistent with CSAR activation resetting barosensitivity. There is substantial evidence that GABA may be the inhibitory neurotransmitter within the NTS. For example, iontophoretic application of GABA markedly reduced or abolished the responses of NTS neurons to stimulation of baroreceptors (3). Inhibitory interneurons in the NTS have become a well-accepted notion to explain baroreflex resetting (7, 25).
In summary, blockade of AT1 receptors significantly improved the depressed baroreflex function evoked by CSAR activation at the level of the NTS in anesthetized rats, suggesting that an NTS-ANG II mechanism plays an important role in processing the interaction between the baroreflex and the CSAR. However, because of the acute nature of the experiments in the present study, it is not known whether the NTS-ANG II mechanism exists in more chronic pathological conditions such as chronic heart failure.
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GRANTS |
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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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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-aminobutyric-acid-mediated inhibition of neurones in the nucleus tractus solitarius of the cat. J Physiol 392: 417–430, 1987.This article has been cited by other articles:
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  I. H. Zucker, H. D. Schultz, K. P. Patel, W. Wang, and L. Gao Regulation of central angiotensin type 1 receptors and sympathetic outflow in heart failure Am J Physiol Heart Circ Physiol, November 1, 2009; 297(5): H1557 - H1566. [Abstract] [Full Text] [PDF]
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Q. Zhang, F. Yao, S. T. O'Rourke, S. Y. Qian, and C. Sun Angiotensin II enhances GABAB receptor-mediated responses and expression in nucleus tractus solitarii of rats Am J Physiol Heart Circ Physiol, November 1, 2009; 297(5): H1837 - H1844. [Abstract] [Full Text] [PDF]
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W.-Z. Wang, L. Gao, H.-J. Wang, I. H. Zucker, and W. Wang Interaction between cardiac sympathetic afferent reflex and chemoreflex is mediated by the NTS AT1 receptors in heart failure Am J Physiol Heart Circ Physiol, September 1, 2008; 295(3): H1216 - H1226. [Abstract] [Full Text] [PDF]
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 P. S. P. Tan, S. Killinger, J. Horiuchi, and R. A. L. Dampney Baroreceptor reflex modulation by circulating angiotensin II is mediated by AT1 receptors in the nucleus tractus solitarius Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2007; 293(6): R2267 - R2278. [Abstract] [Full Text] [PDF]
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W.-Z. Wang, L.-G. Wang, L. Gao, and W. Wang Contribution of AMPA/kainate receptors in the rostral ventrolateral medulla to the hypotensive and sympathoinhibitory effects of clonidine Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2007; 293(3): R1232 - R1238. [Abstract] [Full Text] [PDF]
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, Microinjection sites; 
, barosensitive neurons; 
, nonbarosensitive neurons. AP, area postrema; Gr, gracile nucleus; Cu, cuneate nucleus; Sol, NTS; 10, motor nucleus of vagus nerve; 12, nucleus of hypoglossal nerve.
