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Am J Physiol Regul Integr Comp Physiol 288: R1707–R1715, 2005. First published February 24, 2005; doi:10.1152/ajpregu.00537.2004. NK1 receptor activation in rat rostral ventrolateral medulla selectively attenuates somato-sympathetic reflex while antagonism attenuates sympathetic chemoreflex John M. Makeham,1,2 Ann K. Goodchild,1,2 and Paul M. Pilowsky1,2 1 Hypertension and Stroke Research Laboratory, Department of Physiology, University of Sydney, New South Wales; 2Department of Neurosurgery, Royal North Shore Hospital, Sydney, New South Wales, Australia Submitted 9 August 2004; accepted in final form 15 February 2005 substance P; rostral ventrolateral medulla; baroreflex; brainstem; sympathetic nerve activity; neurokinin 1 THE SYMPATHOEXCITATORY CELLS of the rostral ventrolateral medulla (RVLM) project to the sympathetic preganglionic neurons of the spinal cord that are essential for the maintenance of resting sympathetic tone (5, 21, 40, 44). The RVLM is also essential for the integration of cardiovascular reflexes such as the baroreflex, chemoreflex, and somato-sympathetic reflex, as well as respiratory modulation of the sympathetic outflow (35). Given the importance of these reflexes in cardiovascular regulation, the modulation of the sympathoexcitatory RVLM neurons is of considerable interest. Recently, our laboratory demonstrated that stimulation of ␦-opioid receptors in the RVLM inhibits respiratory-related discharge of lumbar sympathetic nerve activity, while having little effect on splanchnic sympathetic nerve activity (sSNA) and potently attenuating the somato-sympathetic reflex. However, stimulation of ␦-opioid Address for reprint requests and other correspondence: P. M. Pilowsky, Hypertension and Stroke Research Laboratories, Ground Floor, Block 3, Royal North Shore Hospital, St. Leonards, NSW 2065, Australia (E-mail: [email protected]). http://www.ajpregu.org receptors in the RVLM has no effect on the baroreflex or the chemoreflex. Stimulation of RVLM -opioid receptors inhibits the baroreflex, while leaving the somato-sympathetic reflex and chemoreflex unaffected (28). We have also demonstrated that activation of 5-HT1A receptors in the RVLM results in a potent, selective inhibition of the somato-sympathetic reflex but not the baroreflex or chemoreflex (27). The undecapeptide tachykinin substance P and its receptor, the neurokinin 1 (NK1) receptor, have been implicated in the central regulation of the cardiovascular system. Microinjection of substance P into the nucleus of the solitary tract (NTS) modulates the baroreflex (8, 39).The RVLM contains substance P immunoreactive terminals and the NK1 receptor (12, 33). Recently, we have demonstrated that the NK1 receptor is present on some C1 adrenergic neurons of the RVLM (24). Furthermore, substance P immunoreactive terminals are known to contact C1 neurons (26). Microinjection of a stable substance P analog into the RVLM causes powerful pressor responses in vivo (43), and both substance P and the selective NK1 receptor agonist [Sar9, Met (O2)11]SP excite neonatal bulbospinal C1 neurons recorded using patch electrodes in vitro (20). In the present study, we examined the role of substance P and the NK1 receptor in the cardiovascular functions and reflexes mediated by the RVLM. The effects of NK1 receptor agonist and antagonist compounds on the baroreceptor, chemoreceptor, somato-sympathetic reflex, and splanchnic sympathetic nerve activity (sSNA) were examined. MATERIALS AND METHODS All experimental protocols were approved by the Animal Care and Ethics Committees of the Royal North Shore Hospital. General procedures. Male Sprague-Dawley rats (300 –500 g) were initially anesthetized with halothane (2% in 100% O2) followed by an intraperitoneal injection of urethane (1.25–1.3 g/kg) and atropine (90 g ip). The trachea was cannulated, and the right cervical vagus nerve was cut. The right carotid artery was catheterized for arterial blood pressure measurement, and the right jugular vein was catheterized for drug administration. To isolate the aortic depressor nerve (ADN) and phrenic nerve, a longitudinal posterolateral incision was made at the cervico-thoracic junction. After blunt dissection through the subcutaneous fat and diathermy through the parascapular muscles, the scapula was retracted laterally, and the phrenic nerve was located posterior to the carotid sheath at the root of the neck. The ADN was dissected from within the carotid sheath. The splanchnic nerve was isolated 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. 0363-6119/05 $8.00 Copyright © 2005 the American Physiological Society R1707 Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017 Makeham, John M., Ann K. Goodchild, and Paul M. Pilowsky. NK1 receptor activation in rat rostral ventrolateral medulla selectively attenuates somato-sympathetic reflex while antagonism attenuates sympathetic chemoreflex. Am J Physiol Regul Integr Comp Physiol 288: R1707–R1715, 2005. First published February 24, 2005; doi:10.1152/ajpregu.00537.2004.—The effects of activation and blockade of the neurokinin 1 (NK1) receptor in the rostral ventrolateral medulla (RVLM) on arterial blood pressure (ABP), splanchnic sympathetic nerve activity (sSNA), phrenic nerve activity, the somato-sympathetic reflex, baroreflex, and chemoreflex were studied in urethane-anesthetized and artificially ventilated Sprague-Dawley rats. Bilateral microinjection of either the stable substance P analog (pGlu5, MePhe8, Sar9)SP(5–11) (DiMe-SP) or the highly selective NK1 agonist [Sar9, Met (O2)11]SP into the RVLM resulted in an increase in ABP, sSNA, and heart rate and an abolition of phrenic nerve activity. The effects of [Sar9, Met (O2)11]SP were blocked by the selective nonpeptide NK1 receptor antagonist WIN 51708. NK1 receptor activation also dramatically attenuated the somato-sympathetic reflex elicited by tibial nerve stimulation, while leaving the baroreflex and chemoreflex unaffected. This effect was again blocked by WIN 51708. NK1 receptor antagonism in the RVLM, with WIN 51708 significantly attenuated the sympathoexcitatory response to hypoxia but had no effect on baseline respiratory function. Our findings suggest that substance P and the NK1 receptor play a significant role in the cardiorespiratory reflexes integrated within the RVLM. R1708 NK1-RECEPTOR ACTIVATION AND ANTAGONISM IN THE RVLM intermittent electrical stimulation of the tibial nerve; and 4) chemoreceptor activation by a brief period of hypoxia. Drugs were then microinjected into the RVLM bilaterally and the activation of the reflexes (steps 1– 4 above) was repeated. Reflex activation was repeated every 15–20 min until the return of responses to preinjection levels. Because of significant desensitization after microinjection of NK1 receptor agonists into the RVLM, each animal received only one bilateral microinjection of either DiMe-SP or [Sar9, Met (O2)11]SP. Histological procedure. At the end of each experiment, the rats were killed with an intravenous injection of 1 M KCl. The medulla was removed and fixed in 10% formalin in deionized water overnight. The medulla was then sectioned transversely (100 m) on a vibrating microtome. The injection sites were identified using silver intensification of the gold particles (Sigma SE-100 Silver Enhancer Kit). The sections were counterstained using cresyl violet and mounted on glass slides. Data analysis. Data were analyzed during experiments and postacquisition using a CED 1401 data capture system and Spike 4 software (Cambridge, U.K.). The average value over a 20-s period was used to evaluate sSNA and arterial blood pressure. Phrenic frequency and phrenic amplitude were determined using a phrenic nerve triggered waveform average over a 100-s period. The sSNA responses to intermittent ADN stimulation and tibial nerve stimulation were analyzed using peristimulus waveform averaging. The amplitude of the sSNA from ⫺200 to 0 ms before stimulation was taken as the baseline. The maximum response to stimulation was then expressed as a percentage change from the baseline. The response to hypoxia was quantified by comparing the average sSNA for 10 s following the onset of excitation of phrenic nerve discharge caused by the inhaled 100% N2 as a percent change from a control period of 10 s average sSNA before 100% N2 inhalation. Data are expressed as means ⫾ SE. Statistical significance was assessed by paired t-tests to compare the effects before and after injection of a drug. To assess the effect of treatment with DiMe-SP, WIN 51708, and [Sar9, Met (O2)11]SP, a one-way ANOVA followed by multiple t-tests with Bonferroni’s correction (if ANOVA were significant) was conducted. All statistical analysis was performed using GraphPad software. RESULTS Microinjection sites were located between 0 and 500 m caudal to the caudal pole of the facial nucleus, 1.9 and 2.1 mm lateral from the midline, and ventral to the nucleus ambiguus. A typical injection site is seen in Fig. 1. Arterial blood pressure, sSNA, and HR. Microinjection of DiMe-SP (600 pmol, 50 nl) bilaterally into the RVLM resulted in a rise in arterial blood pressure (ABP) of 22 ⫾ 3 mmHg from 96 ⫾ 6 to 119 ⫾ 5 mmHg (n ⫽ 8, P ⬍ 0.001, Fig. 3A) Similarly, bilateral microinjection of [Sar9, Met (O2)11]SP in the RVLM resulted in a rise in ABP of 44 ⫾ 4 mmHg from 115 ⫾ 8 to 156 ⫾ 11 mmHg (n ⫽ 6, P ⬍ 0.001, Figs. 2A and 3A). This rise was maximal at 2–5 min and lasted 30 – 40 min. Splanchnic SNA (sSNA) and HR were also significantly increased after microinjection of DiMe-SP and [Sar9, Met (O2)11]SP. DiMe-SP increased sSNA to 180 ⫾ 7% baseline (n ⫽ 7, P ⬍ 0.001, Fig. 3C) and [Sar9, Met (O2)11]SP increased sSNA to 204 ⫾ 29% baseline (n ⫽ 6, P ⬍ 0.001, Figs. 2A and 3C). DiMe-SP increased HR 24 ⫾ 7 bpm from 384 ⫾ 9 to 408 ⫾ 9 (n ⫽ 7, P ⬍ 0.05, Fig. 3B), whereas [Sar9, Met (O2)11]SP increased HR 13 ⫾ 3 from 428 ⫾ 13 to 441 ⫾ 15 (n ⫽ 6, P ⬍ 0.01, Fig. 3B). Bilateral microinjection of the selective NK1 receptor antagonist WIN 51708 did not significantly change mean arterial pressure [118 ⫾ 7 vs. 124 ⫾ 9, n ⫽ 10, not significant (NS)] AJP-Regul Integr Comp Physiol • VOL 288 • JUNE 2005 • www.ajpregu.org Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017 following a longitudinal paralumbar incision and superomedially directed blunt dissection in the retroperitoneal plane after retraction of the paraspinal muscles and diathermy through the oblique and transverse muscle layers. Nerves were maintained in paraffin oil during recording or stimulation. The right tibial nerve was exposed for stimulation of the somatic afferent nerve fibers. The animals were then secured in a stereotaxic frame, paralyzed with pancuronium dibromide (0.8 mg iv) and artificially ventilated with O2-enriched air. End tidal CO2 was monitored and maintained between 4 and 5% by varying the ventilator frequency. The left cervical vagus nerve was then cut. A partial occipital craniotomy was performed to expose the dorsal surface of the medulla. Adequacy of anesthesia was determined by monitoring the arterial blood pressure and the phrenic nerve discharge. Additional doses of urethane (20 –30 mg iv) and pancuronium dibromide (0.2 mg iv) were given as required to maintain adequate anesthesia and neuromuscular blockade. Rectal temperature was maintained between 36 and 38°C with a heating pad and infrared lamp. Nerve recording. Bipolar silver-wire electrodes were used to record sSNA and phrenic nerve activity. The signals were amplified, filtered (100 –3,000 Hz band pass), full-wave rectified, and integrated using a Paynter filter with a 50-ms time constant. The zero level of sSNA was determined using supramaximal stimulation of the aortic depressor nerve (0.2-ms stimulation, 50 Hz for 5 s). Activation of cardiovascular reflexes. To activate baroreceptor afferent fibers, the ADN was stimulated electrically. Maximal activation of baroreceptor afferents was determined by tetanic ADN stimulation (0.2 ms duration, 50 Hz for 5 s). The stimulation voltage was adjusted to achieve maximal inhibition of sSNA. This was usually 0.5– 4.0 V. To assess baroreceptor function, the average sSNA inhibition in response to intermittent ADN stimulation was determined. The ADN was stimulated (0.2-ms duration, 2 pulses at 2.5-ms interval, 0.5 Hz), and the sSNA response was averaged at least 50 times. Activation of the somato-sympathetic reflex was achieved by stimulating the bipolar silver-wire cuff electrode placed around the right tibial nerve. The nerve was stimulated at 0.5 Hz (1-ms duration, 20 –30 V). Chemoreceptor activation was achieved by a brief period of hypoxia. Animals were ventilated with 100% N2 for 15 s. Microinjections. The stable substance P analog [pGlu5, MePhe8, Sar9]SP(5–11) (DiMe-SP, 600 pmol in 50 nl; Sigma), the highly selective NK1 receptor agonist [Sar9, Met (O2)11]SP [Sar9, Met (O2)11]SP, 600 pmol in 50 nl; Sigma) and the nonpeptide selective NK1 receptor antagonist Win 51708 (5 nmol in 100 nl; Sigma) were prepared for microinjection. The DiMe-SP was dissolved in 10 mM phosphate-buffered saline (0.9%). The pH was found to be 7.4. The [Sar9, Met (O2)11]SP was dissolved in deionized water, and the pH was adjusted to between 7.3 and 7.5. The Win 51708 was solubilized in 1% DMSO, and the pH was adjusted to between 7.3 and 7.5. In the first series of experiments, multibarrel micropipettes were prepared with either DiMe-SP or [Sar9, Met (O2)11]SP in one barrel and albumin-colloidal gold (Sigma) in a second barrel. In the second series of experiments, triple-barrel micropipettes were used, containing DiMe-SP or [Sar9, Met (O2)11]SP, Win 51708 and colloidal gold in the third barrel. The volume of injections was determined by direct observation of the movement of the fluid meniscus in the micropipette. In all experiments the micropipettes were placed bilaterally into the RVLM. Experimental procedures. The pressor region of the RVLM was identified physiologically by microinjection of L-glutamate (50 mM, 50 nl) in a single-barrel micropipette. When a site was identified where L-glutamate microinjection elicited a pressor response of ⬎30 mmHg, the pipette was removed, and multibarrel micropipettes containing drugs were placed stereotaxically in the same sites. Reflexes were tested in the following order: 1) baroreceptor activation by tetanic ADN stimulation; 2) baroreceptor activation by intermittent ADN stimulation; 3) activation of somatic afferent nerve fibers by NK1-RECEPTOR ACTIVATION AND ANTAGONISM IN THE RVLM or HR (436 ⫾ 9 vs. 442 ⫾ 10, n ⫽ 10, NS). There was a small but significant increase in sSNA to 121 ⫾ 6% baseline (n ⫽ 10, P ⬍ 0.01). Five to 10 min after pretreatment with WIN 51708, either DiMe-SP or [Sar9, Met (O2)11]SP was injected into the same site from another barrel of the triple-barrel micropipette. Pretreatment with WIN 51708 failed to fully block the rise in ABP following DiMe-SP, still significantly different from baseline with an increase of 11 ⫾ 3 mmHg from 107 ⫾ 5 to 119 ⫾ 5 mmHg (n ⫽ 5, P ⬍ 0.05, Fig. 3A). However, as shown in Figs. 2B and 3A, pretreatment with WIN 51708 blocked the rise in ABP following [Sar9, Met (O2)11]SP (139 ⫾ 7 vs. 144 ⫾ 11 mmHg, n ⫽ 5, NS). Pretreatment with WIN 51708 blocked the HR response to DiMe-SP (from 454 ⫾ 9 to 458 ⫾ 9 bpm, n ⫽ 5, NS, Fig. 3B) and to [Sar9, Met (O2)11]SP (from 438 ⫾ 18 to 433 ⫾ 17 bpm, n ⫽ 5, NS, Fig. 3B). Pretreatment with WIN 51708 blocked the sSNA response to DiMe-SP (114 ⫾ 6% baseline, n ⫽ 5, NS, Fig. 3C) and to [Sar9, Met (O2)11]SP (112 ⫾ 11% baseline, n ⫽ 5, NS, Figs. 2B and 3C). Phrenic nerve activity. Phrenic frequency was significantly reduced after bilateral DiMe-SP microinjection to 39 ⫾ 7% baseline (n ⫽ 8, P ⬍ 0.001, Fig. 3D). Phrenic nerve activity was consistently abolished following [Sar9, Met (O2)11]SP microinjection. This inhibition lasted 2–5 min (Fig. 2A). There was no significant decrease in phrenic nerve amplitude after DiMe-SP (102 ⫾ 5% baseline, n ⫽ 8) or upon return of phrenic nerve activity after [Sar9, Met (O2)11]SP (104 ⫾ 9% baseline, n ⫽ 6). Bilateral microinjection of WIN 51708 did not significantly alter phrenic frequency (30 ⫾ 2 vs. 31 ⫾ 3/min, n ⫽ 10, NS) or phrenic amplitude (102 ⫾ 6% baseline, n ⫽ 10, NS). Pretreatment with WIN 51708 failed to block the phrenic frequency response to DiMe-SP, reduced to 51 ⫾ 11% baseline (34 ⫾ 4 vs. 19 ⫾ 6/min, n ⫽ 5, P ⬍ 0.05, Fig. 3D) but did block the phrenic frequency response to [Sar9, Met (O2)11]SP (32 ⫾ 5 vs. 22 ⫾ 9/min, n ⫽ 5, NS, Fig. 3D). Somato-sympathetic reflex. The average SNA response to intermittent stimulation of the tibial nerve was tested both before and after microinjection of drugs. Intermittent tibial nerve stimulation resulted in a characteristic two-peaked response in the sSNA with the latencies of 115 ⫾ 2 ms and 211 ⫾ 4 ms (n ⫽ 7, Fig. 4A). Bilateral microinjection of DiMe-SP in the RVLM significantly attenuated the first and second peaks to 27 ⫾ 10% and 1 ⫾ 8% baseline, respectively (n ⫽ 6, P ⬍ 0.001). Bilateral microinjection of [Sar9, Met (O2)11]SP significantly attenuated the first peak to 15 ⫾ 3% baseline (n ⫽ 5, P ⬍ 0.05, Fig. 4, A and C). The second peak of the somato-sympathetic reflex was often absent during baseline stimulation in these experiments. On two occasions, it was shown to be attenuated to 8% and 9% baseline by [Sar9, Met (O2)11]SP microinjection (Fig. 4C). Bilateral microinjection of WIN 51708 did not significantly attenuate either the first or second peak of the somato-sympathetic reflex (83 ⫾ 30%; n ⫽ 10 and 64 ⫾ 19%; n ⫽ 9 baseline, respectively, NS). Pretreatment with WIN 51708 failed to block the response to DiMe-SP of the first peak, still attenuated to 58 ⫾ 11% baseline (n ⫽ 5, P ⬍ 0.05), but the response of the second peak was blocked (82 ⫾ 31% baseline, n ⫽ 4, NS). Pretreatment with WIN 51708 blocked the attenuation of the somato-sympathetic response caused by [Sar9, Met (O2)11]SP for both the first and second peaks (104 ⫾ 43% baseline and 172 ⫾ 56% baseline, respectively; n ⫽ 5, NS, Fig. 4B and C). Baroreflex. Intermittent stimulation of the ADN resulted in a maximal inhibitory potential in the sSNA with a latency of 178 ⫾ 4 ms (n ⫽ 13, Fig. 5C). Bilateral microinjection of DiMe-SP attenuated the amplitude of the inhibitory potential 58 ⫾ 9% (n ⫽ 7, P ⬍ 0.01), whereas [Sar9, Met (O2)11]SP did not significantly attenuate the inhibitory potential (76 ⫾ 11% baseline, n ⫽ 5, NS, Fig. 5C). Bilateral microinjection of WIN 51708 did not significantly alter the response to intermittent ADN stimulation (90 ⫾ 8% baseline, n ⫽ 10, NS). Pretreatment with WIN 51708 blocked the response to both DiMe-SP (89 ⫾ 5% baseline, n ⫽ 5, NS) and [Sar9, Met (O2)11]SP (109 ⫾ 17% baseline, n ⫽ 5, NS). An example is shown in Fig. 5C. Chemoreflex. Stimulation of the chemoreflex resulted in a characteristic increase in sSNA from prestimulus levels of 170 ⫾ 11% (n ⫽ 9, P ⬍ 0.01, Fig. 5A). Expressed as a percentage of the baseline response, microinjection of either DiMe-SP or [Sar9, Met(O2)11]SP failed to significantly alter the chemoreflex (80 ⫾ 13%; n ⫽ 7, NS and 90 ⫾ 24%; n ⫽ 6, NS) baseline, respectively. Bilateral microinjection of WIN 51708, however, resulted in a significant attenuation of the chemoreflex, to 38 ⫾ 5% baseline (n ⫽ 9, P ⬍ 0.001, Fig. 5, A and B). The interval between baseline chemoreflex testing and chemoreflex testing with the NK1 receptor agonist drugs, either DiMe-SP or [Sar9, Met (O2)11]SP, was not significantly different from the interval between baseline chemoreflex testing and that after pretreatment with the NK1 receptor antagonist WIN 51708: 986 ⫾ 63 s (n ⫽ 13) vs. 1116 ⫾ 113 s (n ⫽ 9), NS. DISCUSSION The principal novel findings of this study are 1) activation of NK1 receptors in the RVLM in vivo results in hypertension, AJP-Regul Integr Comp Physiol • VOL 288 • JUNE 2005 • www.ajpregu.org Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017 Fig. 1. Microinjection site in the rostral ventrolateral medulla (RVLM). The arrowhead identifies the microinjection site marked with silver-intensified gold particles. Injections into the RVLM were located caudal to the caudal pole of the facial nucleus and ventral to the compact formation of the nucleus ambiguus. D, dorsal; M, midline. Scale bar ⫽ 500 m. R1709 R1710 NK1-RECEPTOR ACTIVATION AND ANTAGONISM IN THE RVLM tachycardia, and splanchnic sympathoexcitation; 2) activation of NK1 receptors in the RVLM attenuates the somato-sympathetic reflex without affecting other brainstem reflexes such as the baroreflex or chemoreflex, and; 3) blockade of NK1 receptors in the RVLM significantly attenuates the sSNA response to chemoreceptor stimulation. Activation of NK1 receptors in the RVLM was also shown to significantly decrease phrenic frequency. Activation of NK1 receptors in the RVLM in vivo with DiMe-SP has previously been shown to increase ABP and HR (43). Our results agree with these findings. Further, the present study demonstrates a significant increase in sSNA following bilateral NK1 receptor stimulation in the RVLM. This is most likely mediated, in part, by NK1 receptors found on bulbospinal C1 neurons, which are sympathoexcitatory (24). Substance P terminals have been demonstrated within the RVLM and Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017 Fig. 2. A: bilateral microinjection of [Sar9, Met (O2)11]SP into the RVLM (solid arrowheads) elicited a sympathoexcitatory response and a rise in arterial blood pressure (ABP). There was also a complete inhibition of phrenic nerve activity, returning in ⬃400 s. Open arrows indicate tetanic stimulation of the aortic depressor nerve (ADN). Asterisk indicates ventilation with 100% N2 for 15 s. During the sympathoexcitatory period, multiple reflexes were tested, including the baroreflex, somato-sympathetic reflex, and chemoreflex. sSNA, splanchnic sympathetic nerve activity. B: bilateral microinjection of [Sar9, Met (O2)11]SP after pretreatment with WIN 51708 in the RVLM. WIN 51708 had no significant effect on sympathetic or phrenic nerve activity but did cause a small but significant rise in ABP (left). After pretreatment with WIN 51708, [Sar9, Met (O2)11]SP had no significant effect on arterial blood pressure, sympathetic nerve activity, or phrenic nerve activity (right). form synaptic junctions with C1 neurons (18, 26). Substance P and [Sar9, Met (O2)11]SP excite neonatal bulbospinal C1 neurons in vitro, possibly by a reduction in resting potassium conductance (20). Pretreatment of the RVLM with the highly selective NK1 receptor antagonist WIN 51708 attenuated the pressor response to DiMe-SP and blocked the effects of [Sar9, Met (O2)11]SP. This suggests that the pressor response to DiMe-SP may be partially mediated by binding sites that are different to the binding sites activated by [Sar9, Met (O2)11]SP. The carboxyterminal substance P analogs such as DiMe-SP may preferentially activate NK3 receptors (9). NK3 receptors are present within the reticular formation of the medulla (25). Senktide, an NK3 agonist, excited a small number (two out of seven neurons) of neonatal bulbospinal C1 neurons recorded using in vitro patch clamp (20). AJP-Regul Integr Comp Physiol • VOL 288 • JUNE 2005 • www.ajpregu.org NK1-RECEPTOR ACTIVATION AND ANTAGONISM IN THE RVLM R1711 Pretreatment of the RVLM with WIN 51708 blocked the sympathoexcitatory effects of both DiMe-SP and [Sar9, Met (O2)11]SP. There was a small but significant increase in sSNA immediately after administration of WIN 51708. The exact cause of this unknown, but it may be due to a partial agonist activity of WIN 51708, as has been demonstrated previously in other tachykinin antagonists (16, 37). Bilateral microinjection of both DiMe-SP and [Sar9, Met (O2)11]SP into the RVLM markedly attenuated and completely abolished phrenic nerve activity, respectively. Immediately dorsal to the sympathoexcitatory neurons of the RVLM are the Bötzinger neurons of the ventral respiratory group (36, 42). Microinjection of excitatory amino acids in the Bötzinger complex decreases phrenic nerve burst amplitude and frequency (3, 4, 7, 45). This is presumably due to activation of inhibitory expiratory neurons (10, 38). NK1 receptors have also been demonstrated on both spinally and nonspinally projecting neurons within the Bötzinger/RVLM region (11, 24). Activation of NK1 receptors in the Bötzinger region may thus activate glycinergic inhibitory expiratory neurons resulting in an inhibition of phrenic nerve activity. As previous studies have suggested that the majority of NK1 receptor immunoreactive neurons of the ventral respiratory group are glutamatergic (11), the activation of inhibitory expiratory neurons may alternatively occur through an excitatory NK1 receptor immunoreactive interneuron. The fact that NK1 receptor activation within this region resulted in a robust inhibition of phrenic nerve activity, whereas NK1 receptor blockade had no effect, suggests that endogenous NK1 receptor agonists are not constitutively active in the respiratory-related neurons in this region. Activation of NK1 receptors on respiratory-related neurons in the RVLM may occur only under stressful physiological or pathological states. Further studies are required to uncover the mechanism of phrenic nerve activity inhibition by NK1 receptor activation in the Bötzinger region. Electrical stimulation of hindlimb somatic afferent nerves evokes an early and late excitatory response in SNA (27, 31). The excitatory peaks found in this experiment were similar in morphology and latency to those described in previous work from our laboratory and elsewhere (23, 27, 28, 32, 46). This reflex is dramatically attenuated by blockade of, for example, excitatory amino acid receptors in the RVLM (13, 29), and by activation of 5-HT1A (27) and ␦-opioid receptors in the RVLM (28), and by hypercapnia (23). Activation of NK1 receptors in the RVLM by [Sar9, Met (O2)11]SP abolished both peaks of the somato-sympathetic reflex, an effect blocked by WIN 51708. Given that activation of NK1 receptors in the RVLM produces a large sympathoexcitatory response, while leaving the baroreflex unaffected, it is unlikely that NK1 receptor activation abolishes the somatosympathetic reflex via a direct effect on bulbospinal C1 neurons. It may be that NK1 receptor immunoreactive terminals within the RVLM contain inhibitory neurotransmitters such as GABA or glycine and inhibit or selectively gate excitatory somatic inputs. We have previously demonstrated that NK1 receptor immunoreactive terminals exist in the RVLM, and some make close apposition with C1 neurons (24). It is important to note that two forms of the NK1 receptor exist: a long form and a C-terminus-truncated form (19). The commercially available NK1 receptor antibody only recognizes AJP-Regul Integr Comp Physiol • VOL 288 • JUNE 2005 • www.ajpregu.org Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017 Fig. 3. Group data for the effects of DiMe-SP (DiMe), [Sar9, Met (O2)11]SP (SarMet-SP) and WIN 51708 (WIN) within the RVLM on changes in ABP (A), changes in heart rate (HR; B), splanchnic sympathetic nerve activity (sSNA; C) and phrenic frequency (D). Note the increase in ABP, HR, and sSNA and the decrease in phrenic frequency after neurokinin 1 receptor (NK1R) activation with either DiMe or SarMet-SP. The highly selective NK1R antagonist WIN blocked the effects of SarMet-SP but only partially attenuated the effects of DiMe on ABP and phrenic frequency. ⫹P ⬍ 0.05 vs. baseline. *P ⬍ 0.01 vs. baseline. **P ⬍ 0.001 vs. baseline. R1712 NK1-RECEPTOR ACTIVATION AND ANTAGONISM IN THE RVLM the long form, and by definition, anatomical studies using this antibody do not demonstrate immunoreactivity to the short form. In humans and guinea pigs, up to 30% of the NK1 receptor population within the medulla oblongata is of the short form (2, 6). Further studies are required to demonstrate the distribution of the short form of the NK1 receptor within the ventral medulla. Microinjection of a 5-HT1A agonist into the RVLM markedly attenuates the somato-sympathetic reflex while leaving the baroreflex unaffected (27), a result similar to the finding in this study. This indicates the possibility that inhibition of the somato-sympathetic reflex by NK1 receptor activation may occur through a pathway involving 5-HT1A receptors in the RVLM. However, there is evidence that the major nuclei within the brainstem that have serotonergic projections to the RVLM do not display dual labeling for 5-HT and the NK1 receptor (1, 17). Nevertheless, it is possible that within the RVLM, substance P causes activation of presynaptic NK1 receptors on excitatory inputs to serotonergic projections, re- sulting in 5-HT release and subsequent binding to 5-HT1A receptors. This mechanism has previously been demonstrated in the dorsal raphe nucleus in the rat (22). As mentioned previously, it is also possible that serotonergic neurons might express the short form of the NK1 receptor (1, 17). The possibility that the significant increase in sSNA following NK1 receptor activation might itself attenuate the expression of both peaks of the somato-sympathetic reflex must also be considered. This is unlikely to be the case for two reasons. First, the increase in sSNA, though large, is not at the level where no further increase is possible. Following glutamate microinjection into the RVLM, maximum sSNA was found to be 2–3 times as great as the level attained by NK1 receptor stimulation (data not shown). Second, RVLM NK1 receptor activation did not alter the sympathetic baroreflex, a reflex mediated by the same neurons. An interesting finding is the effect of the highly selective NK1 receptor antagonist WIN 51708 on the sSNA chemoreflex response. The consistency of the hypoxic stimulus must be AJP-Regul Integr Comp Physiol • VOL 288 • JUNE 2005 • www.ajpregu.org Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017 Fig. 4. A: electrical stimulation of the tibial nerve (solid arrowheads) produced the two characteristic peaks of the somato-sympathetic reflex in the sSNA recording. Bilateral microinjection of [Sar9, Met (O2)11]SP in the RVLM almost completely abolished this response. This abolition lasted ⬃60 –90 min, followed by partial recovery. B: RVLM pretreatment with WIN 51708 blocked the effects of [Sar9, Met (O2)11]SP on the somato-sympathetic reflex. C: group data for the first and second peaks of the somato-sympathetic reflex. Note the almost complete inhibition of peak 1 and peak 2 after [Sar9, Met (O2)11]SP and the blocking of this effect by WIN 51708. Note also that peak 2 was absent in many experiments, and the effect of [Sar9, Met (O2)11]SP is only measured in two animals; however, in these animals the inhibition of peak 2 was to 8% and 9% baseline. *P ⬍ 0.05 vs. baseline. **P ⬍ 0.001 vs. baseline. NK1-RECEPTOR ACTIVATION AND ANTAGONISM IN THE RVLM R1713 considered. Experiments were conducted in a standardized manner, and animals were ventilated so as to maintain end tidal CO2 between 4 and 5%. Sympathoexcitation occurred rapidly after onset of ventilation with 100% N2, and the measured chemoreflex period occurred wholly within the 100% N2 administration period. Furthermore, the measured chemoreflex period was an average of 10 s immediately following the onset of excitation of phrenic nerve discharge. These factors suggest a consistent hypoxic stimulus. Hypoxia stimulates carotid body chemoreceptors and increases the activity of bulbospinal sympathoexcitatory neurons in the RVLM via the NTS (14, 30, 41). Blockade of excitatory amino acid neurotransmitters in the RVLM also blocks the sympathoexcitation evoked by the chemoreflex (14, 15, 30, 41). There is some evidence that NK1 receptor-containing neurons of the ventral medulla may be chemosensitive or modulate chemosensitivity (34). However, the fact that antagonism of NK1 receptors in the RVLM did not alter baseline respiratory frequency or phrenic nerve amplitude suggests that within this region the NK1 receptor is not involved in basal respiratory function. The NK1 receptor appears to play a role in sympathoexcitation within the RVLM under stressful stimuli, such as the severe hypoxia experienced within this experimental protocol. Substance P may be presynaptically reinforcing the excitatory NTS to RVLM connection (14, 30, 41). Removing this by NK1 receptor blockade would thus reduce the sympathetic response. The apparent failure of either DiMe-SP or [Sar9, Met (O2)11]SP to augment the chemoreflex may be due to the fact that the chemoreceptor facilitation is masked by the general sympathoexcitation evoked by the RVLM NK1 receptor activation. In summary, we find that in anesthetized, vagotomized, paralyzed, and ventilated rats, microinjection of NK1 receptor agonists bilaterally into the RVLM elicits robust increases in ABP, sSNA, and HR. It also results in the abolition of phrenic nerve activity and dramatic attenuation of the somato-sympathetic reflex while leaving the baroreflex unaffected. These effects are due to the selective activation of the NK1 receptor within the RVLM, as evidenced by the blocking of these effects by a highly selective NK1 receptor antagonist. The fact that the selective NK1 receptor antagonist had no significant effect on baseline ABP and HR and a very small effect on sSNA suggests that substance P and the NK1 receptor do not play a significant role in the tonic maintenance of blood pressure and sympathetic outflow. NK1 receptor antagonism also resulted in a dramatic attenuation of the sympathetic chemoreflex response to hypoxia, suggesting a modulation of the chemoreceptor pathway from the NTS to the RVLM by substance P. These results demonstrate that substance P and the NK1 receptor play an integral role in the regulation of cardiorespiratory reflexes within the RVLM. AJP-Regul Integr Comp Physiol • VOL 288 • JUNE 2005 • www.ajpregu.org Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017 Fig. 5. A: 15 s of 100% N2 resulted in robust excitation and rise in ABP (left). This response was not affected by either DiMe-SP or [Sar9, Met (O2)11]SP but was significantly attenuated by bilateral RVLM microinjection of WIN 51708 (right). B: group data for the effects of DiMe-SP, [Sar9, Met (O2)11]SP, and WIN 51708 on the chemoreflex. **P ⬍ 0.001 vs. baseline. C: intermittent stimulation of the aortic depressor nerve (solid arrowheads) resulted in a characteristic inhibitory trough in sSNA with a latency of 178 ⫾ 4 ms. Microinjection of DiMe-SP resulted in attenuation of this inhibition, whereas the highly specific NK1 receptor agonist [Sar9, Met (O2)11]SP and antagonist WIN 51708 had no significant effect. 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