1

NK1 RECEPTOR ACTIVATION IN RAT VENTROLATERAL MEDULLA

SELECTIVELY ATTENUATES SOMATO-SYMPATHETIC REFLEX WHILE

ANTAGONISM ATTENUATES SYMPATHETIC CHEMOREFLEX

John M. Makeham1,2, Ann K. Goodchild1,2 and Paul M. Pilowsky1,2

1Hypertension and Stroke Research Laboratory, Department of Physiology, University of

Sydney, NSW, Australia.

2Department of Neurosurgery, Royal North Shore Hospital, Sydney, NSW 2065, Australia.

Running Head: NK1-receptor activation and antagonism in the RVLM

Text pages: 30

Number of Figures: 5

*Correspondence to: Paul M. Pilowsky Hypertension and Stroke Research Laboratories Ground Floor, Block 3 Royal North Shore Hospital St Leonards, NSW, 2065 Australia. Tel: 02 9926 8080 Fax: 02 9926 6483 Email: pilowsky@med.usyd.edu.au

Articles in PresS. Am J Physiol Regul Integr Comp Physiol (February 24, 2005). doi:10.1152/ajpregu.00537.2004

Copyright © 2005 by the American Physiological Society.

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Abstract

The effects of activation and blockade of the neurokinin 1 (NK1) receptor in the rostral

ventrolateral medulla (RVLM) on arterial blood pressure, splanchnic sympathetic nerve

activity (sSNA), phrenic nerve activity, the somato-sympathetic reflex, baroreflex and

chemoreflex were studied in urethane anaesthetized, and artificially ventilated Sprague-

Dawley rats.

Bilateral microinjection of either the stable substance P analogue (pGlu5, MePhe8, Sar9)-

SP(5-11) (DiMe-SP) or the highly selective NK1 agonist [Sar9, Met(O2)11]-substance P into

the RVLM resulted in an increase in arterial blood pressure, sSNA, and heart rate and an

abolition of phrenic nerve activity. The effects of [Sar9, Met(O2)11]-substance P were

blocked by the selective non-peptide NK1 receptor antagonist WIN 51708. NK1 receptor

activation also dramatically attenuated the somato-sympathetic reflex elicited by tibial nerve

stimulation whilst 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.

Keywords substance P, rostral ventrolateral medulla, baroreflex, brainstem, sympathetic

nerve activity

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Introduction

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, 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 delta opioid receptors in the RVLM inhibits respiratory related discharge of

lumbar sympathetic nerve activity whilst having little effect on splanchnic sympathetic

nerve activity, potently attenuates the somato-sympathetic reflex, but has no effect on the

baroreflex or the chemoreflex. Stimulation of RVLM mu-opioid receptors, however, inhibits

the baroreflex whilst leaving the somato-sympathetic reflex and chemoreflex unaffected

(28). We have also demonstrated that activation of 5-HT1A receptor 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 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

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neurons (26). Microinjection of a stable substance P analogue into the RVLM causes

powerful pressor responses in vivo (43), and both substance P and the selective NK1

receptor agonist [Sar9, Met(O2)11]-substance P 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 effect 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 (300-500g) were initially anaesthetized with halothane (2% in

100% O2) followed by an intraperitoneal injection of urethane (1.25-1.3g/kg) and atropine

(90µg i.p.). The trachea was cannulated and the right cervical vagus nerve cut. The right

carotid artery was catheterised for arterial blood pressure measurement and the right jugular

vein was catheterised for drug administration. To isolate the aortic depressor nerve (ADN)

and phrenic nerve, a longitudinal posterolateral incision was made at the cervico-thoracic

junction. Following 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

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the carotid sheath. The splanchnic nerve was isolated following a longitudinal para-lumbar

incision and supero-medially directed blunt dissection in the retroperitoneal plane following

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 nerves fibres. The

animals were then secured in a stereotaxic frame, paralysed with pancuronium dibromide

(0.8mg i.v.) and artificially ventilated with O2 enriched air. End tidal CO2 was monitored

and maintained between 4-5% 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 anaesthesia was determined by monitoring the arterial blood pressure and the

phrenic nerve discharge. Additional doses of urethane (20-30mg i.v.) and pancuronium

dibromide (0.2mg i.v.) were given as required to maintain adequate anaesthesia and

neuromuscular blockade. Rectal temperature was maintained between 36-38°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 (100-3000 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 seconds).

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Activation of cardiovascular reflexes

To activate baroreceptor afferent fibres, the ADN was stimulated electrically. Maximal

activation of baroreceptor afferents was determined by tetanic ADN stimulation (0.2 ms

duration, 50 Hz for 5 seconds). The stimulation voltage was adjusted to achieve maximal

inhibition of sSNA. This was usually 0.5-4.0 volts. 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

(1ms duration, 20-30 volts).

Chemoreceptor activation was achieved by a brief period of hypoxia. Animals were

ventilated with 100% N2 for 15 seconds.

Microinjections

The stable substance P analogue (pGlu5, MePhe8, Sar9)-SP(5-11) (DiMe-SP, 600pmol in

50nl; Sigma), the highly selective NK1 receptor agonist [Sar9, Met(O2)11]-substance P

([Sar9, Met(O2)11]-SP, 600pmol in 50nl; Sigma) and the non-peptide selective NK1 receptor

antagonist Win 51708 (5nmol in 100nl; Sigma) were prepared for microinjection. The

DiMe-SP was dissolved in 10mM phosphate buffered saline (0.9%). The pH was found to

be 7.4. The [Sar9, Met(O2)11]-SP was dissolved in de-ionised water and the pH adjusted to

between 7.3-7.5. The Win 51708 was solubilized in 1% DMSO and the pH adjusted to

between 7.3-7.5. 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

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(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 (50mM, 50nl) in a single barrel micropipette. When a site was identified where L-

glutamate microinjection elicited a pressor response of >30mmHg, 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 fibres by 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 mins until the return of responses to pre-

injection levels. Due to significant desensitisation following 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.

8

Histological Procedure

At the end of each experiment the rats were killed with an intravenous injection of 1M KCl.

The medulla was removed and fixed in 10% formalin in de-ionised water overnight. The

medulla was then sectioned transversely (100µm) 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 counter stained using cresyl violet and mounted on glass

slides.

Data Analysis

Data were analysed during experiments and post acquisition using a CED 1401 data capture

system and Spike 4 software (Cambridge, U.K.). The average value over a 20 second 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

second period. The sSNA responses to intermittent ADN stimulation and tibial nerve

stimulation were analysed using peristimulus waveform averaging. The amplitude of the

sSNA from –200 to 0 ms prior to 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 seconds

following the onset of excitation of phrenic nerve discharge caused by the inhaled 100% N2

as a percentage change from a control period of 10 seconds average sSNA prior to 100% N2

inhalation.

Data are expressed as means and standard error of means (SEM). Statistical significance

was assessed by paired t-tests to compare effects before and after injection of a drug. To

9

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

significant) was conducted. All statistical analysis was performed using GraphPad software.

Results

Microinjection sites were located between 0 and 500µm 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 (600pmol, 50nl) bilaterally into the RVLM resulted in a rise in

arterial blood pressure (ABP) of 22 ± 3 mm Hg from 96 ± 6 to 119 ± 5 mm Hg (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, 3A). This rise was maximal at 2-5 minutes and lasted 30-40 min

Splanchnic SNA (sSNA) and HR were also significantly increased following the

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, 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, NS) or HR (436 ± 9

10

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 minutes following pre-treatment 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.

Pre-treatment with WIN 51708 failed to fully block the rise in ABP following DiMe-SP,

still significantly different from baseline with an increase of 11 ± 3mm Hg from 107 ± 5 to

119 ± 5mmHg (n=5, P<0.05, Fig 3A). However, as shown in Figs. 2B and 3A, pre-treatment

with WIN 51708 blocked the rise in ABP following [Sar9, Met(O2)11]-SP (139 ± 7 vs. 144 ±

11mmHg, n=5, NS). Pre-treatment 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). Pre-treatment 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, 3C).

Phrenic Nerve Activity

Phrenic frequency was significantly reduced following 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 mins (Fig. 2A).

There was no significant decrease in phrenic nerve amplitude following 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). Pre-treatment with WIN 51708 failed to block the phrenic frequency response to

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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 2-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. 4A&C). The second peak of the somato-

sympathetic reflex was often absent during baseline stimulation in these experiments. On

the 2 occasions it was elicited it was 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). Pre-treatment with WIN 51708 failed to block the response to

DiMe-SP of the first peak, still being 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). Pre-treatment 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).

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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). Pre-treatment 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 pre-

stimulus 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, Figs. 5A 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 pre-treatment

with the NK1 receptor antagonist WIN 51708 (986 ± 63 seconds (n=13) vs. 1116 ± 113

seconds (n=9), NS).

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Discussion

The principal novel findings of this study are 1) activation of NK1 receptors in the RVLM

in vivo results in hypertension, 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 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).

Pre-treatment 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

carboxy-terminal substance P analogues such as DiMe-SP may preferentially activate NK3

receptors (9). NK3 receptors are present within the reticular formation of the medulla (25).

14

Senktide, an NK3 agonist, excited a small number (2 out of 7 neurons) of neonatal

bulbospinal C1 neurons recorded using in vitro patch clamp (20).

Pre-treatment 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 following administration of WIN 51708. The exact cause of this unknown, but

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 non-spinally 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

15

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), by activation of 5-HT1A

(27), and delta-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 whilst leaving

the baroreflex unaffected, it is unlikely that NK1 receptor activation abolishes the somato-

sympathetic 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

recognises 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).

16

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 whilst 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 labelling 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,

resulting 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). Secondly, RVLM

NK1 receptor activation did not alter the sympathetic baroreflex, a reflex mediated by the

same neurons.

17

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 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 seconds 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 nucleus tractus solitarius (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, given 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

pre-synaptically 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.

18

In summary, we find that in anaesthetized, vagotomized, paralysed 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 whilst 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.

19

Acknowledgements and Grants

Work in our laboratory is supported by grants from the National Health and Medical

Research Council of Australia, the National Heart Foundation, The North Shore Heart

Research Foundation, the Doug White AVM Foundation, the Andrew Olle Memorial

Trust,the Garnett Passe and Rodney Williams Memorial Foundation and the High Blood

Pressure Foundation of Australia.

20

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Figure Legends

Figure 1. Microinjection site in the 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 = 500µm.

Figure 2. A: Bilateral microinjection of [Sar9, Met(O2)11]-SP into the RVLM (closed

arrows) elicited a sympathoexcitatory response and a rise in arterial blood pressure. There

was also a complete inhibition of phrenic nerve activity, returning in approximately 400s.

Open arrows indicate tetanic stimulation of the aortic depressor nerve (ADN). Closed star

indicates ventilation with 100% N2 for 15 seconds. During the sympathoexcitatory period,

multiple reflexes were tested, including the baroreflex, somato-sympathetic reflex and

chemoreflex. sSNA, splanchnic sympathetic nerve activity; ABP, arterial blood pressure. B:

Bilateral microinjection of [Sar9, Met(O2)11]-SP after pre-treatment with WIN 51708 in the

RVLM. WIN 51708 had no significant effect the sympathetic or phrenic nerve activity, but

did cause a small but significant rise in arterial blood pressure (left). Following pre-

treatment with WIN 51708, [Sar9, Met(O2)11]-SP had no significant effect on arterial blood

pressure, sympathetic nerve activity or phrenic nerve activity (right).

Figure 3. A-D: 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 arterial blood pressure

(ABP), changes in heart rate (HR), splanchnic sympathetic nerve activity (sSNA) and

phrenic frequency. Note the increase in ABP, HR and sSNA and the decrease in phrenic

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frequency following NK1 receptor activation with either DiMe or SarMet-SP. The highly

selective NK1 receptor 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.

Figure 4. A: Electrical stimulation of the tibial nerve (closed arrowheads) produced the 2

characteristic peaks of the somato-sympathetic reflex in the splanchnic sympathetic nerve

activity (sSNA) recording. Bilateral microinjection of [Sar9, Met(O2)11]-SP in the RVLM

almost completely abolished this response. This abolition lasted approx. 60-90mins,

followed by partial recovery. B: RVLM Pre-treatment 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 following [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 2 animals- however in these animal the inhibition of

peak 2 was to 8% and 9% baseline. (*) P<0.05 vs. baseline. (**) P<0.001 vs. baseline.

Figure 5. A: 15 seconds of 100% N2 resulted in robust excitation-excitation and rise in

arterial blood pressure (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). sSNA, splanchnic sympathetic nerve activity; ABP, arterial blood pressure.

B: Group data for the effects of DiMe-SP, [Sar9, Met(O2)11]-SP and WIN 51708 on the

chemoreflex. sSNA, splanchnic sympathetic nerve activity.(**) P<0.001 vs. baseline. C:

Intermittent stimulation of the aortic depressor nerve resulted in a characteristic inhibitory

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trough in splanchnic sympathetic nerve activity (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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