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Cardiovascular Research 2005 65(4):930-939; doi:10.1016/j.cardiores.2004.11.012
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Copyright © 2004, European Society of Cardiology

Functional interaction between nucleus tractus solitarius NK1 and 5-HT3 receptors in the inhibition of baroreflex in rats

M.-A. Comet, R. Laguzzi, M. Hamon and C. Sévoz-Couche*

Inserm U.288, Faculté de Médecine Pitié-Salpêtrière, 91 Boulevard de l–Hôpital, 75634 Paris Cedex 13, France

* Corresponding author. Tel.: +33 1 40 77 97 14; fax: +33 1 40 77 97 90. Email address: sevoz{at}ext.jussieu.fr

Received 6 September 2004; revised 28 October 2004; accepted 10 November 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: Previous data showed that in the nucleus tractus solitarius (NTS), 5-HT3 receptors are critically involved in the inhibition of cardiac baroreceptor reflex response occurring during the defense reaction. Since stimulation of NTS NK1 receptors has been found to inhibit the baroreflex bradycardia, we examined in this study whether this reflex response is inhibited during the defense reaction via an interaction between NK1 and 5-HT3 receptors.

Methods: For this purpose, we analyzed in urethane-anaesthetized rats the effects of intra-NTS GR205171, a selective NK1 receptor antagonist, on the baroreflex bradycardia inhibition observed either during the defense reaction triggered by electrical stimulation of the dorsal periaqueductal grey matter (dPAG) or after NTS 5-HT3 receptor activation.

Results: Intra-NTS GR205171, reversed, in dose-dependent manner, the inhibitory effect of dPAG stimulation on baroreflex bradycardia. This reversion was of 49% when both sinus carotid and aortic baroreceptors were stimulated by phenylephrine, and of 84% when aortic depressor nerve was stimulated. Similarly, intra-NTS GR205171 reversed partially or almost totally the inhibitory effect of local microinjections of phenylbiguanide, a 5-HT3 receptor agonist, on baroreflex bradycardia induced either by phenylephrine administration or aortic nerve stimulation, respectively.

Conclusion: These results strongly suggest that NK1 receptors contribute downstream to the 5-HT3 receptor-mediated inhibition of the aortic but not carotid cardiac baroreflex response occurring during the defense reaction, therefore implying that baroreceptor afferent inputs may be differentially modulated depending on their origin. This differentiation may be useful for a better understanding of baroreflex dysfunction in disease-induced conditions.

KEYWORDS Baroreflex; Serotonin; NK1; Nucleus tractus solitarius; Autonomic nervous system


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
In mammals, the essential role of arterial baroreceptors is to trigger the baroreflex aimed at buffering any deviation of arterial pressure. Impulses arising from the stimulation of these mechanoreceptors arrive to the nucleus of the tractus solitarius (NTS) [1]. As such, this nucleus plays an essential role in generation of the bradycardiac and hypotensive baroreflex responses. Several studies have shown that, in the NTS, excitatory amino acid afferents and receptors are directly involved in the transmission of baroreceptor signals [2–4]. But, the NTS is not only a relay station of baroreceptor afferent messages. Indeed, at this level, these messages are already integrated to other inputs that modulate (i.e., facilitate or inhibit) the aforementioned cardiovascular baroreflex responses [5]. However, important points concerning the processing of the baroreceptor messages in the NTS remain to be clarified, such as the neurochemical mechanisms involved in their modulations, and whether these modulations are similar depending on the origin of the baroreceptor inputs.

Most of the major neurotransmitters/neuromodulators including monoamines and peptides, such as serotonin (5-HT) and substance P, respectively, have been found in fibers and terminals in the NTS [6]. Some of these neuroactives molecules modulate the processing of arterial aortic and carotid sinus baroreceptor information through the stimulation of specific receptors. In particular, intra-NTS administration of substance P, to stimulate local NK1 receptors [7,8], or of phenylbiguanide [9], a selective 5-HT3 receptor agonist [10], has been shown to inhibit baroreflex bradycardia. Interestingly, other data indicate that prior intra-NTS microinjections of a selective GABAA receptor antagonist, bicuculline, reversed the baroreflex bradycardia inhibition produced by local stimulation of either NK1 [7] or 5-HT3 [9] receptors. Moreover, it has been recently demonstrated that bilateral microinjections into the NTS of selective NK1 and 5-HT3 receptor antagonists attenuate the inhibition of the arterial baroreflex bradycardia produced during somatic nociception [7,8] and stressful conditions like during the defense reaction [11], respectively.

Taken together, these data suggest that a functional link may exist between NK1 and 5-HT3 receptors in the NTS in the cardiac baroreflex modulation, especially during some stressful conditions like the defense reaction. To test this hypothesis, we first determined whether NK1 receptors were involved in the baroreflex inhibition produced during the defense reaction. For this purpose, we analyzed the effects of NTS microinjections of GR205171, a selective nonpeptidergic NK1 receptor antagonist [12], on the inhibition of the carotid and/ or aortic cardiac component of the baroreflex during the defense reaction obtained by direct electrical stimulation of the dorsal periaqueductal grey matter (dPAG). In a second part, to further characterize the possible interaction between NTS NK1 and 5-HT3 receptors, we analyzed the effects of (i) intra-NTS substance P upon the cardiac baroreflex response, before and after local microinjection of granisetron, a selective 5-HT3 receptor antagonist [10], and (ii) prior microinjections of GR205171 upon the inhibition of this baroreflex response normally induced by NTS microinjections of phenylbiguanide, a 5-HT3 receptor agonist.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. General procedures
Experiments were performed on 196 male Sprague–Dawley rats, weighing 330–370 g. Animals were kept under controlled environmental conditions (ambient temperature: 21 ± 1 °C, 60% relative humidity, food and water ad libitum, alternate 12 h:12 h light/dark cycles) for at least 1 week after receipt from the breeding center (CER Janvier, Le Genest-St Isle, France). Procedures involving animals and their care were all conducted in conformity with the institutional guidelines, which are in compliance with national and international laws and policies (Council directive n° 87-848, 19 October 1987, Ministère de l–Agriculture et de la Forêt, Service Vétérinaire de la Santé et de la Protection Animale, permissions n° 75-116 to M.H. and n° 75-117 to R.L.). The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication N°85-23, revised 1996).

Rats were anaesthetized with urethane (1.5 g kg–1, i.p.), and the depth of anaesthesia was regularly assessed by pinching a hindpaw and monitoring the stability of blood pressure and heart rate. In case of withdrawal reflex and/or variations of cardiovascular parameters, a supplementary dose of urethane was given (0.1–0.2 g kg–1 i.v.). A cannula was inserted into the femoral vein for administration of drugs and/or additional doses of urethane. Systemic blood pressure (BP), mean blood pressure (MBP), and heart rate (HR) were monitored (Pressure Processor and DC Amplifier, Gould, Courtaboeuf, France) through a catheter inserted into the femoral artery. Electrocardiogram (ECG) was recorded using stainless steel pins placed subcutaneously into fore- and hindpaws; signals were amplified and filtered (Universal Amplifier, Gould). The R wave of ECG was discriminated with a window discriminator and used to generate pulses. Arterial blood pressure and ECG pulse signals were relayed to a 1401 interface (1401 Plus, CED) connected to a computer running Spike 2 software (CED, Cambridge, UK). Heart rate was automatically computed from R wave pulses and displayed as mean frequency per minute (bin size: 1s). Rectal temperature was maintained at 37 °C with a thermostatically controlled heating blanket.

2.2. Procedures for intra-NTS microinjections and dPAG stimulation
The rats were placed in a stereotaxic frame with the head fixed in horizontal position. A craniotomy was performed, and a bipolar stimulating electrode was lowered into the dPAG using stereotaxic coordinates (P 6.7, L 0.7, V 3.5–4.5) from Paxinos and Watson's atlas [13]. dPAG was identified by observing body reactions and cardiorespiratory responses typical of the defense reaction caused by local electrical stimulation (50 Hz, 1 ms pulse duration, 200 µA, Sévoz-Couche et al., 2003): mydriasis, vibrissae and body movements, tail erection, rise in blood pressure, tachycardia, and a not quantified but visually clear increase in respiratory rate. In some rats, the left aortic depressor nerve was dissected out from the vagus nerve by a lateral approach and placed on silver bipolar hook electrodes for electrical stimulation.

For microinjections of neuroactive substances into the NTS, the dorsal surface of the brainstem was exposed through a limited occipital craniotomy. A single-barrel glass micropipette (<100 µm external diameter), connected to a Hamilton microsyringe filled with drugs or saline, was lowered into the commissural NTS at predetermined coordinates [9]: 0.4 mm rostral and 0.5 mm lateral to the calamus scriptorius, and 0.5 mm beneath the dorsal surface of the medulla. Microinjections (100 nl) were made over 2 s with a pneumatic microinfusion pump, and the micropipette was removed 15 s after the injection. The same micropipette was used for bilateral microinjections of a given drug. The time interval between two symmetrical microinjections was less than 1 min.

2.3. Quantification of the cardiovagal baroreflex response
To eliminate both the influence of the defense reaction-induced sympathetic activation on heart rate and the sympathetic cardioinhibitory component of the baroreflex, all rats used in this study were pretreated with atenolol (1 mg/kg, i.v., 20 min before phenylephrine injection or aortic nerve stimulation), a β-adrenergic receptor antagonist. This treatment produced no significant effect on baroreflex bradycardia, as previously observed [14]. In rats which received phenylephrine (PE; 5 µg/kg, i.v.) to induce activation of both carotid and aortic baroreceptors (n=108), the corresponding PE cardiac reflex response (PECR) was defined as the ratio of the maximal decrease in heart rate ({Delta}HR), expressed as percent change compared with HR baseline value, over the maximal increase in MBP [PECR in mm Hg–1=100 x ({Delta}HR/HR baseline)/{Delta}MBP]. Previous studies demonstrated that this ratio allows quantification of the baroreflex bradycardia as reliable as the slope of the linear decremental portion of the baroreceptor curve obtained by plotting HR as a function of MBP [3].

In rats in which the baroreflex response was triggered by electrical stimulation of the aortic nerve (n=88), the aortic cardiovagal response (ACR) was defined as the ratio of the maximal decrease in heart rate ({Delta}HR) over HR baseline value (ACR={Delta}HR/HR baseline).

2.4. Effects of intra-NTS microinjections of substance P on PECR and ACR
We analyzed the effects of local microinjection of four different doses of substance P on either PECR in four different groups of rats, or ACR in four other groups. PECR or ACR was tested before (10 min) and 2, 12, and 22 min after substance P. Only one given dose of substance P was tested in each rat.

In addition, PECR and ACR were tested in two other groups of rats, before and 2 min after intra-NTS microinjection of substance P (1 and 25 pmol, doses producing the maximal inhibitory effect on PECR and ACR, respectively, see Results), in rats locally pretreated (10 min before substance P) with a NK1 receptor antagonist, GR205171 (50 pmol, the dose producing maximal reduction of the inhibitory effect of dPAG stimulation on PECR or ACR; see Results).

2.5. Analysis of the effects of NTS NK1 receptor blockade on the inhibition of the cardiovagal baroreflex response by dPAG electrical stimulation
Respective PECR and ACR "controls" and "experimental" values were obtained as previously described in Comet et al. [14], with baroreflex triggered independently (controls) or concomitantly with dPAG stimulation (experimental).

As expected from data of a previous study [11], dPAG stimulation deeply reduced both PECR and ACR (see "Results"). Then, the influence of dPAG stimulation upon PECR or ACR was again tested in the same rats after (10 min) intra-NTS microinjections of saline or different doses of GR205171 (NK1 receptor antagonist), precisely at the level of the commissural nucleus [11,14]. Only one dose of GR205171 was tested in each rat. Bilateral intra-NTS microinjections of saline induced no significant MBP and HR changes, as well as on the decrease of PECR or ACR caused by dPAG stimulation.

2.6. Analysis of the functional interaction between NTS 5-HT3 and NK1 receptors on the baroreflex bradycardia inhibition
The effects of intra-NTS GR205171 (NK1 receptor antagonist) on the inhibition of PECR or ACR produced by intra-NTS microinjection of a predetermined [9] dose of phenylbiguanide (10 nmol) were analyzed. Two groups of rats were used in these experiments. In one group, PECR was tested before and 3 min after phenylbiguanide alone. In the other group of rats, PECR was tested before and 3 min after microinjections of a 5-HT3 receptor agonist, phenylbiguanide (10 nmol) into the NTS in rats pretreated locally (10 min before phenylbiguanide) with GR205171 (50 pmol). The same protocols were used in two other groups in which baroreflex was triggered by aortic nerve stimulation.

Finally, to analyse the effects of NTS 5-HT3 receptor blockade upon the inhibition of baroreflex bradycardia produced by substance P, PECR and ACR were tested before and 2 min after microinjection of substance P (1 and 25 pmol, respectively) into the NTS in rats pretreated locally (10 min before substance P) with a predetermined [11] dose of a 5-HT3 receptor antagonist, granisetron (250 pmol).

2.7. Statistical analyses
Absolute values are expressed as means ± S.E.M of n rats. One-way analysis of variance (ANOVA) followed by multiple comparison Scheffé's test were used to compare the effects of intra-NTS microinjections of different doses of substance P on PECR and ACR, and of different doses of GR205171 on PECR or ACR inhibition during dPAG stimulation. Other comparisons were made using Student's paired or unpaired t-test as appropriate.

2.8. Histology
At the end of experiments, electrolytic lesions (50 Hz, 4 mA, 20 s) were made at dPAG stimulation sites, and methylene blue (0.1 µl) was microinjected into the injection sites within NTS. Rats were then perfused intracardially, and brain coronal sections (60 µm) were cut and stained with neutral red.

2.9. Drugs
Atenolol base (Sigma-Aldrich, Saint Quentin-Fallavier, France), [3(s)-(2-methoxy-5 (5-trifluromethyltetrazol-1-yl)-phenylmethylamino)-2(s)-phenylpiperidine] (GR205171) and granisetron (Glaxo, Smith Kline-Beecham, Harlow, UK), 1-phenylbiguanide (Aldrich-Chemie, Steihem, Germany), phenylephrine chlorhydrate Chibret (Merck Sahrp and Dohme-Chibret, Paris, France), substance P (Sigma-Aldrich) were dissolved in saline. The pH of all solutions microinjected into the NTS was adjusted to 7.4.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
Baseline values of MBP and HR in atenolol-pretreated urethane anaesthetized rats were 83 ± 12 mm Hg and 333 ± 9 bpm, respectively (n=196).

3.1. Effects of dPAG stimulation on the reflex bradycardia induced by phenylephrine or aortic nerve stimulation
As previously reported [11,14], electrical stimulation of dPAG (Fig. 1A) produced an important inhibition of the baroreflex bradycardia normally evoked by phenylephrine (0.67 ± 0.04 vs. 0.16 ± 0.01 mm Hg–1, –75%, n=32, P<0.05) or by aortic nerve stimulation (0.50 ± 0.04 vs. 0.09 ± 0.01, –82%, n=24, P<0.05).


Figure 1
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  		Fig. 1 Coronal brain sections showing the localization of sites where electrical stimulation and microinjections were performed in dPAG and NTS. (A) Camera lucida drawing displaying the area (in dark grey) where electrical stimulations of dPAG (50 Hz, 200–300 µA, 1 ms) produced inhibition of phenylephrine (n=108)- or aortic nerve (n=88)-induced baroreflex bradycardia. (B) Section displaying the area (in black) in the NTS where bilateral microinjections of GR205171 (12.5–100 pmol, n=93), substance P (0.1–50 pmol, n=76), or phenylbiguanide (10 pmol, n=26) were performed. Anteroposterior levels (in mm) are from bregma, according to Paxinos and Watson's atlas [13]. Amb: ambiguus nucleus; AP: area postrema; cc: central canal; Cu: cuneate nucleus; DG: dentate gyrus; Gr: gracile nucleus; LRt: lateral reticular nucleus; mPAG: medial part of the periaqueductal grey matter; py: pyramidal tract; Sol: nucleus tractus solitarius; Sp5C:caudal spinal trigeminal nucleus; tfp: transverse fibers pons; 10: dorsal motor nucleus of the vagus; 12: hypoglossal nucleus.

 
3.2. Effects of intra-NTS microinjections of substance P on the baroreflex bradycardia
Bilateral intra-NTS microinjection of substance P (n=52), in a range of predetermined [7] doses (0.5–50 pmol/0.1 µl), produced an increase in MBP and a bradycardia. The maximal effects were obtained with the dose of 25 pmol. Indeed, this dose produced an immediate and brief (6–7 min) increase in MBP (+25 ± 2 mm Hg from a baseline of 84 ± 1 mm Hg, P<0.05) and a concomitant decrease in HR (–45 ± 7 bpm from a baseline of 334 ± 5 bpm, P<0.05).

Bilateral microinjections of substance P into the NTS at the abovementioned doses (n=52) also produced a dose-dependent inhibition of PECR (n=28) or ACR (n=24) tested 2 min after substance P administration (Tables 1 and 2Go, respectively). Data in Table 1 showed that the maximal inhibitory effect of substance P on PECR (–52%) was observed with the dose of 1 pmol (Fig. 2A.1), and that highest doses of the peptide did not further inhibit PECR. In contrast, substance P at the dose of 25 pmol nearly completely blocked ACR (–91%; Table 2; Fig. 2A.2). At all the doses tested, the effect of substance P was totally reversed after 32 min. Data in Tables 1 and 2Go also showed that in rats pretreated with GR205171 (NK1 receptor antagonist, 50 pmol), the inhibitory effects of substance P on PECR at the dose of 1 pmol (Table 1) or on ACR at the dose of 25 pmol (Table 2) were completely suppressed.


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  		Table 1 Cardiac baroreflex responses triggered by phenylephrine administration before and after microinjection into the NTS of substance P alone (SP) or after prior local microinjection of GR205171 (GR+SP)

 

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  		Table 2 Cardiac baroreflex responses triggered by aortic nerve stimulation before and after microinjection into the NTS of substance P alone (SP) or after prior local microinjection of GR205171 (GR+SP)

 

Figure 2
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  		Fig. 2 Effect of NTS microinjections of substance P and GR205171 on baroreflex bradycardia and defense reaction-induced inhibition of this reflex response, respectively. (A) Representative tracings showing the maximal inhibitory effect of the baroreflex bradycardia by intra-NTS administration of substance P when baroreflex was triggered by phenylephrine administration (A.1; PE, 5 µg/kg i.v., arrow), or by aortic depressor nerve stimulation (A.2; AS, horizontal bar). Note that this maximal inhibitory effect was approximately of 50% (with 1 pmol substance P) in (A.1), and almost total (with 25 pmol substance P) in (A.2). (B): (B.1) Representative tracings showing that phenylephrine (PE, arrow)-induced bradycardia (control) was reduced by electrical stimulation of the dPAG (horizontal bar, experimental). (B.2) The inhibition of phenylephrine-evoked baroreflex bradycardia normally observed during dPAG stimulation was reduced by prior bilateral microinjections into the NTS of GR205171 (50 pmol, 10 min before dPAG stimulation).

 
3.3. Effects of intra-NTS microinjections of a specific NK1 receptor antagonist
Prior (15 min) bilateral intra-NTS microinjections (see Fig. 1B) of a specific NK1 receptor antagonist, GR205171, at predetermined doses [15], prevented the inhibitory effect of dPAG stimulation on PECR (n=32) and ACR (n=24) in a dose-dependent manner (12.5 to 100 pmol; Figs. 3 and 4Go). In both cases, the maximal effect was achieved with 50 pmol of GR205171. This effect corresponded to a 49% reduction in PECR inhibition (Fig. 2B) and a 84% reduction in ACR inhibition as compared to those evoked by dPAG stimulation alone. The preventive effect of all doses of GR205171 upon the inhibition of PECR and ACR induced by dPAG stimulation lasted approximately 30–40 min. Control experiments showed that intra-NTS administration of GR205171 (50 pmol) produced a transient increase in blood pressure ({Delta}MBP:+25 ± 5 mm Hg from a baseline of 84 ± 6 mm Hg, n=12, P<0.05) which lasted 5 to 8 min but did not affect PECR or ACR tested 15 min later (Tables 1 and 2Go, respectively).


Figure 3
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  		Fig. 3 Dose-dependent effect of intra-NTS administration of GR205171 on the inhibition of phenylephrine-evoked baroreflex bradycardia by dPAG stimulation. Histograms showing that phenylephrine-evoked cardiac response (PECR control, n=40) was inhibited during dPAG stimulation (PE/dPAG, 200–300 µA), and that this inhibition was not affected after (10 min) intra-NTS bilateral microinjections of saline (n=8, grey bar) but was partially prevented in a dose-dependent manner by prior (10 min) intra-NTS administration of GR 205171 (25, 50, 75, and 100 pmol, n=8 for each dose, hatched bars). *P<0.05 compared with control, **P<0.05 compared with saline, ***P<0.05 compared with inferior dose. ns: not significant.

 

Figure 4
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  		Fig. 4 Effect of intra-NTS GR205171 on the inhibition of aortic baroreflex bradycardia by dPAG stimulation. Aortic cardiac response (ACR) elicited by aortic nerve stimulation (AS control) was deeply inhibited during dPAG stimulation (AS/dPAG, 200–300 µA, n=30). This inhibition was not affected after (10 min) intra-NTS bilateral microinjections of saline (n=6, grey bar) but was almost totally reversed in a dose-dependent manner by prior (10 min) microinjections of GR 205171 (12.5–100 pmol, n=6 for each dose, hatched bars) into the NTS. *P<0.05 compared with control, **P<0.05 compared with saline, ***P<0.05 compared with inferior dose. ns: not significant.

 
3.4. Analysis of NK1 and 5-HT3 receptor interactions in the NTS
We first confirmed, as already shown in a previous study [9], that baroreflex cardiac response was inhibited by prior (3 min) microinjections of a specific 5-HT3 receptor agonist, phenylbiguanide (10 nmol), into the NTS (Table 3). Interestingly, the inhibitory effect of phenylbiguanide was significantly reduced by prior (15 min) intra-NTS microinjections of GR205171 (NK1 receptor antagonist, 50 pmol; Table 3).


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  		Table 3 Cardiac baroreflex responses triggered by phenylephrine administration or by aortic nerve stimulation before and after microinjection into the NTS of phenylbiguanide (PBG) alone or after prior local microinjection of GR205171 (GR+PBG)

 
On the contrary, prior (15 min) intra-NTS microinjections of granisetron (250 pmol), a 5-HT3 receptor antagonist, did not modify the inhibition of PECR or ACR elicited by local administration of substance P (PECR after substance P, 1 pmol, in granisetron-pretreated rats was 0.30 ± 0.06 mm Hg–1, n=6; ACR after substance P, 25 pmol, in granisetron-pretreated rats was 0.05 ± 0.05, n=6, to be compared with data in Tables 1 and 2Go).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
The present data show for the first time that NTS NK1 receptors are involved in the defense reaction-induced inhibition of the aortic baroreflex bradycardia through interaction with local 5-HT3 receptors.

The existence of a functional link between 5-HT3 and NK1 receptors has been proposed at the gastrointestinal level [16,17]. In line with the existence of such receptor/receptor interaction, it is well established that both NK1 and 5-HT3 receptors expressed by gastrointestinal vagal afferents are involved in the emetic responses induced by cytotoxic anticancer drug treatments [18,19]. Clearcut evidence also supports the idea that interactions between NK1 and 5-HT3 receptors do exist as well in the NTS. Indeed, a GABAergic-dependent inhibition of the cardiac baroreflex response was observed after stimulation of either 5-HT3 or NK1 receptors in this nucleus [7–9]. Furthermore, both 5-HT3 and NK1 receptors in the NTS have been shown to play a role in the cardiac baroreflex response inhibition occurring during two different stressful conditions: the defense reaction [11,14] and somatic nociception [7], respectively. We presently found that, like the intra-NTS administration of granisetron [11,14], a selective 5-HT3 receptor antagonist, the bilateral local microinjection of GR205171, a selective NK1 receptors antagonist, attenuated, in a dose-dependent manner, the inhibition of the cardiac baroreflex response triggered by phenylephrine administration (PECR) or by aortic nerve stimulation (ACR) produced during the defense reaction elicited by dPAG electrical stimulation. However, at the difference of what was obtained with granisetron which reversed the dPAG-induced PECR and ACR inhibitions in the same manner, the reversion found after GR205171 microinjections was only partial (approximately 50%) when baroreflex was triggered by phenylephrine, which induces activation of both aortic and carotid baroreceptors, and almost total when aortic nerve was stimulated. NTS microinjections of GR205171 (NK1 receptor antagonist) produced also the same respective quantitative reversion of PECR and ACR inhibition obtained by microinjection of the specific 5-HT3 receptor agonist (i.e., phenylbiganide). These results strongly suggest that aortic but not carotid sinus baroreflex bradycardia is modulated by NTS NK1 receptor activation. In support with this hypothesis, we also found that substance P inhibited the baroreflex bradycardia also differently depending on whether the baroreflex was triggered by aortic nerve stimulation (approximately 80%) or by phenylephrine administration (approximately 45%). Thus, these data clearly show for the first time the involvement of NTS NK1 receptors in the inhibition of the cardiac component of the baroreflex occurring during the defense reaction. In addition, present data strongly suggest that different pathways are involved in the inhibition of either aortic (NK1 receptor-dependent) or carotid (NK1 receptor-independent) baroreflex messages. The latter conclusion implies that most NTS cells involved in the aortic baroreflex bradycardia are different from those contacted by carotid sinus afferents, and that they can be differently modulated. Hence, NTS cells responsible for the aortic baroreflex bradycardia may be contacted by substance P interneurons activated during the defense reaction, at the difference of neurons in the NTS receiving carotid baroreflex afferents. This idea is supported by the fact that the vast majority of NTS neurons receiving inputs from carotid sinus baroreceptors do not receive inputs from aortic nerve [20]. However, we are aware that the differential effect of substance P on inhibition of the baroreflex bradycardia either induced by aortic stimulation or phenylephrine could also be the result of some effect of phenylephrine on the baroreflex function. Indeed, by rising blood pressure and then possibly increasing the blood–brain barrier permeability [21], phenylephrine may act on central alpha 1-adrenoceptors, which are reported to inhibit the cardiac baroreflex response [22]. Although the fact that UK-52,046, a potent alpha 1-adrenoceptor antagonist, was found to have no effect on the phenylephrine-induced baroreflex sensitivity [23] does not support this possibility, other means to induce carotid baroreflex (i.e., activation of carotid sinus baroreceptors) would be necessary to exclude this hypothetical effect of phenylephrine on the degree of baroreflex bradycardia inhibition induced by NTS NK1 receptors.

It is also important to note that facilitation of cardiac baroreflex by NTS NK1 receptor activation has also been suggested in other studies. Indeed, (1) ablation of NK1 receptors in the rat nucleus tractus solitarius attenuates the cardiac baroreflex response triggered by phenylephrine administration [24], and (2) Seagard et al. [25] concluded that substance P in the NTS actually enhances the baroreflex sensitivity triggered by changes in pressure at the level of an isolated carotid sinus in anesthetized dogs. Concerning the apparent discrepancy between the facilitatory (abovementioned data) and inhibitory (present and previous [7,8] data) effects of substance P in the NTS upon the baroreflex, it is important to stress that different substance P-ergic systems within the NTS have been described. Indeed, the NTS contains substance P interneurons [26], as well as substance P afferents coming both from aortic and carotid sinus regions [27]. The existence of these local and peripheral substance P-ergic systems in the NTS agrees with the idea that NK1 receptor activation in this nucleus may produce facilitation as well as inhibition of the baroreflex. Hence, taking into account all these considerations, it seems that, on one hand, the phasic inhibition (i.e., during the defense reaction) of NTS aortic baroreceptor neurons is mediated through the activation, by substance P release after 5-HT3 receptor activation, of GABAergic interneurons endowed with one functional pool of NK1 receptors. On another hand, the release of substance P from baroreceptor afferents may directly activate all NTS baroreceptors neurons endowed with another pool of NK1 receptors to trigger a facilitatory modulation of the baroreflex bradycardia (Fig. 5). There is also the possibility that, instead of two functional pools, two subtypes of NK1 receptors would be in fact involved in the apparent dual effects of substance P on baroreflex bradycardia. Indeed, in support with this latter possibility, the existence of three different subtypes of NK1 receptors in the brain has recently been demonstrated [28]. However, the analysis of such a possibility awaits the development of specific antagonists for each subtype of NK1 receptors.


Figure 5
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  		Fig. 5 Functional interaction between NTS glutamatergic, 5-HT3, GABAA, and NK1 receptors in the medullary pathways involved in the baroreflex inhibition associated with the defense reaction. It is known that arterial carotid sinus and aortic afferents excite baroreceptor NTS cells (pale grey and white circle, respectively) through the activation of excitatory amino acid (EAA) receptors (small hatched square) to induce the vagal baroreceptor reflex bradycardia. Our previous [11,14] and present findings show that, during the defense reaction triggered by dPAG stimulation, release of serotonin (5-HT), presumably from neurons originating in medullary raphe nuclei, produces the activation of 5-HT3 receptors (small black circle), localized presynaptically on vagal glutamatergic afferents in the NTS. Our present data suggest that, depending on the origin (i.e., aortic or carotid sinus) of baroreceptor afferents, baroreceptor NTS cells are modulated by different pathways. Hence, we propose that, in the pathway involving modulation of NTS cells (white circle) receiving aortic afferents, the local release of glutamate, elicited by the stimulation of 5-HT3 receptors, provokes the excitation of substance P interneurons then of GABAergic interneurons endowed with NK1 receptor (small white circle). In a second pathway involving modulation of NTS cells (pale grey circle) receiving carotid sinus afferents, the abovementioned glutamate release directly excites GABAergic interneurons. In both pathways, the resulting GABAergic activation, finally produces the inhibition of the baroreflex bradycardia through the stimulation of GABAA receptors (small grey circle) present on each type of baroreceptor NTS cells. This hypothetical model takes also into account data suggesting that substance P (SP) released from baroreceptor afferents may facilitate baroreflex bradycardia through the activation of a second subgroup of NK1 receptors (small white dotted circle) probably located on baroreceptor NTS cells. +: excitatory effect; –: inhibitory effect.

 
Concerning the NTS transmission of inputs involved in the inhibition of the NK1 receptor-dependent aortic cardiac baroreflex component, data reported above indicate that such an inhibition produced by intra-NTS administration of phenylbiguanide (5-HT3 receptor agonist) was antagonized by prior local microinjections of GR205171. On the contrary, we observed that prior microinjections of granisetron (selective 5-HT3 receptor antagonist) into the NTS did not prevent the inhibition of the aortic baroreflex bradycardia produced by local substance P administration. These data mean that the aortic cardiac baroreflex inhibition is actually the consequence of the activation of 5-HT3 receptors that produces the release of substance P to induce NK1 receptor stimulation, and not the opposite (Fig. 5). In addition, previous data showed that (1) stimulation of presynaptic NTS 5-HT3 receptors triggers the local release of glutamate [29], (2) stimulation of NTS glutamate ionotropic receptors triggers the local release of substance P [30], and (3) prior microinjections of bicuculline, a GABAA receptor antagonist, into the NTS, suppress the inhibitory effects of both intra-NTS administration of substance P [7] and phenylbiguanide [9] upon the baroreflex. Consequently, from all these data, it can be proposed that NTS 5-HT3 receptor stimulation (i.e., during the defense reaction) produces, via the release of glutamate, the activation of substance P to, in turn, inhibit cardiac aortic baroreceptor neurons via the activation of local GABAergic interneurons endowed with NK1 receptors (Fig. 5). The data of previous studies showing that (1) in some structures, NK1 receptors are located on GABAergic interneurons [31,32], and (2) NTS contains high densities of both GABAergic interneurons [33] and NK1 receptors [34,35], agree with this hypothesis. In addition, it is possible that, in a second independent pathway, responsible for the NK1 receptor-independent inhibition of the cardiac carotid baroreflex bradycardia, GABAergic interneurons, directly activated by 5-HT3 receptor-induced glutamate release during the defense reaction, would not be in relation with substance P interneurons and would inhibit cells receiving cardiac carotid sinus inputs (Fig. 5). An overview of the resulting aortic and carotid baroreflex modulation by glutamatergic, 5-HT3, GABAA, and NK1 receptors within the NTS, taking into account present data and those in the relevant literature, is schematically represented in Fig. 5.

In conclusion, the results reported herein provide strong evidence that, in the NTS, NK1 receptors, through a functional interaction with local 5-HT3 receptors, participate in the inhibition of the reflex control of heart rate during the defense reaction, which is one of the essential cardiovascular adaptations during this behaviour assuring the animal survival. Present data also strongly suggest, that in the NTS, baroreflex cardiac afferent inputs are differentially modulated depending on their origin.

From a therapeutic point of view, the importance of these data appears by the fact that the cardiovascular adaptations (i.e., modulation of the baroreflex bradycardia) associated with emotional stressful conditions are linked to various cardiovascular dysfunctions in humans, including hypertension, cardiac arrhythmias, and myocardial infarction.


   Acknowledgements
 
This research has been supported by grants from INSERM. We are grateful to Glaxo Smith-Kline labs Beecham (Hertfordshire, UK) for generous gifts of granisetron and GR205171.


   Notes
 
Time for primary review 23 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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