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. 1999 Apr 15;516(Pt 2):471–484. doi: 10.1111/j.1469-7793.1999.0471v.x

The rostral ventrolateral medulla mediates the sympathoactivation produced by chemical stimulation of the rat nasal mucosa

Paul F McCulloch *, W Michael Panneton *, Patrice G Guyenet *
PMCID: PMC2269263  PMID: 10087346

Abstract

  1. We sought to outline the brainstem circuit responsible for the increase in sympathetic tone caused by chemical stimulation of the nasal passages with ammonia vapour. Experiments were performed in α-chloralose-anaesthetized, paralysed and artificially ventilated rats.

  2. Stimulation of the nasal mucosa increased splanchnic sympathetic nerve discharge (SND), elevated arterial blood pressure (ABP), raised heart rate slightly and inhibited phrenic nerve discharge.

  3. Bilateral injections of the broad-spectrum excitatory amino acid receptor antagonist kynurenate (Kyn) into the rostral part of the ventrolateral medulla (RVLM; rostral C1 area) greatly reduced the effects of nasal mucosa stimulation on SND (-80 %). These injections had no effect on resting ABP, resting SND or the sympathetic baroreflex.

  4. Bilateral injections of Kyn into the ventrolateral medulla at the level of the obex (caudal C1 area) or into the nucleus tractus solitarii (NTS) greatly attenuated the baroreflex and significantly increased the baseline levels of both SND and ABP. However they did not reduce the effect of nasal mucosa stimulation on SND.

  5. Single-unit recordings were made from 39 putative sympathoexcitatory neurons within the rostral C1 area. Most neurons (24 of 39) were activated by nasal mucosa stimulation (+65·8 % rise in discharge rate). Responding neurons had a wide range of conduction velocities and included slow-conducting neurons identified previously as C1 cells. The remaining putative sympathoexcitatory neurons were either unaffected (n= 8 neurons) or inhibited (n= 7) during nasal stimulation. We also recorded from ten respiratory-related neurons, all of which were silenced by nasal stimulation.

  6. In conclusion, the sympathoexcitatory response to nasal stimulation is largely due to activation of bulbospinal presympathetic neurons within the RVLM. We suggest that these neurons receive convergent and directionally opposite polysynaptic inputs from arterial baroreceptors and trigeminal afferents. These inputs are integrated within the rostral C1 area as opposed to the NTS or the caudal C1 area.

Stimulation of the upper respiratory tract, including the nasal mucosa, produces a powerful functional reorganization of the cardiovascular and respiratory systems (Daly, 1984; Widdicombe, 1986). This complex autonomic response usually includes apnoea in the expiratory position, bradycardia, and an alteration of peripheral vascular tone resulting in a redistribution of cardiac output (Angell-James & Daly, 1972; McRitchie & White, 1974; White et al. 1974; Gieroba et al. 1994). Generally, peripheral vasoconstriction is selectively increased so that there is a redistribution of cardiac output away from hypoxia-tolerant tissues. Fractional blood flow decreases to skeletal muscle, liver, spleen, intestines and other tissues that are capable of withstanding a temporary period of hypoxia (Zapol et al. 1979; Heieis & Jones, 1988; Ollenberger et al. 1998). In contrast, cerebral blood flow increases or remains nearly constant, and myocardial blood flow is reduced in proportion to the decrease in cardiac work that results from the bradycardia (Blix et al. 1976; Zapol et al. 1979; Yu & Blessing, 1997). The opposing effects of the increase in total peripheral resistance and decrease in cardiac output generally maintain mean arterial pressure at pre-stimulation levels. Essentially the cardiovascular responses are a defence mechanism to conserve oxygen by protecting against the developing hypoxia resulting from the asphyxic apnoea that prevents noxious substances or water from entering the lungs (Butler & Jones, 1982; Blix & Folkow, 1984; Daly, 1984; Widdicombe, 1986). However, an inappropriately produced or exaggerated response, especially in newborn animals, can lead to cardiac death, and may be a contributing factor in sudden infant death syndrome (Widdicombe, 1986; Sant'Ambrogio & Sant'Ambrogio, 1991).

Initiation of this response results primarily from stimulation of receptors on trigeminal afferent fibres, including those in the anterior ethmoidal nerve (Dutschmann & Herbert, 1997) that innervate the face and nasal passages. The central circuitry that mediates the cardiorespiratory responses to upper respiratory tract stimulation has been the object of very few investigations, none of which included electrophysiological recordings. Using Fos expression as a measure of neuronal activation, Gieroba et al. (1994) suggested that nasopharyngeal stimulation in conscious rabbits may stimulate catecholaminergic neurons within the ventrolateral medulla. Others have suggested that the dorsolateral pons or the nucleus tractus solitarii (NTS) may be involved (Dutschmann & Herbert, 1997, 1998).

Because neurons in the rostral C1 area of the ventrolateral medulla (RVLM) are an important source of tonic excitatory drive to sympathetic vasomotor neurons controlling peripheral vasculature, and are an essential component of many cardiovascular reflex pathways (Guyenet, 1990; Dampney, 1994; Spyer, 1994), we further examined their involvement in this trigeminally induced response. We first characterized the medullary pathway responsible for their activation by injecting kynurenate (Kyn), a general glutamate receptor antagonist, into the RVLM and other medullary nuclei important for controlling sympathetic tone. Our hypothesis that barosensitive bulbospinal neurons within the RVLM are involved in this response was then verified by single-unit extracellular recordings.

METHODS

All procedures were approved by local Animal Research Committees.

General procedures

Male Sprague-Dawley rats (n= 15) weighing 275-359 g were intubated after initial induction with 5 % halothane. The rats were artificially ventilated, usually with 100 % oxygen, for the rest of the experiment (approximately 60 breaths min−1, tidal volume (Vt) approximately 1 ml (100 g body weight)−1). Rectal temperature was maintained at 37 ± 0·5°C using a rectal thermocouple probe and ventral and dorsal heating sources. Halothane anaesthesia (1-2 %) was used for the remaining surgical procedures. A nasopharyngeal tracheal cannula was inserted as far as the choana. The right femoral artery and vein were cannulated for measurement of arterial blood pressure (ABP) and infusion of drugs, respectively. Heart rate was determined from ABP pulse pressures using a tachometer. Splanchnic sympathetic nerve discharge (SND), and in some animals phrenic nerve discharge (PND), were recorded with bipolar silver electrodes insulated with silicone dental impression material (Carlisle Laboratories, Rockville Centre, NY, USA). The left splanchnic sympathetic nerve was approached retroperitoneally and recorded, uncut, distal to the suprarenal ganglion (Koshiya & Guyenet, 1996). The right phrenic nerve was isolated via a dorsolateral approach and cut distally in the lower neck (Koshiya & Guyenet, 1996).

A concentric bipolar stimulating electrode (diameter, 250 μm; tip separation, 500 μm) was placed in the fascia around the mandibular branch of the facial nerve (Brown & Guyenet, 1985; Haselton & Guyenet, 1989). Monopolar stimulation (100 ms, 1-2 mA, 1 Hz) of the nerve orthodromically activated muscles underlying the vibrissae, providing an index for successful nerve stimulation. Nerve stimulation also generated a field potential within the facial nucleus recorded by a glass microelectrode (filled with 2 M NaCl; 4-8 MΩ impedance). We used these field potentials to map the co-ordinates of the caudal pole of the facial nucleus and thus to determine in each animal the precise location of the ventrolateral medulla and other brainstem locations (see below). A second identical stimulating electrode was implanted in the ipsilateral spinal cord (T3-T4) to antidromically activate reticulospinal neurons (Brown & Guyenet, 1985; Morrison et al. 1988; Haselton & Guyenet, 1989). The tip of the electrode was placed 1·0-1·5 mm below the dorsal surface of the spinal cord in the approximate location of the intermediolateral cell column. The exposed surface of the spinal cord was immersed in mineral oil.

After completion of the surgical procedures, halothane was discontinued and replaced with α-chloralose (65 mg kg−1i.v.; Sigma). The rats were then paralysed with pancuronium bromide (Pavulon; 1 mg kg−1i.v.). To maintain adequate anaesthesia after administration of the paralytic agent, supplemental doses of α-chloralose (20 mg kg−1i.v.) were given hourly, or if a strong nociceptive stimulus (toe pinch) produced a pressor response of more than 10 mmHg.

To stimulate the nasal mucosa, a gentle suction was applied to the free end of the nasopharyngeal tube. A cotton swab soaked in 100 % ammonia was placed immediately in front of the rats' nares for 5 s. This drew ammonia vapour through the nasal passages and over the nasal mucosa.

All physiological variables were monitored on a chart recorder and simultaneously stored on a videocassette recorder via a digitizer interface (Vetter 3000A, frequency range: DC-22 kHz) for subsequent computer analysis. Nerve activities were filtered (10-3000 Hz bandpass plus 60 Hz notch filter), full-wave rectified and averaged in 1 s bins. The residual noise following intravenous injection of phenylephrine that raised mean arterial blood pressure above the saturation point of the baroreflex was taken as 0 SND units and the mean activity at resting arterial pressures at the beginning of the experiment was arbitrarily defined as 100 SND units (Sun & Guyenet, 1987; Huangfu & Guyenet, 1991).

Baroreflex curves were generated by plotting SND (averaged over 1 or 2 s intervals) versus ABP (averaged during the same intervals) using dedicated computer software (Guyenet et al. 1987; Sun & Guyenet, 1987; Huangfu & Guyenet, 1991). In these experiments ABP was manipulated with i.v. injections of phenylephrine (1-4 μg kg−1) and/or sodium nitroprusside (5-10 μg kg−1) as required. In order to quantify the sympathetic baroreflex, two SND measurements were made from each plot, one at high (150-165 mmHg, SNDhigh) and the other at low (100-110 mmHg, SNDlow) ABP. The measurements represented the average of four consecutive 1 s bins. The baroreflex was quantified by the ratio of SNDhigh divided by SNDlow (baroreflex index). A value of 1 for this index indicates complete blockade of the reflex. In the absence of microinjected drugs, the high range of ABP was close to the saturation of the reflex, and the index had a value of between 0·05 and 0·15.

Injection of kynurenate into the brainstem

The broad-spectrum excitatory amino acid receptor antagonist kynurenate (25 mM kynurenic acid (Sigma) in artificial cerebrospinal fluid (mM): 124 NaCl, 26 NaHCO3, 1 NaH2PO4, 2·5 KCl, 2 MgSO4, 2 CaCl2, 10 glucose, pH 7·35 with NaOH), was injected bilaterally into several medullary locations (Guyenet et al. 1987; Sun & Guyenet, 1987; Huangfu & Guyenet, 1991). A glass pipette (tip, 20 μm) glued to a microlitre Hamilton syringe was used to deliver 50-100 nl Kyn per injection site. The injectate also contained 5 % rhodamine-tagged microbeads (20-100 nm in diameter) (Guyenet et al. 1987). For Kyn injections into the caudal C1 area (n= 4 rats), raphe obscurus (n= 5) and RVLM (n= 1), a dorsal transcerebellar approach was used (Huangfu & Guyenet, 1991). For Kyn injections into the NTS (n= 5), 1 mm ventral to the caudal C1 area (n= 2) and RVLM (n= 5), the head of the rat was flexed ventrally and the atlanto-occipital membrane opened (Guyenet et al. 1987). The microsyringe was advanced into the brainstem at an angle (usually 40 deg), either visually in relation to the calamus scriptorius (NTS) or sterotaxically in relation to the caudal pole of the facial nucleus determined with field potentials.

At the conclusion of the experiment, the rats were deeply anaesthetized with urethane, and then perfused transcardially with phosphate-buffered saline, followed by a 10 % formaldehyde solution. The brains were removed and postfixed with the same formaldehyde solution. The brainstems were cut coronally at 40 μm on a freezing microtome. A one-in-three series was Nissl-stained (Thionin), coverslipped (Krystalon; Harleco, Saddle Brooks, NJ, USA) and viewed with a fluorescence microscope to determine the location of the microbeads. The injection sites and major nuclei were plotted with the aid of a computer-driven stage and the Neurolucida software (Microbrightfield, Colchester, VT, USA).

RVLM unit recordings

We recorded single units from the RVLM using glass microelectrodes (WPI; filled with 2 M NaCl at pH 7·5; impedances: 4-8 MΩ, measured at 400 Hz), as described previously (Brown & Guyenet, 1985; Haselton & Guyenet, 1989). The recordings were filtered (bandpass: 200-4000 Hz), monitored on an oscilloscope, and recorded using a videocassette recorder and Vetter digitizer. Neuronal activity was also recorded as integrated rate histograms using 1 or 2 s time bins. We focused on the ventrolateral medullary region that contains the largest concentration of putative sympathoexcitatory neurons (rostral C1 area). This region is located 0-500 μm caudal to the posterior edge of the facial motor nucleus, 1·7-2 mm lateral to the mid-line, and within 400 μm of the ventral surface. Sympathoexcitatory neurons were identified as described before by their barosensitivity, the pulse synchrony of their discharges (obvious only in the most active cells), their relatively modest respiratory entrainment and, in most cases, by their projection to the thoracic cord (Brown & Guyenet, 1985; Morrison et al. 1988; Haselton & Guyenet, 1989). Spinal projections were identified by antidromic activation (T3, 0·2-2·0 mA, 200 ms, 1·0 Hz) and collision test. Conduction velocity was calculated by dividing the antidromic latency by the straight line distance between the stimulation and recording sites (typically 35 mm). Within this region, we also recorded from non-barosensitive neurons with activity that displayed a clear silent period during a particular phase of each respiratory cycle (Koshiya & Guyenet, 1996). These neurons with respiratory-related activity were commonly found 200-300 μm dorsal to barosensitive neurons (Brown & Guyenet, 1985; Haselton & Guyenet, 1989). A few were identified as Bötzinger neurons based on their discharge pattern during late expiration and their projection to the contralateral lateral reticular formation (Otake et al. 1987, 1988).

Statistics and presentation

Baseline values of ABP, SND and unit activity were determined by averaging 15 to 20 1 s bins preceding the onset of each trial (nasal stimulation). Peak values during each trial were measured as the average of the two highest 1 s bins, which typically occurred 4-6 s after the onset of the stimulus. Differences between baseline and peak values were compared with Student's paired t tests. One-way repeated measures ANOVAs were used to determine the effect of Kyn injections on the dependent variables (ABP, SND and baroreflex index at rest, peak ABP and peak SND during trials, and differences between peak and resting values). The significance level was set at P < 0·05. In the case of a significant ANOVA F value, Tukey's post hoc testing determined which groups were significantly different from each other. A computer program (SigmaStat; Jandel) was used for all statistical analyses. All values are presented as means ± standard error of the mean.

RESULTS

Cardiorespiratory responses to stimulation of the nasal mucosa

Drawing ammonia vapour through the nasal passages produced stereotyped cardiorespiratory effects consisting of an increase in SND, an elevation of ABP, a slight increase in heart rate and usually complete cessation of respiration (Fig. 1). The remainder of the study was designed to identify the circuits responsible for the sympathoactivation produced by nasal stimulation.

Figure 1. Cardiovascular and respiratory response to stimulation of the nasal mucosa or baroreceptors.

Figure 1

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Ammonia vapour was drawn over the nasal mucosa during the period indicated by the horizontal bar. Nasal stimulation resulted in phrenic apnoea, as evidenced by inhibition of the integrated phrenic nerve discharge rate (PND, integrated, top trace), an increase in splanchnic sympathetic nerve discharge (SND), an increase in arterial blood pressure (ABP) and an increase in heart rate (HR). Vertical scales for SND and PND are arbitrary.

Effects of brainstem injections of kynurenate on the sympathetic responses to stimulation of the nasal mucosa

Kyn, a broad-spectrum inhibitor of ionotropic glutamate receptors, was injected bilaterally into various brainstem locations to assess its effect on the sympathoactivation produced by stimulation of the nasal mucosa with ammonia. Three structures (the rostral C1 area, the caudal C1 area, sometimes called ventrolateral medullary depressor area, and dorsolateral NTS) were targeted because they are important relay nuclei in the baroreflex and potential targets of trigeminal subnuclei activated by nasal afferents. The exact co-ordinates of the rostral or caudal C1 area were identified in each rat by first locating the point at the caudal and ventral margin of the facial nucleus with antidromic field potentials (Haselton & Guyenet, 1989). The microinjections were made 0·2 mm (rostral C1 area) and 1·5 mm (caudal C1 area) caudal to this point. Control injections were made into structures that exert no or minor control over the splanchnic sympathetic outflow under anaesthesia, namely the raphe obscurus and the ventrolateral medulla ventral to the caudal C1 area (Fig. 2).

Figure 2. Kynurenate injection sites.

Figure 2

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Computer-assisted coronal drawings through the medulla oblongata showing the locations of Kyn injection sites. All injections (except those in the raphe obscurus) were bilateral and symmetrically located. Only one side is shown for simplicity. Injection sites that strongly attenuated sympathoexcitation in response to nasal stimulation are represented by filled circles. Sites in which injection of Kyn blocked the baroreflex but did not decrease sympathoexcitation in response to nasal stimulation are represented by open circles. Open diamonds represent injection sites which changed neither baroreflexes nor sympathoexcitation in response to nasal stimulation. Nomenclature and millimetric co-ordinates relative to bregma after atlas of Paxinos & Watson (1998). NTS, nucleus tractus solitarii; NA, nucleus ambiguus; pyr, pyramidal tract; XII, hypoglossal motor nucleus; LRN, lateral reticular nucleus; Sp5I, spinal trigeminal nucleus; IO, inferior olive; AP, area postrema.

Rostral C1 area (level -11·8 mm according to atlas of Paxinos & Watson, 1998)

Before injection of Kyn, stimulation of the nasal mucosa produced large increases in both SND and ABP (Fig. 3, mean data in Table 1). After Kyn injection, stimulation of the nasal mucosa produced significantly reduced effects on both SND and ABP (Fig. 3 and Table 1). The normalized response (sympathoactivation expressed as a percentage of the level of SND that existed before the stimulus) was reduced by 80 % by Kyn (Fig. 4). The effects of Kyn were reversible within 30 min (Fig 3 and Fig 4). Kyn injection into the rostral C1 area did not affect resting SND, ABP (Fig. 3A and Table 1) or the baroreflex (Fig. 3A and B and Table 2).

Figure 3. Sympathetic response to stimulation of the nasal mucosa after kynurenate injections into the RVLM.

Figure 3

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A, before injection of kynurenate (Control), ammonia stimulation of the nasal mucosa (NH3) produced an increase in SND and mean arterial blood pressure (MABP). Phenylephrine (PE, short arrow) increased MABP and decreased SND (baroreflex). Sodium nitroprusside (SNP, long arrow) decreased MABP and increased SND (baroreflex). Injection of kynurenate into the RVLM (Kyn) attenuated the increase in SND and MABP caused by nasal stimulation, while the baroreflex responses to PE and SNP were unaffected. After recovery from the effects of Kyn (Recovery), nasal stimulation, PE and SNP produced the same SND and MABP responses as in Control. B, the baroreflex curve (SND versus MABP) was unaltered by Kyn injection, although after recovery the maximum SND was slightly elevated.

Table 1.

Effects produced by stimulating the nasal mucosa with NH3 vapour following injections of kynurenate into various brainstem nuclei

Arterial blood pressure (mmHg) Sympathetic nerve activity (units)
Pre-NH3 NH3 Change Pre-NH3 NH3 % change
Rostral C1 (n= 6)
 Control 127 ± 5 167 ± 5* 40 ± 3 113 ± 13 283 ± 55* 142 ± 20
 Kyn 118 ± 8 133 ± 8*† 15 ± 5†‡ 130 ± 14 168 ± 23*† 29 ± 5†‡
 Recovery 122 ± 7 152 ± 9* 31 ± 3 110 ± 15 232 ± 44* 105 ± 14
Caudal C1 (n= 4)
 Control 133 ± 3 159 ± 4* 26 ± 4 117 ± 4 269 ± 36* 136 ± 36
 Kyn 175 ± 5§‖ 188 ± 3*§‖ 13 ± 3 407 ± 59§ 809 ± 95*§‖ 104 ± 13
 Recovery 145 ± 4§ 170 ± 6*§ 25 ± 6 318 ± 41§ 615 ± 72*§ 95 ± 6
NTS (n= 5)
 Control 132 ± 6 164 ± 8* 32 ± 6 124 ± 18 247 ± 61* 89 ± 23
 Kyn 162 ± 7§‖ 190 ± 6*§ 28 ± 5 169 ± 35§ 363 ± 128 91 ± 33
 Recovery 137 ± 9 169 ± 14* 32 ± 6 164 ± 35 307 ± 90 73 ± 19
Raphe (n= 5)
 Control 134 ± 5 157 ± 3* 23 ± 3 229 ± 38 429 ± 79* 89 ± 15
 Kyn 133 ± 4 154 ± 3* 22 ± 3 225 ± 39 400 ± 73* 81 ± 11
 Recovery 131 ± 5 142 ± 6* 22 ± 2 241 ± 41 405 ± 73* 71 ± 13
Ventral to caudal C1 (n= 2)
 Control 135 ± 5 168 ± 13 33 ± 8 116 ± 25 302 ± 114 154 ± 48
 Kyn 160 ± 1 199 ± 1 39 ± 2 166 ± 33 494 ± 214 183 ± 73
 Recovery 140 ± 0 177 ± 6 38 ± 6 170 ± 53 365 ± 153 107 ± 25
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Values are means ± s.e.m.; n, number of rats. Mean arterial blood pressure and integrated splanchnic sympathetic nerve discharge were recorded before (Pre-NH3) and during (NH3) stimulation of the nasal mucosa with ammonia vapour. Measurements were made before (Control), 5 min after (Kyn) and 30 min after (Recovery) Kyn injections into the RVLM (Rostral C1), caudal ventrolateral medulla (Caudal C1), dorsolateral nucleus tractus solitarii (NTS), raphe obscurus (Raphe) and 1 mm ventral to the caudal C1 area. Using Student's paired t test

*

significantly greater than pre-NH3. Using one-way repeated measures ANOVA

†

significantly less than control

‡

significantly less than recovery

§

significantly greater than control

‖

significantly greater than recovery.

Figure 4. Normalized sympathetic responses to stimulation of the nasal mucosa: effect of Kyn injections into rostral and caudal C1 area, NTS and raphe obscurus (Raphe).

Figure 4

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The normalized responses represent the percentage increase in SND caused by nasal stimulation relative to the baseline activity measured during the 2 min preceding the stimulus. Furthermore, in each case the sympathoactivation before administration of Kyn was also normalized as 100 %. This presentation illustrates the fact that Kyn caused a significant proportional decrease in SND only when it was injected into the rostral C1 area. In all other areas, nasal stimulation produced the same proportional increase in SND as in the control condition despite large changes in baseline SND.

Table 2.

Effect of brainstem kynurenate injections on baroreflex index

Control Kyn Recovery
Rostral C1 0.146 ± 0.068 0.104 ± 0.059 0.075 ± 0.039
Caudal C1 0.023 ± 0.008 0.931 ± 0.062*†‡ 0.011 ± 0.004
NTS 0.106 ± 0.071 0.769 ± 0.023*†‡ 0.205 ± 0.133
Raphe 0.043 ± 0.036 0.016 ± 0.009 0.050 ± 0.047
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Integrated SND was averaged over 4 s intervals during periods of high (150–165 mmHg; SNDhigh) and low (100–110 mmHg; SNDlow) ABP. ABP was brought into these high and low ranges by I.V. infusion of phenylephrine and/or sodium nitroprusside, as necessary. Measurements were made before (Control), 5 min after (Kyn) and 30 min after (Recovery) Kyn injections into the RVLM (Rostral C1), caudal ventrolateral medulla (Caudal C1), dorsolateral NTS or raphe obscurus (Raphe). The baroreflex index represents the ratio of SNDhigh divided by SNDlow. The baroreflex was not affected by Kyn injection into the RVLM or raphe obscurus, but was dramatically attenuated after injections into the caudal C1 and NTS (baroreflex index close to 1). Values are means ± s.e.m. (n values as in Table 1). Using one-way ANOVA

*

significantly greater than control or recovery

†

significantly greater than rostral C1

‡

significantly greater than raphe obscurus.

Caudal C1 area (level -13 mm)

Before injection of Kyn, stimulation of the nasal mucosa greatly increased both SND and ABP (Fig. 5A and Table 1). Kyn injection significantly increased the baseline levels of SND and ABP (Fig. 5A and Table 1). These injections also blocked the baroreflex as indicated by the flat relationship between SND and ABP (Fig. 5B) and a baroreflex index near 1 (Table 2). Kyn injections reduced the magnitude of the rise in ABP caused by stimulation of the nasal mucosa and increased the associated sympathoactivation (+402 units versus+152 units; Fig. 5A and Table 1). However, when the sympathoactivation was expressed as a percentage of the level of SND that existed before the stimulus (normalized response), the effect of nasal stimulation on SND was unchanged by Kyn (Fig. 4). The effects of Kyn were reversible within 30 min (Fig 4 and Fig 5).

Figure 5. Sympathetic responses to stimulation of the nasal mucosa after kynurenate injections into the caudal C1 area.

Figure 5

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A, before injection of Kyn (Control), ammonia stimulation of the nasal mucosa (NH3) produced an increase in both SND and MABP. Injection of Kyn into the caudal C1 area produced a large increase in baseline MABP, and an increase in baseline SND. In the presence of Kyn, nasal stimulation produced a larger increase in SND and a smaller pressor response. All responses recovered after 30 min (Recovery). B, the plot of SND versus MABP (baroreflex curve) became flat after injection of Kyn indicating that the baroreflex was blocked.

NTS

Before injection of Kyn into the NTS, stimulation of the nasal mucosa greatly increased both SND and ABP (Fig. 6A and Table 1). Kyn elevated the baseline levels of SND and ABP (Fig. 6A and Table 1) and virtually abolished the baroreflex (Fig. 6B and Table 2). Kyn reduced slightly the magnitude of the rise in ABP caused by stimulation of the nasal mucosa and increased the associated sympathoexcitatory response (+194 units versus+123 units; Fig. 6 and Table 1). However, when the sympathoactivation was expressed as a percentage of the level of SND that existed before the stimulus (normalized response), the effect of nasal stimulation on SND was unchanged by Kyn injection into the NTS (Fig. 4). The effects of Kyn were reversible within 30 min (Fig 4 and Fig 6).

Figure 6. Sympathetic responses to stimulation of the nasal mucosa after kynurenate injections into the NTS.

Figure 6

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A, before injection of Kyn (Control), ammonia stimulation of the nasal mucosa (NH3) produced an increase in both SND and MABP. Injection of Kyn into the NTS increased baseline SND and MABP. However, nasal stimulation still produced a further increase in both SND and MABP. After recovery from the effects of Kyn (Recovery), nasal stimulation again produced an increase in both SND and MABP. B, the baroreflex curves (SND versus MABP) indicate that Kyn injection into the NTS reversibly attenuated the baroreflex.

Control injections

Kyn injections into the raphe obscurus did not change the increase in ABP and SND caused by stimulation of the nasal mucosa (Fig. 4 and Table 1). These injections did not change resting SND or ABP (Table 1) nor the baroreflex (Table 2). Finally, Kyn injections 1 mm ventral to the caudal C1 area were also ineffective (Fig. 4 and Table 1).

Extracellular single-unit recordings within the RVLM

Stable long-term extracellular recordings (> 20 min) were made from 55 tonically active neurons within the rostral C1 area. Thirty-nine neurons were barosensitive (i.e. were silenced by i.v. injections of phenylephrine) and were activated (10-60 %) by lowering ABP with sodium nitroprusside (Figs 7A and Fig 8). Ten units had an on-off pattern of activity that was respiratory related and were not affected by altering ABP (i.e. Fig. 9). Six neurons were unidentified, having neither barosensitivity nor respiratory-related activity, and were excluded from further analysis.

Figure 7. Identification of a sympathoexcitatory neuron and its response to stimulation of the nasal mucosa.

Figure 7

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A, effect of phenylephrine (PE) on ABP (bottom trace) and simultaneously recorded single-unit activity (top trace), integrated PND (second trace) and SND (third trace). Note that inhibition of the neuron mirrored that of SND while PND was unaffected by the rise in ABP. Vertical scales for SND and PND are arbitrary. B, ammonia stimulation of the nasal mucosa (NH3) activated neuronal activity (same neuron as in A), increased SND and produced phrenic apnoea.

Figure 8. Pattern of response of sympathoexcitatory neurons to stimulation of the nasal mucosa.

Figure 8

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A, example of a neuron whose kinetics of response to ammonia (NH3) closely approximated that of SND (sustained increase group). Top trace: unit activity (integrated rate histogram; bin size, 2 s). Middle trace: simultaneously recorded SND (integrated; bin size, 2 s). Bottom trace: MABP. B, example of one neuron that responded transiently to ammonia stimulation (NH3, *; transient increase group). C, example of one neuron whose activity remained unchanged during nasal stimulation. D, example of one neuron that was inhibited by nasal stimulation, probably reflexly due to the increase in ABP. In all cases the response to i.v. phenylephrine (PE) and/or sodium nitroprusside (SNP) is shown as evidence of the barosensitivity of these cells.

Figure 9. Axonal conduction velocities and resting firing rates of sympathoexcitatory neurons.

Figure 9

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Scatter plots of resting discharge rate versus conduction velocity of bulbospinal sympathoexcitatory neurons tested for responsiveness to nasal mucosa stimulation. A shows neurons that were activated (Responders). These cells were divided into two groups. Filled circles represent cells that were activated with kinetics similar to those of the splanchnic response (sustained increase; e.g. Fig. 8A). Open squares indicate cells that were more transiently activated (transient increase; e.g. Fig. 8B). B shows neurons that did not increase their basal firing rate (Non-responders) in response to stimulation of the nasal mucosa. Filled circles denote neurons that were unaffected (e.g. Fig. 8C). Open squares denote neurons that were passively inhibited (e.g. Fig. 8D).

Barosensitive neurons

Most barosensitive neurons (24 of 39; 61·5 %) were activated by nasal stimulation (Fig. 8A). In this group of 24 cells, nasal stimulation increased SND by 101·9 %, ABP by 28 ± 2 mmHg and neuronal discharge rate by 65·8 %. Most commonly (19 of 24 cells) the kinetics of neuronal activation reproduced faithfully that of the sympathetic response (‘sustained increase’ group; Fig. 8A). However, in a minority of cases (5 of 24), neuronal activation was more transient than the splanchnic response (‘transient increase’ group; Fig. 8B) though the peak response of the cells (+78·0 % increase in discharge rate) was very similar to that of the sustained increase group (+64·4 %). The five cells with a transient response to nasal stimulation were unusually sensitive to baroreceptor activity as suggested by their especially large activation following nitroprusside injection (Fig. 8B). Therefore we assume that this type of cell responded transiently to nasal stimulation simply because its activation by trigeminal afferents was rapidly counteracted by baroreflex inhibition as soon as ABP started to rise.

Eighteen of the 24 responders (75·0 %) were identified as bulbospinal on the basis of antidromic responses from the thoracic spinal cord and time-controlled collisions between spontaneous and evoked spikes. Of the remaining six neurons, four could not be antidromically stimulated, and two were not tested for antidromic activation. Bulbospinal neurons that were activated by nasal stimulation (sustained and transient responders) had a mean basal firing rate of 15·7 ± 2·7 spikes s−1 and a mean conduction velocity of 2·7 ± 0·5 m s−1. Conduction velocity fell into two clusters (Fig. 9A). One cluster (8 of 18; 44·4 %) had a mean conduction velocity of 5·1 ± 0·2 m s−1. These neurons had a basal firing rate of 26·1 ± 2·9 spikes s−1. All of these neurons were from the sustained increase subgroup. The second cluster (10 of 18; 55·5 %) had a mean conduction velocity of 0·8 ± 0·1 m s−1 and contained both types of neurons (8 sustained increase neurons, 2 transient increase neurons). These neurons had a basal firing rate of 7·3 ± 1·6 spikes s−1.

The rest of the barosensitive neurons (15 of 39; 38·5 %) were either unaffected by nasal mucosa stimulation (n= 8 neurons, Fig. 8C) or were inhibited (n= 7 neurons, Fig. 8D). The cells that were inhibited appeared to respond reflexly to the increase in ABP caused by nasal stimulation as their inhibition was time-locked to the rise in ABP and of a magnitude commensurate to their response to i.v. phenylephrine. These cells were otherwise undistinguishable from the cells that were activated by nasal stimulation. In particular, the same proportion (11 of 15; 73·3 %) were identified as bulbospinal and their discharge rate and conduction velocity fell within the normal range (Fig. 9B).

Respiratory-related units

Single-unit extracellular recordings were obtained from ten neurons that exhibited on-off respiratory-related activity. These cells were recorded 200-300 μm dorsal to the barosensitive neurons within the rostral C1 area. All respiratory-related neurons were profoundly inhibited by stimulation of the nasal mucosa, most neurons being completely silenced. A Bötzinger neuron was fully identified by its projection to the contralateral ventrolateral medulla (Fig. 10A) and its late expiratory pattern of discharge in relation to the phrenic nerve discharge (Fig. 10B). Nasal stimulation decreased the discharge of this neuron in a manner proportional to the effect on the phrenic nerve (Fig. 10C).

Figure 10. Extracellular single-unit recording of a putative Bötzinger neuron.

Figure 10

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A, antidromic stimulation from the contralateral ventrolateral medulla (level: caudal C1; see Fig. 2). Arrow points to electrical stimulation artifact. The stimulation was triggered at variable times after spontaneous spikes (s). Note collision of the antidromically stimulated spike (a) in the bottom two traces. B, single-unit recording (top trace) showing that the neuron was active only during late expiration, as determined by the integrated phrenic neurogram (bottom trace). C, stimulation of nasal mucosa (NH3) silenced phrenic nerve discharge (bottom trace) and neuronal activity (top trace). In B and C, vertical scales for PND are arbitrary.

DISCUSSION

This study suggests that the sympathoactivation produced by chemical stimulation of the nasal mucosa is mediated predominantly by activation of bulbospinal sympathoexcitatory neurons located within the RVLM, including the C1 cells themselves. The sympathoexcitatory neurons are activated via the release of glutamate within the surrounding neuropil. The pathway from the trigeminal nucleus to the sympathoexcitatory neurons appears not to involve the NTS or more caudal regions of the ventrolateral medulla. We suggest that this pathway could involve a direct projection from the trigeminal nucleus to the RVLM.

Technical considerations and anatomical nomenclature

In the present experiments, stimulation of the nasal mucosa produced only two of the three classic physiological responses, namely an increase in sympathetic tone and inhibition of respiration. We did not observe bradycardia, but rather a mild tachycardia. Bradycardia was not observed perhaps because of the vagolytic effect of the paralysing agent pancuronium, and the powerful inhibitory effect of α-chloralose on vagal tone and our stringent criteria for anaesthesia (less than 10 mmHg increase in ABP in response to a strong nociceptive stimulation of the hindpaw).

Anatomical subdivision of the ventrolateral medulla into regions is disturbingly imprecise. For the sake of accuracy we have chosen in the present work to simply refer to millimetric co-ordinates in relation to clear cut anatomical landmarks such as the facial motor nucleus and the lateral reticular nucleus. Furthermore, our computer-assisted maps were matched to specific plates of a well-known atlas (Paxinos & Watson, 1998) to avoid any confusion regarding the medullary levels investigated. The C1 area extends from the caudal pole of the facial motor nucleus to the first few hundred micrometres of the rostral lateral reticular nucleus. The rostral half of the C1 area (0-700 μm caudal to the caudal end of the facial motor nucleus) coincides with the ventrolateral pressor area or RVLM. It is characterized by the presence of C1 and other cells with projections directed to the spinal cord (Tucker et al. 1987). These neurons are believed to have a sympathoexcitatory function (Dampney, 1994; Spyer, 1994; Sun, 1995; Guyenet et al. 1996). The caudal C1 area (700 μm caudal to the facial motor nucleus to the rostral tip of the lateral reticular nucleus), is characterized by the presence of C1 cells with projections to the hypothalamus and basal forebrain but not to the spinal cord (Tucker et al. 1987). The caudal C1 area has also been called the depressor area or CVLM and also contains the bulk of the propriomedullary GABAergic neurons that mediate the sympathetic baroreflex by inhibiting the sympathoexcitatory neurons of the RVLM (Dampney, 1994; Spyer, 1994; Sun, 1995; Guyenet et al. 1996).

Activation of C1 and other putative sympathoexcitatory neurons of the RVLM area by nasal stimulation

In agreement with previous studies, we failed to find any tonically active neuron in the rostral C1 area that was activated by raising arterial pressure. All the pressure-sensitive neurons were inhibited and had properties typical of previously described neurons with putative sympathoexcitatory function (Schreihofer & Guyenet, 1997).

Nasal stimulation activated a majority of the putative sympathoexcitatory neurons of the RVLM irrespective of their axonal conduction velocity or baseline discharge rate. Those with conduction velocities of less than 1 m s−1 and a low basal discharge rate have been recently identified as C1 neurons (Schreihofer & Guyenet, 1997). Thus our neurophysiological data suggest that nasal stimulation activates a significant proportion of the bulbospinal C1 neurons. This interpretation agrees with a prior study on awake rabbits in which exposure of the nasal passages to formaldehyde vapour produced Fos expression in many tyrosine hydroxylase-immunoreactive neurons of the RVLM (Gieroba et al. 1994). In addition, the present data show that nasal stimulation also prominently activates the barosensitive neurons with higher conduction velocity and high resting rate of discharge (Fig. 9A), which, in their majority, do not express catecholaminergic enzymes (Lipski et al. 1995; Schreihofer & Guyenet, 1997). These neurons are presumed to be glutamatergic. The pattern of activation of the putative sympathoexcitatory neurons was somewhat variable, activation being more transient in a minority of the responsive neurons (Fig. 8). These differences did not segregate according to phenotype (Fig. 9). Our preferred interpretation is that some neurons responded transiently to nasal stimulation because of an especially strong baroreceptor feedback that developed as soon as ABP started to rise. In any case, roughly a third of the putative sympathoexcitatory neurons were either unaffected or inhibited by nasal stimulation (Fig. 8C and D). Since the sympathoexcitatory neurons of the RVLM are believed to be organized in an organ-specific pattern (McAllen, 1986; McAllen & Dampney, 1990; McAllen et al. 1995), this result supports the notion that nasal stimulation activates sympathetic outflow differentially according to the vascular bed, as suggested by blood flow studies in awake animals (Zapol et al. 1979; Heieis & Jones, 1988; Ollenberger et al. 1998).

Neural pathway responsible for the activation of C1 and other putative sympathoexcitatory neurons

The demonstration that C1 and other putative sympathoexcitatory neurons are activated is necessary but not sufficient to establish the importance of the RVLM in mediating the sympathoexcitation elicited by nasal stimulation. A number of other cells may also be affected elsewhere in the medulla. To address this issue we used a more global approach consisting of microinjection of Kyn into the rostral C1 area. Kyn is a broad-spectrum specific glutamate receptor antagonist that blocks all classic excitatory amino acid receptor subtypes (kainate, AMPA and NMDA) (Collingridge & Lester, 1989). The usefulness of this technique for the identification of central nervous system pathways has been established previously (Gordon, 1987; Guyenet et al. 1987; Sun & Guyenet, 1987; Huangfu & Guyenet, 1991; Koshiya et al. 1993; Panneton & Yavari, 1995). The selectivity of Kyn injections has been well established and therefore control injections of vehicle or of an inactive analogue of Kyn (xanthurenic acid) were not repeated in the present experiments (Guyenet et al. 1987; Sun & Guyenet, 1987).

In agreement with prior studies (Guyenet et al. 1987; Sun & Guyenet, 1986, 1987; Koshiya et al. 1993), injection of Kyn into the RVLM produced no change in resting sympathetic tone, ABP or the baroreflex (Fig. 3 and Tables 1 and 2). This result has been variously interpreted. One theory based on cellular work in slices suggests that the resting discharge of the sympathoexcitatory neurons is due to intrinsic pacemaker properties and neurohormones, rather than glutamatergic inputs (Guyenet et al. 1996). Another theory suggests that the basal activity of these cells in vivo is due to the convergence of glutamatergic and local GABAergic inputs. This second theory assumes that both excitatory and inhibitory inputs could be reduced in equal proportion when glutamatergic receptors are blocked locally, since GABAergic neurons are usually also driven by excitatory inputs (Lipski et al. 1996; Ito & Sved, 1997). In any case, Kyn injection into the rostral C1 area dramatically reduced (by 80 %) the sympathoactivation caused by nasal mucosa stimulation. The most parsimonious interpretation is that this stimulus shifts the balance of inputs to the sympathoexcitatory cells towards more excitation and glutamate release. Although this experiment clearly shows that the RVLM plays an essential role in causing the sympathoexcitation, the exact synaptic mechanisms responsible for the activation of the bulbospinal neurons remain to be determined. The only secure conclusion is that glutamate receptor blockade by injections of Kyn must be very limited spatially because injections within the raphe obscurus, i.e. 1·7 mm away, produced no effect. Also injections into the caudal C1 area 1·3 mm from the rostral sites produced completely distinct effects and injections 1 mm below the caudal C1 area were also without effect. This leads one to conclude that the brain area affected by Kyn is likely to be a sphere of substantially less than 1 mm in diameter.

The ventral part of the medullary dorsal horn (MDH) is critical for the cardiorespiratory depression after nasal stimulation (Panneton, 1991b; Panneton & Yavari, 1995). Primary afferent fibres innervating the nasal cavity project here (Lucier & Egizii, 1986; Panneton, 1991a), and neurons in the ventral MDH show an increase in Fos after nasal stimulation (McCulloch & Panneton, 1997). Recent neuroanatomical studies have shown projections to the NTS and to both the rostral and caudal C1 area (Panneton & McCulloch, 1996; Panneton et al. 1997; Sun & Panneton, 1998). The densest projections from the MDH to the NTS are to neurons surrounding the solitary tract just below the obex, the same location as that of the Kyn injections in the present study. Yet our data suggest that neither the NTS nor the caudal C1 area play a major role in activating the sympathetic system when the nasal mucosa is stimulated since injections of Kyn into either of these areas failed to decrease the sympathoactivation produced by nasal stimulation (Fig. 4 and Table 1). This suggests that the trigeminal projections from MDH to the NTS and caudal C1 area may regulate a function other than sympathetic vasomotor outflow, perhaps respiration or the bradycardic response, which was not observed under the present anaesthetic conditions.

In cases in which Kyn was injected into either the NTS or caudal C1 area, large increases in resting ABP were produced. This confirms many previous observations and is explained in large part by the blockade of the baroreflex (Guyenet et al. 1987; Sun & Guyenet, 1987). After blockade of synaptic transmission in NTS with Kyn, the rise in ABP due to nasal stimulation was smaller than that before synaptic blockade. A similar reduction has been observed and interpreted as evidence that the NTS is involved in the pressor response seen after stimulation of the anterior ethmoidal nerve (Dutschmann & Herbert, 1998). We disagree with this interpretation and believe that the reduction in the magnitude of the pressor effect is probably due to the reduced vascular reactivity triggered by a large rise in circulating catecholamines when synaptic transmission in the NTS is impaired. Our view is supported by the neurally recorded sympathoexcitation caused by nasal stimulation that is increased in absolute terms by Kyn injection into the NTS (Table 1). This increase may be attributable to the loss of the buffering effect of the baroreflex (Table 2). When the effect of nasal stimulation was expressed as a percentage of the resting SND, Kyn injection into the NTS or caudal C1 area did not affect the response (Fig. 4). We interpret this result as evidence that synaptic blockade within NTS or the caudal C1 area prevents the buffering of the trigeminal response by the baroreflex but is not essential for the activation of the sympathoexcitatory neurons.

Summary and conclusion: neural pathway responsible for activation of the sympathetic system by stimulation of the nasal mucosa

The circuit responsible for the sympathetic activation caused by stimulation of the nasal mucosa is tentatively represented in Fig. 11. The peripheral receptors in part are located in the anterior portion of the nasal mucosa which is innervated by the anterior ethmoidal nerve, a branch of the ophthalmic division of the trigeminal nerve (Read, 1908; Greene, 1963). Although there are minor projections directly from the anterior ethmoidal nerve to the ventrolateral medulla (Panneton, 1991a), these projections apparently are not sufficient for the cardiorespiratory response to nasal stimulation (Panneton, 1991b; Panneton & Yavari, 1995). Rather, secondary trigeminal neurons in the superficial laminae of the ventral MDH project directly to the RVLM (Panneton & McCulloch, 1996; Panneton et al. 1997; Sun & Panneton, 1998). Here we characterize this part of the circuit by showing that stimulation of the nasal mucosa increases sympathetic nerve activity by activating the sympathoexcitatory neurons of the RVLM. The connection between the MDH and the sympathoexcitatory neurons could be direct, or, alternatively, indirect via as yet unidentified medullary interneurons. However, in any case, this connection recruits neither the brainstem circuitry of the baroreflex (NTS and caudal C1 area (Guyenet, 1990)) nor the raphe obscurus. The present data suggest that the baroreflex and the sympathoexcitatory effects produced by stimulation of trigeminal afferents may be integrated at the level of the rostral C1 sympathoexcitatory neurons or by interneurons located in their immediate vicinity.

Figure 11. Proposed circuit diagram.

Figure 11

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The proposed circuit consists of a multisynaptic input from the ethmoidal nerve to the rostral C1 area via the medullary dorsal horn (MDH). Activation of C1 and other sympathoexcitatory neurons that project to the sympathetic preganglionic neurons in the interomediolateral cell column of the spinal cord (IML) would cause the rise in SND. Differential activation of functional subgroups of sympathoexcitatory neurons would create the characteristic pattern of sympathoactivation triggered by nasal stimulation. Trigeminal and baroreceptor influences would be integrated primarily in the rostral C1 area, as opposed to the NTS or the caudal C1 area which contain two relays essential for the baroreflex but not for the trigeminal pressor response. CN IX, glossopharyngeal nerve.

Acknowledgments

This work was supported by a Grant-in-Aid to P. F. M. from the American Heart Association, Missouri Affiliate, NIH grant HL38471 to W. M. P., and NIH grant HL 28785 to P. G. G.

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