Rostra1 ventrolateral integration in rats

medulla

and sympathorespiratory

PATRICE G. GUYENET, ROBERT A. DARNALL, AND TINA A. RILEY Departments of Pharmacology and Pediatrics, University of Virginia School of Medicine, Charlottesville, Virginia 22908

GUYENET,PATRICEG.,ROBERT A. DARNALL,AND TINA A. RILEY. Rostra1 ventrolateral medulla and sympathorespiratory integration in rats. Am. J. Physiol. 259 (Regulatory Integrative Comp. Physiol. 28): R1063-R1074, 1990.-The respiratory modulation of the lumbar sympathetic nerve discharge (LSND) was examined in halothane-anesthetized, paralyzed, and vagotomized rats by means of phrenic nerve discharge (PND)triggered histograms. The respiratory modulation was 1) proportional to PND amplitude during chemoreceptor activation with CO, and 2) reduced at elevated arterial pressure. Bilateral injections of bicuculline [y-aminobutyric acid (GABA)* receptor antagonist, n = 51 into the rostra1 ventrolateral medulla (RVLM), but not into medullary raphe, reversibly increased mean arterial pressure (MAP) and resting LSND, decreased the baroreflex, reduced PND amplitude and central respiratory rate, and greatly magnified the respiratory modulation of LSND. Injections of strychnine (glycine receptor antagonist, n = 5) or phaclofen (GABAB receptor antagonist, n = 2) into RVLM were without effect. Injections of kynurenic acid (excitatory amino acid receptor antagonist) into RVLM (n = 8), but not raphe (n = 3), reduced PND amplitude, increased central respiratory rate, reduced MAP, elevated resting LSND slightly, and greatly reduced the respiratory modulation of LSND. These data suggest that the rostra1 tip of the ventrolateral medulla represents a critical link between the central respiratory rhythm generator and the vasomotor outflow. Also, it indicates that the respiratory modulation of SND does not involve a gating of the activity of the medullary neurons that convey baroreceptor information to RVLM sympathoexcitatory neurons. medulla oblongata; central control of arterial pressure; sympathetic nerve activity; central respiratory rhythm generation; excitatory amino acids; y-aminobutyric acid; glycine

PONTOMEDULLARY NETWORK (central respiratory rhythm generator; CRG) responsible for the rhythmic generation of the various respiratory outflows (phrenic, abdominal, intercostal, laryngeal, pharyngeal, etc.) and for their central chemosensitivity is believed to consist of a rhythm generator, perhaps made up of conditional bursting cells (8, 26), a pattern generator, and premotoneurons activating specific motoneuronal pools (see Ref. 8 for review). The sympathetic vasomotor outflow also represents one of the many targets of this respiratory rhythm-pattern generator, judging from the pronounced CRG-related rhythm of the mass or unit discharge of sympathetic nerves, which persists in vagotomized animals with or without sinoaortic baroreceptor denervation (1, 2, 5, 11, 12, 16, 24; for review see Ref. 29). THE

0363-6119/90

$1.50

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In va.gotomized rats, the CRG seems to shape the activity pattern of SYmP athetic vasomotor pre- or postganglionic cells into one of only two patterns. The-first consists of an inspiratory depression followed by a postinspiratory peak, and the second is characterized by a peak of activity coinciding with the middle of the inspiratory phase (7, 15, 25). These two patterns are found simu.ltaneously in the same preparation, but the proportion of units exhi .biting one or the other pattern varies in different sympathetic nerves (25). Although the CRG modulation of the sympathetic outflow is often reported to be species specific (7), the same two characteristic respiratory patterns are also simultaneously exhibited (albeit in different proportion) by separate reticulospinal sympathetic premotoneurons of the rostra1 ventrolateral medulla (RVLM) in both cat, rat, and rabbit subjected to vagotomy with or without additional barodenervation (15, 23, 34). Because, at least under anesthesia, these RVLM cells contribute one of the most important excitatory drives to sympathetic preganglionic cells, it is reasonable to theorize that they could be responsible for conveying most if not all of the CRG-related information to the sympathetic outflow. This hypothesis is also supported by the experiments of Connelly and Wurster (5), who demonstrated that the respiratory modulation of the sympathetic outflow descends via fibers located in the dorsolateral part of the lateral funiculus, which contains the axons of RVLM sympathoexcitatory neurons but not those of respiratory premotoneurons. The purpose of the present experiments was to determine how critical the RVLM actually is in the generation of the CRG modulation of the vasomotor outflow. This issue is approached by determining whether the CRG modulation of the sympathetic vasomotor outflow can be significantly altered by antagonists to the most common neurotransmitters [excitatory amino acids and y-aminobutyric acid (GABA)] when they are introduced into the RVLM in the immediate vicinity of the sympathetic premotoneurons. Another goal of the study was to determine whether the CRG modulation of the sympathetic outflow might be caused by periodic gating by the CRG of the activity of medullary neurons involved in the baroreflex. MATERIALS

AND

METHODS

General procedures. Male Sprague-Dawley rats (weighing 350-390 g) were tracheostomized under ether anesthe

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thesia, which was then maintained with 1.4% halothane in 100% oxygen during surgery. The tidal volume initially was set at 1 cm3/100 g, and the pump rate was adjusted to maintain end-expiratory CO, around 4.5% (Beckman infrared analyzer). Both vagal nerves were sectioned caudal to the confluence of the superior laryngeal nerves. The cervical sympathetic chain was left intact. It is likely that the aortic depressor nerves were also left intact, although these very fine nerves were not routinely identified. The femoral artery and veins were cannulated on one side for arterial pressure determination and fluid (sterile normal saline with 5% glucose) or drug administration. The left phrenic nerve was isolated as described previously and placed on a bipolar silver electrode for recording (15). The right half of the lumbar sympathetic chain was dissected at or immediately rostra1 to the origin of the iliolumbar artery and, after being cut distally, was placed on a bipolar recording electrode. Both nerves were immersed in Wacker Sil-Gel. The occipital plate was partially removed for a dorsal transcerebellar approach to the medulla oblongata. The right marginal mandibular branch of the facial nerve was identified through a skin incision, and a bipolar stimulation electrode was placed in the surrounding fascia to evoke antidromic activation of facial motoneurons (for details see Ref. 3). Thus the final preparation consisted of the rat in a prone position in the stereotaxic frame with its lower body rotated ~150’ to gain access to the lumbar sympathetic chain. Before recording, the halothane concentration was reduced to 0.05% above the of the minimum at which barely detectable withdrawal distal phalanges could be triggered by strong nociceptive stimulation of the hind paws. After 30 min of stabilization at this concentration (0.85-0.90% halothane), the animals were paralyzed with pancuronium bromide (l.O1.5 mg/kg iv). When appropriate, arterial pressure was raised or lowered by infusion of phenylephrine (PE; 0.2 mg/ml) or sodium nitroprusside (SNP; 0.5 mg/ml) into the femoral vein via a syringe pump. Recordings. Phrenic nerve discharge (PND) and lumbar sympathetic nerve discharge (LSND) were recorded with differential amplifiers using a band pass of 30-3,000 Hz plus a 60-Hz “notch” filter. Both signals were fullwave rectified and stored together with the arterial pressure and end-tidal CO2 on magnetic tape (3-dB band width O-1,125 Hz) for later analysis. Previously described custom-developed software written for an IBM-PCXT microcomputer outfitted with a Metrabyte DASH-16 data acquisition board was used to generate PND-triggered or femoral pulse-triggered averages of the LSND (13, 15). Sampling rate was 1 kHz, and a minimum of 100 consecutive sweeps of 2,500-ms length were used. Although some aliasing was expected to occur at the sampling rate used, its effect on the final histograms was minimized by the summation of 100 sweeps. Indeed, PND-triggered averages were not noticeably different when the same or closely overlapping segments of taped data were reanalyzed several times consecutively. Bin regrouping (20 ms/bin) was done to generate the final histograms shown in Figs. 1-12. The noise level for LSND was determined at the end of the experiment by constructing in each experiment a PND-triggered aver-

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age of the residual electrical signal recorded immediately after the intravenous administration of a large dose of clonidine (200 pg/kg). The hypertension (~140 mmHg) and consequent baroreceptor activation plus the central sympathoinhibitory effect of this drug resulted in the elimination of sympathetic activity for 5-10 min. The voltage corresponding to the computer-averaged noise was subtracted from pulse or PND-triggered averages of LSND to quantify the relative amplitude of the mass LSND at each point during the phrenic or cardiac cycle. Microinjections and histology. The injection devices and the electrophysiological means by which these can be accurately targeted to the RVLM or elsewhere have been previously described (33). All drugs were dissolved in phosphate (20 mM)-buffered saline (pH 7.3) and contained 9% (vol/vol) of a commercial suspension of rhodamine-tagged microbeads (Lumafluor, New City, NY). Final concentrations of drugs in the injectate were (in mM) 45 kynurenate, 4.5 bicuculline or strychnine, and 14 phaclofen (a GABAB antagonist). Injection volumes were 50 nl except where indicated. After aldehyde fixation, the brains were cut with a vibratome (40 pm). Every fourth section had a cover slip put on with Krystalon for inspection under fluorescent light, and a series of alternate sections were Nissl stained. Injection sites were plotted on a series of standardized sections derived from our own atlas. The nuclear nomenclature is borrowed from the atlas of Paxinos and Watson (27), except- as pertains to the RVLM. Drugs and chemicals. Strychnine, kynurenic acid, and bicuculline methiodide were obtained from Sigma, phaclofen from Research Biochemical, and pancuronium bromide from Astra Pharmaceutical. Statistics. Most statistical analyses were performed using a commercially available statistics package (Systat, Evanston, IL). Paired t tests were used to compare the mean effects of various pharmacological and physiological manipulations. Regression analyses were performed with a multivariate general linear model and analysis of variance. In some cases the slopes of the regressions were compared using a small-sample t test for parallelism (17). All results are expressed as means t SE. RESULTS

Effect of arterial pressure on cardiorespiratory integration. At resting arterial pressure (104.9 t 4.3 mmHg) and in the presence of high levels of end-tidal CO2 (7-8%), PND-triggered LSND averages exhibited one of two slightly different but distinct patterns illustrated in Fig. 1. The dominant pattern (Fig. 1A) consisted of an inspiratory depression (latency of nadir = 95-170 ms after PND onset) followed by a single postinspiratory peak (latency = 314-505 ms after PND onset). In a few cases (Fig. 1B) an additional well-discriminated but smaller peak occurred during late inspiration (latency = 145-360 ms after PND onset). This peak was often reduced to a shoulder on the ascending phase of the larger postinspiratory peak (not illustrated). The latency of the postinspiratory peak increased linearly with the duration of the phrenic burst, whereas that of the inspiratory nadir was relatively constant (Fig. 1C). The latency of the inspir-

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macological treatments. Preinspiratory LSND level was determined by computer-averaging LSND over a ZOO-ms period preceding the onset of the second PND waveform of the histogram. To determine the effect of baroreceptor feedback on the respiratory modulation of SND, in a series of 10 animals, the variables illustrated in Fig. 2 were measured at rest [mean arterial pressure (MAP) = 104.9 t 4.3 mmHg], during PE infusion (MAP = 123.3 t 4.3 mmHg, P c O.OOl), and again during infusion with SNP (MAP = 73.3 t 4.0 mmHg, P < 0.001, vs. both control and PE periods). End-tidal CO2 was maintained at 7.58% throughout each experiment. The typical effect of these manipulations is illustrated in Fig. 3, and the overall data presented in Table 1. Under conditions of reduced systemic pressure (i.e., with SNP), there was no significant change from resting state in any of the variables measured, although mean PND tended to be elevated (mean = 118% control) as well as preinspiratory LSND (mean = 138% control). When pressure was elevated

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atory peak also exhibited a positive but weaker correlation with PND duration. Figure 2 represents idealized patterns of sympathetic nerve discharge (SND) and PND, in which several characteristic variables of the dominant pattern are defined. The variables, preinspiratory LSND level (PreIL), postinspiratory peak (PostIP), inspiratory depression (Idep), total modulation (TOTmod), inspiratory nadir (Inadir), PND amplitude (PNDamp), phrenic cycle time (Ttot), and phrenic inspiratory time (Ti), were systematically measured to quantify waveform changes during physiological or phar-

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PND DURATION (ms) FIG. 1. Respiratory modulation of lumbar sympathetic nerve discharge (SND) in vagotomized rats. A and B: 2 alternate configurations of respiratory modulation of lumbar SND. In a minority of cases (B), an early inspiratory peak of activity preceded ubiquitous peak during postinspiratory phase. SND and phrenic nerve discharge (PND) were full-wave rectified and digitally averaged (100 sweeps) with trigger (tine 0) set at start of phrenic burst. Long dashed line (noise) represents background nonneuronal signal recorded from lumbar nerve after intravenous administration of a high dose of clonidine. All vertical scales are arbitrary. C: relationship between PND duration and latency of inspiratory nadir (Inadir) and inspiratory (InspP) and postinspiratory (PostIL) peaks (results acquired in 16 rats in presence of various CO* concentrations in inspired gas).

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TIME (ms) FIG. 2. Definitions for variables of PND and PND-triggered lumbar SND averages that were selected to quantify waveform changes resulting from physiological or pharmacological manipulations. A: definitions of variables relating to timing of PND. Ttot, total cycle time; Ti, neuronal inspiratory time; and PNDamp, amplitude of integrated phrenic burst. Te (phrenic expiratory time, not shown), Ttot - Ti. B: definitions of variables related to signal averaged SND histograms. PreIL, mean baseline nerve activity; PostIP, amplitude of postinspiratory peak of activity relative to baseline (SND noise, arbitrarily set to 0 mv in Fig. 2); Idep, amplitude of inspiratory depression of activity relative to baseline; TOTmod, maximum activity (PostIP) relative to minimum activity (Idep); and Inadir, level of minimal activity during inspiration relative to baseline.

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Values are means & SE. PND, phrenic nerve discharge; LSND, lumbar sympathetic nerve discharge; MAP, mean arterial pressure; PNDamp, PND amplitude; PreIL, preinspiratory LSND; TOTmod, total modulation; PostIP, postinspiratory peak; Idep, inspiratory depression; Inadir, inspiratory nadir. * Significantly different from resting; t significantly different from phenylephrine.

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TIME (ms) FIG. 3. Effect of baroreceptor activation on respiratory modulation of lumbar SND (LSND). Typical example of PND-triggered LSND generated at resting pressure [mean arterial pressure (MAP) = 104.9 k 4.3 mmHg; A], during an infusion of phenylephrine (MAP = 123.3 t, 4.3 mmHg; B), and during infusion of vasodilator sodium nitroprusside (MAP = 73.3 2 4.1 mmHg; C). Note reduction in respiratory modulation at elevated pressure (B) and very small effect on PND. SND noise is represented by long dashed line.

(i.e., with PE), PND was modestly reduced; however, this reduction (9.5%) did not quite reach our significance cut off (P = 0.063). All SND variables were significantly reduced by a similar percentage. When comparisons were made between reduced pressure (SNP) and elevated pressure conditions (PE), PND amplitude and all LSND parameters were significantly different. The total respiratory modulation of the LSND signal and the preinspiratory LSND level were highly correlated when examined at all three arterial pressure (AP) levels, and as illustrated in Fig. 4, the two variables appeared to be linearly related (r = 0.783, P < 0.001). In contrast, there was little correlation between PND amplitude and the total respiratory modulation of LSND (r = 0.498, P = 0.036). Effect of chemoreceptor activation on relationship between respiratory modulation of sympathetic discharge

and amplitude of phrenic discharge. In this series of experiments, the rats were hyperventilated to near apnea, and then COz was added to the breathing mixture in amounts designed to produce stepwise increments in the PND. For each experiment, PND-triggered averages of LSND were constructed at two to six different levels of PND, and the variables depicted in Fig. 2 were collected. The values of these variables measured at the highest level of PND corresponding to the highest level of end-expiratory CO2 (7-8%) were taken as lOO%, and all other values were expressed as a percentage of the high PND control values. The total modulation of the LSND, as well as the amplitude of the postinspiratory peak, were linearly related to PND amplitude over a wide range of PND (Fig. 5). Figure 5, in which each line represents a different animal (n = 8), illustrates the individual variability in the relationship between SND modulation and PND amplitude. On average, however, there was proportionality between PND amplitude and the total modulation or postinspiratory peak (see also Fig. 9). Also, there was a significant correlation between PND and the preinspiratory level (r = 0.600, P c 0.001) and the inspiratory nadir (r = 0.777, P c 0.001). In addition, MAP showed a direct correlation with PND (r = 0.540, P = 0.017). Injections of kynurenic acid into RVLM: effects on respiratory and sympathetic outflows. The PND-related pattern of LSND and that of the sympathoexcitatory cells of the RVLM are very similar (15, 23, 34). This suggests 1) that these cells may be responsible for relaying the respiratory pattern of LSND from medulla to cord and 2) that their respiratory modulation might be due to a CRG-related excitatory drive during the early expiratory phase and possibly to a separate inhibitory input during phrenic inspiration. Because of the prevalent role of excitatory amino acid transmission in medullary sympathetic networks (33), kynurenic acid, a nonselective antagonist of excitatory amino acid receptors (33), was microinjected into the RVLM of 12 rats (bilateral 50-nl injections, each containing 2.25 nmol of kynurenate) to address the possibility that the release of an excitatory amino acid might be involved in the expiratory drive of RVLM sympathoexcitatory neurons. All rats were ventilated with a concentration of CO, in the breathing mixture producing a maximal PND amplitude, and this concentration was maintained throughout the pharmacological challenge. The effects of kynurenate on

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PND (X control) FIG. 4. Respiratory modulation of LSND during baroreceptor activation: correlation with preinspiratory SND level and PND. A: correlation between total modulation of SND (TOTmod) and preinspiratory level (PreIL; for definition of terms, see Fig. 2). PreIL is taken as index of nonrespiratory modulated component of SND. B: correlation between TOTmod and PND amplitude. All variables are expressed as percent of values observed at resting arterial pressure. Note highly significant correlation between TOTmod and PreIL but very weak correlation between TOTmod and PND amplitude.

MAP, PND (amplitude, rate, and duration), and LSND (all variables described in Fig. 2) were determined at various times after the injection. All values are expressed as percentage of the control (predrug) values. In eight cases, injection sites were located very rostrally 1.n the ventrolateral medulla or in the caudal end of the facial motor nucleus (Figs. 6A and 7). A typical example taken from this group of eight rats is illustrated in Fig. 8. Kynurenic acid injection produced a 42% reduction in PND amplitude and phrenic tachypnea [largely due to reduced phrenic expiratory time (Te), i.e., phrenic cycle time (Ttot) minus phrenic inspiratory time (Ti)] and a massive reduction in the respiratory modulation of LSND. In this group of animals, MAP generally decreased by a modest amount (mean = 15 mmHg), but the preinspiratory level of LSND was slightly elevated. All parameters returned toward control values within 40 min. A summary of the results of these eight experiments is presented in Table

2. In Fig. 9, the CRG modulation of LSND recorded immediately after kynurenic injection (TOTmod and

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FIG. 5. Relationship between respiratory modulation and amplitude of phrenic burst. In A and B, 1 regression line represents 1 animal in which respiratory modulation was measured at 2-6 different levels of PND achieved by altering concentration of COs in inspired gas. All results are expressed as percent of maximum values recorded with highest CO, concentration used (typically generating an end-tidal COz concentration of 74%). For definitions of PostIP and TOTmod, see Fig. 2. Note that respiratory modulation and PND amplitude are, on average, proportional (see also Fig. 9) but exhibit significant intersubject variability.

PostIP) is plotted against PND amplitude (each point represents one experiment in one animal, n = 8). The same graph also reproduces the data from Fig. 4 describing the relationship between LSND modulation and PND amplitude determined at various levels of chemoreceptor activation in the absence of pharmacological treatment (2 or more points per experiment). Note that in the presence of kynurenic acid the respiratory modulation of LSND was still linearly related to PND amplitude, but the slope of the curve was significantly reduced compared with the chemoreceptor activation curves (P = 0.001). Also note that, in four animals, kynurenic acid drastically reduced the respiratory modulation of LSND while producing minimal effects on PND amplitude. When present (3 of these 8 cases), the inspiratory peak of the SND was attenuated by kynurenate to the same extent as the postinspiratory peak. In four additional rats, the kynurenic acid injections were located 200 pm or more caudal to the caudalmost pairs of injection sites depicted on Fig. 6. In these cases, the antagonist completely eliminated PND for 30-40 min, thus precluding the study of the CRG modulation of LSND. After PND recovery, the CRG modulation of

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FIG. 6. Anatomic maps of drug injection sites in the rostra1 medulla. A: distribution of 8 bilateral injections of kynurenate (black circles, numbered 1 to 8), which produced sympathorespiratory uncoupling. Diamonds represent 3 injections into the medullary raphe, which produced no effect on respiration or SND. Each section is 180 pm apart (most rostra1 on top). 23: map of 5 bilateral microinjections of strychnine. C: map of 5 bilateral microinjections of bicuculline. AC, compact subdivision of nucleus ambiguus; F, facial motornucleus; Giv, gigantocellular field, ventral subdivision; ro, raphe obscurus; pyr, pyramidal tract.

LSND quickly recovered to a level directly proportional to PND amplitude (results not shown). Kynurenic acid (two 50-nl injections for a total of 4.5 nmol) was also microinjected into the medullary raphe in three rats (diamonds in Fig. 6A). These microinjections produced no significant effect on any of the cardiorespiratory variables measured (MAP 100 t 7.3 mmHg before drug, 97 t 9.5 after drug; postdrug PND amplitude 94.4 t 11.77o of predrug level, total modulation 103 t 9% of predrug level, postinspiratory peak 106 t 5.5% of predrug control). Injections of GABA and glycine receptor antagonists into RVLM: effect on respiratory modulation of lumbar sympathetic discharge. All the experiments described in this section were conducted in the presence of -7% COZ in the inspired mixture (balance oxygen) to produce the largest possible PND under resting (predrug) conditions. Bicuculline was bilaterally injected into the RVLM in five rats (225 pmol/side, injection sites shown in Fig. 6). PND-triggered LSND averages were analyzed in the usual manner. In addition, pulse-triggered histogram of LSND were constructed to determine the extent to which the baroreflex was inhibited by the drug. The result of a typical experiment is represented in Figs. 10 and 11.

PND amplitude was slightly reduced, and PND rate was slowed considerably. LSND was greatly increased, and its CRG-modulation became greatly exaggerated. More precisely, the preinspiratory level was increased, the inspiratory depression was unaffected, and the postinspiratory peak was greatly increased in magnitude and especially duration. Characteristically, bicuculline also triggered the appearance of a late expiratory trough (nadir = 1,558.4 t 116.7 ms after PND onset or 313.6 t 56.8 before PND onset), which was never observed in the absence of drug. Note also in this particular example that the amplitude of the inspiratory peak, barely detectable before bicuculline, is greatly magnified by the drug (Fig. 1OB). In these experiments, the pulse synchrony of LSND (apex-nadir divided by apex, a sensitive index of baroreflex integrity) was measured at various levels of MAP (using different rates of PE infusion) before bicuculline administration. The PE infusion was then stopped and pulse-triggered LSND averages were determined before and at various times after injection of bicuculline into the medulla (Fig. 11). As indicated in Fig. 12, the pulse synchrony of LSND was linearly related to MAP from baroreceptor threshold (-70 mmHg in this case) to 125

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mmHg. Above 125 mmHg, pulse synchrony remained maximal at 95-100% (data not obtained in this particular rat), In the presence of bicuculline, pulse synchrony was reduced to 20% despite the large increase in arterial pressure and thus presumably in the activity of baroreceptor afferents. All variables gradually returned to control levels over 30-45 min. The average numerical data of five experiments are presented in Table 3. However, the usual waveform descriptors (Fig. 2) do not adequately convey the magnitude of the effect of bicuculline because of the major qualitative change in the respiratory pattern. Control injections of the same total dose of bicuculline (450 pmol) into the midline medulla (raphe pallidus, rz = 2) or 1 mm above (n = 1) at the level of the caudal end of the facial motor nucleus produced no detectable effect on any cardiovascular variables (data not shown). In addition, the GABAn receptor antagonist phaclofen was bilaterally injected into the RVLM (0.7 nmol/side, n = 2). This compound produced no (90%) of units with a pattern of discharge identical to the prevalent pattern of the mass discharge

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1200 1000

400

I

800 600 400 200

'

200

z -

0 -200

L1 I -500

0

I

I

I

500

1000

1500

'

7 &, 0 g

l-200

2000

TIME (ms) FIG. 10. Effect of bicuculline microinjection into rostra1 ventrolatera1 medulla (RVLM) on sympathorespiratory coupling. A: control histogram. B: histogram obtained 5-15 min after bilateral injection of bicuculline into RVLM. C: recovery after 1 h. D: histogram obtained in same animal during phenylephrine infusion (MAP 128 mmHg) and in absence of bicuculline. Note attenuation of respiratory modulation, in contrast to B, where arterial pressure is elevated as a result of central bicuculline administration.

of the lumbar nerves (Fig. lA), which is likely to represent that of muscle vasoconstrictor units (12). However, a small proportion of barosensitive, hence presumably vasomotor, units (both pre- and postganglionic) exhibited a different pattern characterized by a slow incremental discharge during late expiration, a sharp peak during midinspiration, and a depression in early expiration. The inspiratory peak of activity of this second type of barosensitive neuron occurred -300 ms after PND onset for an average PND duration of 450 ms. This is almost identical to the latency of the small inspiratory peak occasionally observed in the mass discharge of the lumbar chain in the present study (Fig. 1C). This heterogeneity of pattern at the single cell level suggests that

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CRG

-

AND SYMPATHETIC

SND

m-m-BP -noise

OUTFLOW loor

1800 1

1000

600 200 0400

00

R1071 CONTROL

80

E

-TzE n

n m

VI

0

1000

1

60

800 600 400 200 0

n z m

200 800 150

E E

600 400

100

i

800

n

m

I 100

80

I 120

I 140

I 160

1 180

MAP (mmHg) 12. Effect of bicuculline on pulse synchrony of LSND (see legend of Fig. 11). Pulse synchrony is plotted against MAP (digital average over 1 cardiac cycle computed from records like those of Fig. 11). Open circles, measurements obtained at resting pressure or during infusion of either phenylephrine or sodium nitroprusside. Closed circles, 5 sequential measurements of pulse synchrony in same animal performed at various times after a bilateral injection of bicuculline into rostra1 ventrolateral medulla. FIG.

TABLE 3. Effect of RVLM bicuculline injection on PND and LSND

-

n 200 z v) 0

50

0’

TE

-200

f

1

600

;

400 200

n z

0

tn

TIME (ms) FIG. 11. Effect of bicuculline on arterial pressure (BP) and pulse synchrony of LSND. Pulse synchrony was defined as apex minus nadir divided by apex, determined from curves such as those of Fig. 11 and expressed as a percentage. A-D represent pulse-triggered digital averages of rectified LSND (400 sweeps, 200-ms duration) and averages of trigger signal (i.e., femoral arterial pressure). A: before bicuculline. B: 5-15 min after bicuculline. C: after 1 h. D: histogram obtained during intravenous infusion of phenylephrine in absence of bicuculline.

the amplitude of the inspiratory-related reduction in whole nerve discharge is likely to be somewhat attenuated by the near-simultaneous peak in the activity of the minority of inspiratory-related units also present in the nerve. It is still unknown whether this inspiratory “depression” represents inhibition of preganglionic neurons or a lapse in the respiratory-related excitatory drive to these cells. The latter view is consistent with the fact that a similar inspiratory-related depression also occurs in the unit activity of a large component of the sympathetic premotoneurons of the RVLM (15). Because injections of strychnine, bicuculline, or phaclofen into the RVLM did not produce any discernible effect on the

Variable

MAP, mmHg Ttot, ms Ti, ms PNDamp, % PreIL, % TOTmod, % TOTmodz, % PostIP, % Idep, %

Control

103.5&6.7 1,372*80 310*14 100.0 100.0 100.0 100.0 100.0 100.0

Bicuculline

130.9-e11.I3* 1,739&137” 545t38* 67.0t9.9* 141.0&16.0* 110.2t21.0 139.0t19.0 13O.Ok17.8 93.0t33.0

Recovery

105.0t12.0 1,208_+184t 378t23t 90.1&6.7t 120.0k12.7 90.0*19.5 98.0t31.5 99.3k29.0 87.Ok7.0

Values are means * SE. See Tables 1 and 2 for definitions. TOTmod2, difference between postinspiratory peak and nadir (inspiratory nadir for control and recovery, late expiratory nadir for bicuculline). * Significantly different from resting; “r significantly different from bicuculline.

TABLE 4. Effect of RVLM strychnine injection on PND and LSND Variable

MAP, mmHg Ttot, ms Ti, ms PNDamp, % PreIL, % TOTmod, % PostIP, % Idep, %

Control

Strychnine

Recovery

112.0t5.5 1,218+49 322t19 100.0 100.0 100.0 100.0 100.0

116.6*6.5* 1,062+49* 276212 97.8k1.9 106.Ok3.4 109.2t6.7 102.7t7.1 115.028.6

107.5t8.4t 1,062*24* 33Ok-22 107.8k3.7t 125.Ok7.5”r 105.8t3.8 94.2t11.0 132.Ok20.8

Values are means t SE. See Tables 1 and 2 for definitions. * Significantly different from resting; t significantly different from strychnine.

inspiratory inhibition of LSND, the present results provide no evidence that either GABA or glycine release in the RVLM plays a role in the inspiratory-related depression of the sympathoexcitatory cells and LSND. Thus the present results suggest that the inspiratory-related depression of the activity of the lumbar nerve could simply represent a period of relative disfacilitation of RVLM premotoneurons with consequent relative disfacilitation of their targets, the preganglionic cells. Alter-

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R1072

CRG

AND

SYMPATHETIC

natively, this depression could be caused by a distinct heretofore unknown descending inspiratory-related inhibitory input to spinal preganglionic cells or it could be mediated in the rostra1 medulla by a yet unidentified inhibitory transmitter. Potential sources of descending inhibitory inputs to preganglionic cells include the raphe and the dorsolateral pons (29). Combined central and peripheral chemoreceptor stimulation with COz produced a pressor effect and an increase in the respiratory modulation of LSND. On average, the respiratory modulation was proportional to PND amplitude, as previously noted, in a rat preparation identical to the present, with the exception that rats from the previous study were barodenervated (15). Similar results have been reported previously in other species under different anesthetic conditions (1, 4, 24, 35). In animals with intact carotid sinus nerves, both peripheral and central chemoreceptors contribute to the sympathoexcitatory response to COz inhalation with a preponderant effect of central chemoreceptors at high levels of co* (14).

The literature is somewhat divided on the issue of how chemoreceptor inputs (both peripheral and central) reach the sympathetic outflow. One view is that the chemoreceptor input is entirely mediated by activation of the CRG and thus manifested solely by increases in the respiratory-modulated component of the sympathetic outflow (24). The predominant view is that chemoreceptor activation can also activate the sympathetic outflow in a tonic manner independent of the effects on the CRG (6, 14, 21). Some of the discrepancy may be due to the exclusive use of the PND as index of CRG activity (31) and possibly to regional differences in the sympathetic outflows (19, 20). Nevertheless, it is clearly established that RVLM sympathoexcitatory neurons contribute to the effect of peripheral (34) and central (15, 23) chemoreceptor activation on the sympathetic outflow. Effect of baroreceptor activation on respiratory modulation of LSND. The overall pattern of the PND-trig-

gered lumbar sympathetic discharge was qualitatively unchanged when MAP was changed from 73 to 123 mmHg. Thus the respiratory modulation cannot derive from respiratory fluctuations in the degree of baroreceptor feedback. In fact, far from being amplified by baroreceptor feedback (23), the amplitude of the modulation was maximal at reduced pressure and appeared linearly and almost proportionately related to the preinspiratory level of SND (Fig. 4A). The preinspiratory level of LSND is almost identical to the mean SND averaged over one respiratory cycle (data not shown), and its decrease can be taken as an index of the degree of overall baroreceptor feedback on the nerve activity. The correlation between respiratory modulation and overall level of sympathetic discharge suggests that the baroreceptor input might gate or at least influence the respiratory input to medullary sympathetic-related cells. This gating could be due in part to baroreceptormediated inhibition of the CRG itself (28). Indeed, when the effects of nitroprusside and PE are compared (Table l), PND amplitude was significantly reduced in the hypertensive state (28% compared with hypotensive situation), and the total respiratory modulation of the signal

OUTFLOW

was also significantly reduced by a comparable amount (34%). However, we only found a weak correlation between PND amplitude and respiratory modulation over the range of MAP recorded during a control period and after PE and SNP infusions (Fig. 4B), which suggests the existence of other contributing factors. One such contributing factor could simply be a nonlinear summation of baroreceptor and respiratory-related inputs on rostra1 ventrolateral medullary sympathoexcitatory neurons (see Ref. 32 for an example of nonlinear summation between excitatory effects of glutamate and inhibitory barorereceptor input). In any case, the present study does not support the alternate hypothesis, namely that the central respiratory modulation of the sympathetic discharge might operate via a periodic gating by the CRG of the negative baroreceptor feedback. Effect of bicuculline on cardiorespiratory coupling. The large increase in MAP produced by bilateral microinjection of bicuculline into the RVLM confirms similar results described previously (9, 36). Some of the bicuculline-related increase in MAP may be related to an attenuation of the sympathetic baroreflex (32,33, 36). This is corroborated by the present observation of a marked reduction in the pulse synchrony of LSND in the face of a large increase in baroreceptor drive. However, it is likely that a large fraction of the increase in MAP is not related to this effect, since in our preparation resting arterial pressure (104 mmHg) is usually no more than 20 mmHg above baroreflex threshold. Furthermore, total baroreceptor unloading produced by decreasing arterial pressure to 72 mmHg with SNP increased mean preinspiratory LSND by only a nonsignificant 38% (Table 1). The present results suggest that a large fraction of the bicuculline-induced increase in sympathetic outflow may in fact be due to an increased excitatory drive from elements of the CRG (increased sympathorespiratory “coupling”). This interpretation relies on two observations: 1) the large increase in the respiratory modulation of LSND after bicuculline and 2) the lack of an associated increase in PND amplitude. Also, the central respiratory modulation pattern of the LSND was altered in a qualitative sense by bicuculline. Although the inspiratory depression and postinspiratory peak remained present, a new respiratory depression appeared during the late expiratory phase. Single-unit experiments will be needed to determine whether these qualitative changes in the pattern of the lumbar nerve mass discharge are reflected at the level of individual pre- or postganglionic units or whether the overall pattern change is due to an altered proportion of active cells exhibiting distinct respiratory-related patterns. Finally, because the respiratory modulation of the sympathetic outflow can be increased by bicuculline at the same time as the baroreflex is being impaired, this again suggests that the baroreceptor and CRG-related inputs to RVLM sympathoexcitatory cells are largely independent of each other. The results of the kynurenic acid experiments (to be discussed below) further support this point of view, since this agent, when injected in the RVLM, can virtually eliminate the CRG modulation of the sympathetic nerve discharge without affecting either the basal sympathetic tone (present study) or the sym-

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CRG

AND

SYMPATHETIC

pathetic baroreflex (34). Effect of kynurenic acid injection on cardiorespiratory coupling. When introduced into the very rostra1 tip of the ventrolateral medulla, the nonselective excitatory amino acid antagonist, kynurenic acid, produced variable degrees of impairment of the phrenic nerve output, a slight decrease in MAP, and a very large reduction in the CRG modulation of the sympathetic outflow. The decrease in MAP was associated with a slight increase in the preinspiratory LSND, which could be related to baroreceptor unloading, since kynurenic acid does not impair the sympathetic baroreflex when introduced at this level of the medulla (33). A decrease in blood pressure unaccompanied by a reduction in lumbar sympathetic outflow could also signify that the antagonist produces selective inhibition of other sympathetic outflows such as those to the heart or the splanchnic area. Other interpretations include a reduction in the release of vasopressor hormones such as vasopressin and possibly angiotensin II (the release of which is in part regulated by the RVLM) (30) or the loss of the respiratory patterning of the sympathetic outflow, which may contribute up to 25% of the vasoconstrictor effect of the SND. Also, kynurenate generally produced a reduction in PND amplitude and a considerable central tachypnea (associated with a prominent decrease in Te). The increase in rate represents an effect opposite to that of bicuculline, which indicates that the RVLM may contain cells involved in controlling the duration of the expiratory phase. It should be noted that the effect of kynurenate is different from that produced in the neonatal brain stem preparation by bath application, where the major effect is a rate decrease (8). However, judging from the widespread effect of medullary injections of glutamate on the respiratory outflow, it is likely that excitatory amino acids have effects at many different sites within the CRG (8, 10, 18, 22). The effects of kynurenic acid on the phrenic nerve outflow were more pronounced when the injections were more caudally located in the RVLM. In fact, it is essential to stress that a large dissociation between the effect of kynurenate on phrenic discharge and CRG modulation of the sympathetic activity could only be observed when the injections were made at the very rostra1 tip of the RVLM at the level of the caudal end of the facial motonucleus. In a few cases, the respiratory modulation of the lumbar nerve was almost completely eliminated, whereas PND was only slightly reduced (see Fig. 9). These occurrences suggest that, although kynurenic acid interferes with both the generation of the phrenic output and with the coupling between the CRG and the sympathetic outflow, these effects probably occur at ventrolateral medullary sites that, although geographically close, are nevertheless distinct. Indeed, preliminary evidence suggests that central respiratory rhythm generation may critically depend on the activity of neurons located in the RVLM perhaps only a few hundred microns caudal to the area that contains the highest density of sympathetic premotoneurons (26). In conclusion, the present study suggests that the RVLM is a critical link between the CRG and the svm-

R1073

OUTFLOW

pathetic vasomotor outflow and that both excitatory amino acids and GABA are involved in coupling these two networks. The results do not demonstrate, however, that RVLM sympathetic premotoneurons derive their respiratory modulation directly from monosynaptic inputs that utilize either GABA or glutamate, because these two transmitters could be involved at some earlier interneuronal step in the circuitry. This work was supported by National Heart, Lung, and Blood Institute Grants HL-28785 and HL-39841 to P. G. Guyenet. Address for reprint requests: P. G. Guyenet, Dept. of Pharmacology, University of Virginia School of Medicine, Charlottesville, VA 22908. Received

26 March

1990; accepted

in final

form

6 July

1990.

REFERENCES 1. BAINTON, C. R., D. W. RICHTER, H. SELLER, D. BALLANTYNE, AND J. P. KLEIN. Respiratory modulation of sympathetic activity. J. Auton. Nerv. Syst. 12: 77-90, 1985. 2. BARMAN, S. M., AND G. L. GEBBER. Basis for synchronization of sympathetic and phrenic nerve discharges. Am. J. Physiol. 231: 1601-1607,1976. 3. BROWN, D. L., AND P. G. GUYENET. Electrophysiological study of cardiovascular neurons in the rostra1 ventrolateral medulla in rats. Circ. Res. 56: 359-369, 1985. 4. BUDZINSKA, K., C. VON EULER, F. F. KAO, T. PANTALEO, AND Y. YAMAMOTO. Effects of graded focal cold block in rostra1 areas of the medulla. Acta Physiol. Stand. 124: 329-340, 1985. 5. CONNELLY, C. A., AND R. D. WURSTER. Spinal pathways mediating respiratory influences on sympathetic nerves. Am. J. Physiol. 249 (Regulatory Integrative Comp. Physiol. 18): R91-R99, 1985. 6. CONNELLY, C. A., AND R. D. WURSTER. Sympathetic rhythms during hyperventilation-induced apnea. Am. J. Physiol. 249 (Regulatory Integrative Comp. Physiol. 18): R424-R431, 1985. 7. CZYZYK, M. A., L. FEDORKO, AND A. TRZEBSKI. Pattern of the respiratory modulation of the sympathetic activity is species-dependent: synchronization of the sympathetic outflow over the respiratory cycle in the rat. In: Organization of the Autonomic System, edited by J. Ciriello, F. R. Calaresu, L. P. Renaud, and C. Polosa. New York: Liss, 1987, p.143-152. 8. FELDMAN, J. L., AND J. C. SMITH. Cellular mechanisms underlying modulation of breathing pattern in mammals. Modulation of defined vertebrate neural circuits. Ann. NY Acad. Sci. 563: 114-130, 1989. 9. GATTI, P. J., A. M. DASILVA, AND R. A. GILLIS. Cardiorespiratory effects produced by injecting drugs that affect GABA receptors into nuclei associated with the ventral surface of the medulla. Neuropharmacology 26: 423-431, 1987. 10. GATTI, P. J., W. P. NORMAN, A. M. TAVEIRA DASILVA, AND R. A. GILLIS. Cardiorespiratory effects produced by microinjecting Lglutamic acid into medullary nuclei associated with the ventral surface of the feline medulla. Brain Res. 381: 281-288, 1986. 11. GILBEY, M. P., N. YOSHINOBU, AND K. M. SPYER. Discharge pattern of cervical sympathetic preganglionic neurons related to central respiratory drive in the rat. J. Physiol. Land. 378: 253-266, 1986. 12. GREGOR, M., W. JANIG, AND L. WIPRICH. Cardiac and respiratory rhythmicities in cutaneous and muscle vasoconstrictor neurones to the cat’s hindlimb. Pfluegers Arch. 370: 299-302, 1977. 13. GUYENET, P. G., AND D. L. BROWN. Nucleus paragigantocellularis lateralis and lumbar sympathetic discharge in the rat. Am. J. Physiol. 250 (Regulatory Integrative Comp. Physiol. 19): R1081R1094,1986. 14. HANNA, B. D., F. LIOY, AND C. POLOSA. Role of carotid and central chemoreceptors in the CO, response of sympathetic preganglionic neurons. J. Auton. Nerv. Syst. 3: 421-435, 1981. 15. HASELTON, J. R., AND P. G. GUYENET. Central respiratory modulation of medullary sympathoexcitatory neurons in rat. Am. J. Physiol. 256 (Regulatory Integrative Comp. Physiol. 25): R739R750,1989. 16. HUANG, W., S. LAHIRI, A. MOKASHI, AND A. K. SHERPA. Relationship between sympathetic and phrenic nerve responses to periph-

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R1074

CRC

AND

SYMPATHETIC

era1 chemoreflex in the cat. J. Auton. Nerv. Syst. 25: 95-105, 1988. 17. KLEINBAUM, D. G., AND L. L. KUPPER. Applied Regression Analysis and Other Multivariable Methods. North Scituate, MA: Duxbury, 1978. 18. LAWING, W. L., D. E. MILLHORN, D. A. BAYLISS, J. B. DEAN, AND A. TRZEBSKI. Excitatory and inhibitory effects on respiration of L-glutamate microinjected superficially into the ventral aspects of the medulla oblongata in cat. Brain Res. 435: 322-326, 1987. 19. LIOY, F., M. T. BLINKHORN, AND C. GARNEAU. Regional haemodynamic effects of changes in Pace, in the vagotomized, sinoaortic deafferented rat. J. Auton. Nerv. Syst. 12: 301-313, 1985. 20. LIOY, F., B. D. HANNA, AND C. POLOSA. Cardiovascular control by medullary surface chemoreceptors. J. Auton. Nerv. Syst. 3: l-7, 1981. 21. LIOY, F., AND A. TRZEBSKI. Pressor effects of CO, in the rat: different thresholds of the central cardiovascular and respiratory responses to COZ. J. Auton. Nerv. Syst. 10: 43-54, 1984. 22. MCALLEN, R. M. Location of neurones with cardiovascular and respiratory function, at the ventral surface of the cat’s medulla. Neuroscience 18: 43-49, 1986. 23. MCALLEN, R. M. Central respiratory modulation of subretrofacial bulbospinal neurones in the cat. J. Physiol. Lond. 388: 533-545, 1987. 24. MILLHORN, D. E. Neural respiratory and circulatory interaction during chemoreceptor stimulation and cooling of ventral medulla in cats. J. Physiol. Lond. 370: 217-23 1, 1986. 25. NUMAO, Y., N. KOSHIYA, M. P. GILBEY, AND K. M. SPYER. Central respiratory drive-related activity in sympathetic nerves of the rat: the regional differences. Neurosci. Lett. 81: 279-284, 1987. 26. ONIMARU, H., A. ARATA, AND I. HOMMA. Firing properties of respiratory rhythm generating neurons in the absence of synaptic transmission in rat medulla in vitro. Exp. Brain Res. 76: 530-536, 1989. 27. PAXINOS, G., AND C. WATSON. The Rat Brain in Stereotaxic

OUTFLOW

Coordinates. New York: Academic, 1982. 28. RICHTER, D. W., AND H. SELLER. Barorereceptor effects on medullary respiratory neurones of the cat. Brain Res. 86: 168-171,1975. 29. RICHTER, D. W., AND K. M. SPYER. Cardiorespiratory control. In: Central Regulation of Autonomic Function, edited by A. D. Loewy and K. M. Spyer. New York: Oxford Univ. Press, 1990, p. 189-207. 30. Ross, C. A., D. A. RUGGIERO, D. A. PARK, T. H. JOH, A. F. SVED, J. FERNANDEZ-PARDAL, J. M. SAAVEDRA, AND D. J. REIS. Tonic vasomotor control by the rostra1 ventrolateral medulla, effect of electrical and chemical stimulation of the area containing Cl adrenaline neurons on arterial presuure, heart rate, and plasma catecholamines and vasopressin. J. Neurosci. 4: 474-494, 1984. 31. ST. JOHN, W. M., AND T. A. BLEDSOE. Comparison of respiratoryrelated trigeminal, hypoglossal and phrenic activities. Respir. Physiol. 62: 61-78, 1985. 32. SUN, M.-K., AND P. G. GUYENET. GABA-mediated baroreceptor inhibition of reticulospinal neurons. Am. J. Physiol. 249 (Regulatory Integrative Comp. Physiol. 18): R672-R680, 1985. 33. SUN, M.-K., AND P. G. GUYENET. Arterial baroreceptor and vagal inputs to sympathoexcitatory neurons in rat medulla. Am. J. Physiol. 252 (Regulatory Integrative Camp. Physiol. 21): R699-R709, 1987. 34. TERUI, N., Y. SAEKI, AND M. KUMADA. Barosensory neurons in the ventrolateral medulla in rabbits and their responses to various afferent inputs from peripheral and central sources. Jpn. J. Physiol. 36: 1141-1164, 1986. 35. VAN LUNTEREN, E., J. MITRA, N. R. PRABAKHAR, M. A. HAXHIU, AND N. S. CHE~NIACK. Ventral medullary surface inputs to cervical sympathetic respiratory oscillations. Am. J. Physiol. 252 (Regulatory Integrative Comp. Physiol. 21): R1032-R1038, 1987. 36. YAMADA, K. A., R. M. MCALLEN, AND A. D. LOEWY. GABA antagonists applied to the ventral surface of the medulla oblongata block the baroreceptor reflex. Brain Res. 297: 175-180, 1984.

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