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(Circulation. 1997;95:1813-1821.)
© 1997 American Heart Association, Inc.

Articles

Origin of Respiratory Sinus Arrhythmia in Conscious Humans

An Important Role for Arterial Carotid Baroreceptors

Massimo Piepoli, MD, PhD; Peter Sleight, MD; Stefano Leuzzi, MD; Felice Valle, MD; Giammario Spadacini, MD; Claudio Passino, MD; Jim Johnston, MS; Luciano Bernardi, MD

From the Department of Cardiovascular Medicine (M.P., P.S., J.J.), John Radcliffe Hospital, University of Oxford, UK, and the Dipartimento di Medicina Interna (S.L., F.V., G.S., C.P., L.B.), IRCCS S. Matteo, University of Pavia, Italy.

Correspondence to Peter Sleight, MD, Department of Cardiovascular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK.

	Abstract

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Background We investigated whether respiratory sinus arrhythmia (RSA) in healthy humans originated from central neuronal oscillations or from peripheral baroreceptors responding to respiratory changes in venous return.

Methods and Results During subjects' controlled breathing we used sinusoidal neck suction to influence RSA (spectral analysis of RR interval). In 11 subjects, 20-second apnea greatly reduced RSA, which was restored by neck suction at the frequency of respiration. Counteracting the respiration-induced cycles of carotid blood pressure decreased RSA in 13 subjects (from 2136±682 to 1372±561 ms², P<.01). The critical phase of this neck suction was constant for each subject at around the phase shift (with regard to respiration-related fluctuations of blood pressure) best for smoothing respiratory (mechanical) changes in blood pressure. Suction of a nonbaroreceptor area (the thigh) did not affect RSA. In 4 subjects, to separate the effects of peripheral baroreceptor afferents from respiration-entrained central oscillation (15 breaths/min), we cycled the neck suction at 12 cycles/min. Increasing neck suction from -7 to -30 mm Hg increased the ratio of the power of the 12 cycles compared with the 15-cycle RSA oscillation in RR interval spectral analysis from 0.26 to 2.57. A 12-cycle/min suction of an area other than the neck had little effect on the RR interval spectrum.

Conclusions RSA can be mimicked or reduced by stimulation of arterial baroreceptors with cycles of appropriately phased neck suction at the frequency of respiration. This suggests an important influence of the arterial baroreceptors in the generation of RSA in conscious humans.

Key Words: blood pressure • arrhythmia • respiration • baroreceptors

	Introduction

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Respiration-related variations in cardiovascular parameters have been increasingly studied not only as indices of autonomic activity but also because they convey information about pathophysiological processes and their prognosis. For example, in healthy¹ and diabetic² subjects, respiration-related fluctuations in RR-interval RSA are used as an index of the gain of the baroreceptor cardiac vagal reflex responses. Acute reductions of fetal RSA during labor predict fetal death,³ and chronic reductions of HR variability after myocardial infarction are a marker for subsequent sudden cardiac death.^{4 5}

Central to this problem is the origin of RSA. Several theories have been proposed. According to some studies, mostly animal-based,^{6 7 8 9} RSA originates from respiration-entrained oscillations in medullary neuron firing rates ("central oscillator")¹⁰ ; this medullary neuronal network shows regular oscillations in firing rates even when all afferent input is interrupted.^{11 12} These oscillations, when entrained by afferent stimuli from receptors in the lungs and thoracic wall in time with respiration, may result in an HR oscillation called an RSA.¹³

These autonomic changes may also be due to baroreflex stimulation caused by changes in arterial pressure due to the regular inspiratory increase in venous return to the heart.^{14 15 16 17} In addition, other reflexes originating from the cardiopulmonary receptors may be implicated in the origin of RSA.^{13 18} Whether and to what extent all these reflexes contribute to RSA is still controversial. From a practical point of view, RSA is a relatively simple index of the gain of the arterial baroreflex^{19 20} that can give important predictive clinical information.^{3 4 5}

The aim of the present study was to establish the role played by the arterial baroreceptors in the origin of RSA in conscious humans. We posed three questions. (1) During apnea, can RSA be mimicked by stimulating the carotid baroreceptors by rhythmic neck suctions cycled at the frequency of the previous respiration? (2) Is it possible to reduce RSA by neck suction phased so as to smooth out the BP waves caused by respiration? (3) Is it possible to separate the effects of respiration from those of carotid baroreceptor input by examination of RR variability when neck suction is applied at a frequency close to, but distinct from, the respiratory frequency? We show below that the answers to all three questions were affirmative.

Arterial baroreceptors can be selectively stimulated by pressure changes in an applied neck chamber²¹ and can cause reflex RR interval changes: this methodology has been used in healthy subjects^{22 23} and patients.¹⁵ Neck suction has been used to assess the presence and type of delayed reinnervation in human heart transplant recipients.²⁴ In the present study, we used the neck chamber to test for reflex HR and BP responses in normal humans.

	Methods

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Subjects and Measurements
The study was approved by the local ethics committees; the participants gave written informed consent. The study group comprised 13 healthy male volunteers aged 27.5±5.9 (range, 22 to 43) years. Because the studies were performed in two different centers, Oxford and Pavia, the men were not all subjected to the full protocol. All subjects were familiarized with the laboratory, all the equipment, and the measurement technique before the actual day of investigation; however, in order not to influence their behavior during the recordings, they were unaware of the specific aims of the study. They were studied in the sitting position after a 10- to 15-minute rest, when recordings of ECG (lead II), respiration, noninvasive BP, and neck chamber pressure were obtained. The respiratory signal was obtained simultaneously from thorax ECG electrodes (modified Frank system) by means of an electrical impedance pneumograph (with a flat frequency response from 0 to 25 Hz) designed in our laboratory.²⁵ Noninvasive BP recordings were obtained by using a Finapres unit (Model 2300, Ohmeda) and a conventional sphygmomanometer technique. The neck-suction technique was applied by means of a molded lead collar connected with a vacuum cleaner whose power was modulated by a function generator (Krohn-Hite model 5200) to generate a sinusoidal suction. Neck pressure was continuously monitored by a Statham P23d pressure transducer. During all recordings the controlled respiration was maintained at 0.25 Hz (15 breaths/min) by visual synchronization with an oscilloscope displaying a sinusoidal waveform. The subjects were instructed to maintain a steady, quiet respiration and to avoid hyperpnea: this was visually checked by the supervisor of the test to minimize any intrasubject change in tidal volume, as this parameter was not controlled.

Protocols
Three sets of experiments were performed.

Spontaneous RR Interval Fluctuations and Induction of RSA During Apnea
First, spontaneous RR interval fluctuations during apnea and induction of RSA-like HR changes ("SA") during apnea caused by neck suction were analyzed in 11 subjects. After a 2-minute recording of controlled breathing the subject held his breath for 20 seconds starting at end expiration. The breath-hold maneuver was then repeated but with continuous sinusoidal neck suction set at the same frequency as the respiration, for the period before, during, and 2 minutes after apnea. The neck pressure swing was from 0 to -15 mm Hg. Finally, the recordings were repeated with the neck suction only during apnea, starting at the onset and ending when the subject resumed respiration.

Suppression of RSA by Varying Phase of Neck Suction
Next, suppression of RSA was obtained by varying the phase of neck suction in 13 subjects. After a 4-minute baseline recording obtained, as before, during quiet, controlled breathing, recordings were taken for 2 minutes while the neck suction was set at the same frequency as that of respiration, swinging from 0 to -15 mm Hg. Four recordings were obtained in each subject at progressive phase differences between respiration and neck suction, ie, with neck suction in phase with respiration and then with progressive 90° shifts out of phase with inspiration. The phase shift (in 90° steps) that maximally suppressed RSA was then noted. In order to determine that this phase was also the phase that best smoothed out respiratory BP fluctuations, in 10 subjects we measured the phase relationship between neck suction and Finapres BP (we assumed Finapres BP bears a constant phase relation with carotid BP).

To assess the reproducibility of this test, 6 of the 13 subjects repeated the same protocol 1 month and 24 months later. To exclude a nonspecific effect of suction (ie, not mediated by the baroreflex) in suppressing RSA, suctions from 0 to -30 mm Hg of a nonbaroreflex area (thigh or leg) were performed in 6 subjects: four recordings were performed in each subject following the same protocol (above) to suppress RSA.

Baroreceptor- Versus Respiration-Induced SAs
Finally, baroreceptor- versus respiration-induced SAs were compared in four subjects. While respiration was maintained at 0.25 Hz (15 breaths/min), neck suction was set at a similar but distinct frequency, ie, 0.20 Hz (12 cycles/min). The carotid baroreceptor stimulation lasted 2 minutes, during which recordings were obtained. Three sets of recordings were obtained by setting the pressure within the neck to swing from 0 to -7 mm Hg, from 0 to -15 mm Hg, and from 0 to -30 mm Hg. As a control for nonbaroreflex stimulation, suction from 0 to -30 mm Hg was also applied to the thigh or leg at 0.20 Hz in the same four subjects during controlled breathing at 0.25 Hz.

Data Acquisition and Analysis
The data were digitized on line by a 12-bit analogue-to-digital converter (NB-MIO-16 board, National Instruments) at a sampling rate of 500 samples/s for each channel. The converter was connected to a Macintosh II computer (Apple Inc). A C language program identified all the QRS complexes in each sequence and then located the peak of each R wave. The RR interval, SBP, DBP, neck suction, and respiration time series were obtained from these data. The respiratory time series was obtained from the thoracic impedance signal that occurred at the peak of the R wave and was expressed in arbitrary values. For each step of the protocol, all the RR intervals obtained were analyzed. The very few premature beats were interactively identified and corrected by linear interpolation with the previous and following beats. The original signals and RR interval data were then stored for further analysis, including mean RR interval and HR variability (evaluated as the variance of RR interval).

PS Analysis
We applied PS analysis to RR interval, respiratory, SBP, DBP, and neck-suction signals by using an autoregressive model.²⁵ In most cases a model order of 11 to 13 was adequate. Spectral components were obtained by using a decomposition method to measure the area below each spectral peak. According to the autoregressive model, each peak identifies the presence of an oscillatory component; however, to distinguish between signal and noise, components below 5% of total variability were ignored. Two frequency bands were considered: the so-called LF band (from 0.03 to 0.15 Hz) and the HF band around the respiratory frequency (from 0.18 to 0.35 Hz). Because the HF oscillations are due to the effect of respiration, in the present study the respiration-related oscillations in the RR interval spectrum (index of RSA) were more precisely identified by their correspondence with the oscillations in the respiratory spectrum by using coherence analysis. During the 0.20-Hz neck-suction stimulation (experiment 3), the HF band was further divided into two components: the respiratory component at 0.25 Hz (identified by significant coherence with respiration, as in the rest of the study) and the neck-suction component at 0.20 Hz (identified by coherence with the neck-suction signal and absence of coherence in frequency with the respiratory signal).

Analysis of Apnea by Time-Varying PS Analysis
The insertion of a necessarily short period of apnea in a recording introduces several practical problems in the estimation of variability. Spectral analysis, which is the method of choice, assumes there are no changes during the period of recording and so is no longer valid. One applicable method uses so-called time-varying spectral analysis algorithms. The Wigner-Ville transform allows continuous measurement of the spectral components for each beat of recording²⁶ and can be plotted in three-dimensional figures to show time-varying changes in the various spectral components (see "Results"); average power can be obtained for each band.²⁷ We obtained moving windows of 128 heartbeats and used a 5-beat moving average of the spectra to remove the "interference artifacts" inherent in the Wigner-Ville transform.²⁸ The results are summarized as average LF and HF power before apnea, during the first and second half of apnea, and after apnea.

Statistical Analysis
All results are given as mean±SEM. A paired t test was used to evaluate the differences within tests; when more than one trial was performed, repeated-measures ANOVA was used. As we were interested in the absolute amount of RSA and not the relative proportion of RSA with respect to other sources of variability (as in terms of sympathovagal balance), we did not feel it appropriate to evaluate RSA in relative or normalized (percent) units.

	Results

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The subjects completed the tests without discomfort. In particular, they did not present any difficulty in performing the apnea or during the controlled respiration.

Analysis of Spontaneous RR Interval Fluctuations and Induction of RSA During Apnea
No changes were observed in the mean RR interval during apnea tests. During undisturbed apnea, SA (HF) was much reduced and at a rhythm similar to, but generally slower than, that of respiration before apnea (Table 1). After the apnea, RSA increased as a result of the initial compensatory increase in ventilation. During the first part of undisturbed apnea the LF components of the RR interval were markedly decreased, but during the second half they increased above resting values. As a result, the LF components did not show any significant differences with respect to baseline (Table 1). In addition, the BP fluctuations at the respiratory frequency were reduced, which accounted for the decreases in RR interval fluctuations during apnea. These patterns were evident on both the time series (Fig 1) and in the three-dimensional continuous spectral plots (Wigner-Ville transforms) (Fig 2). When the apnea was performed during neck suction and cycled at the same frequency as the prior respiration, SA remained unchanged, and the onset and end of the apnea were almost indistinguishable in the RR interval patterns (Fig 3) or in the Wigner-Ville plots (Fig 4). SA also remained unchanged when the neck suction was delivered only during apnea. In the pressure signals the HF fluctuations persisted during apnea with neck suction but were attenuated (Figs 3 and 4).

View this table: [in this window] [in a new window]   Table 1. Changes of Spontaneous RR Interval Fluctuations During Apnea

View larger version (22K): [in this window] [in a new window]   Figure 1. Time series of RR interval, respiratory, SBP, and neck-suction signals before, during, and after 20 seconds of held expiration (apnea) without baroreflex stimulation by neck suction. Apnea substantially reduced the fluctuations of RR interval and BP signals. a.u. indicates arbitrary unit.

View larger version (54K): [in this window] [in a new window]   Figure 2. Three-dimensional continuous spectral plots (Wigner-Ville transforms) of RR interval, respiratory, SBP, and neck-suction signals before, during, and after 20 seconds of apnea. For comments, see Fig 1. n.beats indicates number of heart beats.

View larger version (33K): [in this window] [in a new window]   Figure 3. Time series of RR interval, respiratory, SBP, and neck-suction signals before, during, and after 20 seconds of apnea with baroreflex stimulation by neck suction cycled at the respiratory frequency during the whole recording. During apnea, HF fluctuations persisted unchanged in the RR interval, while they were attenuated in the BP signal. a.u. indicates arbitrary unit.

View larger version (57K): [in this window] [in a new window]   Figure 4. Three-dimensional continuous spectral plots (Wigner-Ville transforms) of RR interval, respiratory, SBP, and neck-suction signals before, during, and after 20 seconds of apnea with continuous baroreflex stimulation by neck suction. For comments, see Fig 3. n.beats indicates number of heartbeats.

Analysis of Suppression of RSA by Varying Phase of Neck Suction
In all subjects we were able to reduce RSA by cycling the neck suction with the phase shifted but at the same frequency as respiration. The phase difference between respiration and neck suction that best reduced RSA differed among the subjects. Repetition of the maneuver in six subjects after 1 and 24 months yielded similar results, ie, RSA could be reduced only if the individuals' specific phase relationship was chosen. HF was reduced from 1152±594 to 602±284 ms² after 24 months (P<.01). An in-phase relationship (shift 0°) was effective in 3 of 13 subjects, in-phase opposition (shift 180°) was effective in 5 of 13 subjects, a 270° phase shift between neck suction and respiration was effective in 5 of 13, and a 90° phase shift was effective in none. RR variance was reduced, and the LF component of the RR interval variability was also slightly but significantly reduced. The mean RR interval was unchanged during the test. No significant changes were observed in BP oscillations, although there was a trend to reduction in all values (Table 2). Fig 5 shows, in a representative subject, the considerable suppression of the RR interval fluctuations (particularly HF) induced by counteracting baroreceptor activity with neck suction. Suction of a nonbaroreflex area (thigh or leg) at 0.25 Hz did not affect any of the PS analysis parameters with respect to baseline. Changing the phase relationship of the nonbaroreflex area suction with respect to respiration did not affect the power of the RSA (whereas in the neck, a specific phase shift was more influential in counteracting RSA). In particular, suction on the leg timed at the individuals' specific phases that reduced RSA when performed on the neck produced the following results: HF 1152±594 (baseline) versus 1652±1117 (suction) ms² and LF 551±352 (baseline) versus 703±290 (suction) ms² (P=NS).

View this table: [in this window] [in a new window]   Table 2. Suppression of Spontaneous RR Interval Fluctuations by Neck Suction

View larger version (23K): [in this window] [in a new window]   Figure 5. PS and time series of RR interval during control baseline and counteraction of the baroreflex activity by neck suction cycled at respiratory frequency but timed to be at a phase that reduced RSA. Note greatly reduced HF oscillations (bottom right) and power in the HF band in the right-hand peak (upper right).

When we examined in 10 subjects the phase relationship between neck suction at 0.25 Hz and BP, we observed that the optimal phases for counteracting RSA were suction around 0° phase shift (Fig 6A). At this phase we would expect the neck suction to augment baroreceptor activity at a time when it was falling due to a trough in the BP caused by the mechanical variations in venous return due to respiration (Fig 6B and 6C). We did not expect an exact match with 0° phase shift, since our 90° steps in phase change were relatively coarse. In addition, there would also be a small individual correction for the pulse wave transmission between the Finapres BP (measured in the finger) and the earlier pressure wave at the carotid.

View larger version (19K): [in this window] [in a new window]   Figure 6. Diagram shows phase relationship between neck suction and BP (at 0.25 Hz) at the phase shift that best suppressed RSA for each subject (A). Optimum phase shift is scattered around 0° (arrow indicates the mean value of -29.2°). Since the phase steps were rather large (90°), and since there would be a time difference between carotid and finger BP, one would not expect to see a very close grouping; at a phase shift of 0° the decreased baroreceptor firing during a BP trough would be counteracted by a stretch of the carotid sinus by neck suction (B and C).

Comparisons of Baroreceptor- Versus Respiration-Induced SAs
Neck suction at 0.20 Hz induced a distinct oscillation in the RR interval PS, the amplitude of which was proportional to the degree of suction, whereas the respiration-induced 0.25-Hz component remained unaffected (Table 3). As a consequence, the ratio of the two components increased from 0.26±0.04 to 0.78±0.10 to 2.57±0.45 as the neck-suction amplitude was increased from 7 to 15 to 30 mm Hg (P<.001, ANOVA for repeated measures): ie, at 15 mm Hg neck suction the two components were approximately similar in power, but at 30 mm Hg the baroreceptor-induced component was largely predominant (Fig 7). The mean RR interval did not change significantly during the different phases of the test. Thigh or leg suction at 0.20 Hz induced only a small effect on RR interval spectrum, which was only 13% with respect to the respiration-induced 0.25-Hz component: 84±155 (at 0.20 Hz) versus 644±218 (at 0.25 Hz) ms² (P<.05). As previously noted, RSA was not altered by thigh or leg suction at 0.25 Hz.

View this table: [in this window] [in a new window]   Table 3. Comparison of Baroreceptor-Induced (0.20-Hz) vs Respiration-Induced (0.25-Hz) Components of RR Interval Variability PS

View larger version (30K): [in this window] [in a new window]   Figure 7. Time series and PS of RR interval, SBP, DBP, respiratory, and neck-suction signals during cyclical neck suction at near respiratory frequency to separate effects of peripheral baroreceptor afferents from respiration-entrained central oscillation. Note in the RR interval spectrum (top right) the presence of a peak coincident with the respiratory oscillation and a peak coincident with the neck pressure. AR indicates autoregressive; au, arbitrary unit.

	Discussion

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Background
Despite many past studies, the precise mechanisms of respiration-induced SA are still debated. Two main theories have been proposed that are not mutually exclusive. The peripheral or "baroreflex" theory postulates that mechanically induced respiratory BP fluctuations stimulate the arterial baroreceptors to produce reflex changes in the circulation.^{14 15} Inspiratory augmentation of venous return, which is delayed and damped during passage through the lungs, leads to a regular rise and fall of peripheral arterial pressure; these respiration-related changes in venous return to the right atrium induce changes in left ventricular volume that are sufficient to reduce or increase stroke volume. Moreover, inspiration "afterloads" the left ventricle (by decreasing intrathoracic pressure) and momentarily decreases cardiac output and BP. This cycle of BP change is sensed by the arterial baroreceptors, which then modulate HR and determine RSA. This hypothesis is supported by evidence in humans that shows that respiration-induced changes in central hemodynamics elicit reflexes that control HR.¹⁸ However, to our knowledge, no direct evidence of the role of the arterial baroreceptors in RSA has been presented.

According to the "central" theory, RSA originates from respiratory oscillations in medullary neuron firing rates. This old hypothesis,^{29 30} which has been adopted by several authors,^{6 7 8 9 11 31} is supported by animal experiments, mostly performed in paralyzed or open-chest animals,^{7 9} which show that RSA may persist in the absence of any respiratory movements and that HR acceleration is still related to inspiratory activity in the phrenic nerve.^{7 8 32} These studies also show that the amplitude of RSA is positively correlated with the PCO₂ level of the arterial blood.⁷ The "central" theory is also sustained by the evidence of the LF and HF components in the discharge variability of brain stem neurons recorded in cats with sinoaortic deafferentation.³³ However, studies on humans that aimed to separate respiratory movements from central nervous respiratory activity by assisting ventilation (external intermittent negative pressure ventilation) and that measured changes in arterial PCO₂ failed to identify any central origin of RSA.¹⁷ The discrepancy with animal work has been ascribed to species difference, but more probably might be due to the fact that the animals were anesthetized.^{7 32}

Other theories have been proposed. Taha et al¹³ have suggested that RSA could result from reflexes other than the baroreflex, eg, cardiopulmonary reflexes, including those from lung stretch reflexes. Studies investigating the relationship between lung expansion and the amplitude of RSA, independent of simultaneous changes of intrathoracic pressure, have excluded the role of reflexes from the lungs or the thoracic wall.¹⁸ Another hypothesis is that local stretch of the sinus node causes changes in the spontaneous depolarization rate.³⁴ This seems unlikely during physiological pressure conditions because RSA is dependent on an intact vagus nerve,^{30 35 36} and the SA seen in transplanted (denervated) hearts is of very small amplitude.^{13 24}

Practical Considerations on the Genesis of RSA
Most of these theories have experimental support, so it is likely that most or all of these mechanisms contribute to RSA. Thus, redundancies may explain why it has been so difficult to identify which specific mechanism is actually generating RSA. Indeed, it seems likely that any mechanism, either central or peripheral, could entrain other structures, such as a medullary cardiorespiratory neuronal network, making it virtually impossible to identify a real "origin" of RSA. Nevertheless, the relative importance of different factors can be investigated by using suitable methodology. We performed three different experiments devised to evaluate the relative contribution of different mechanisms.

Observations on Simple Apnea
If RSA were mainly due to a respiration-entrained central oscillator, it should persist despite respiratory apnea, especially at first; conversely, if respiration acting at any level were the main source of RSA, then RSA would be abolished by apnea. In fact, during apnea not only is respiration stopped, but other complex changes occur that involve chemoreceptors and cardiopulmonary receptors. After 15 to 20 seconds of apnea an increase in sympathetic efferent activity is often observed in microneurographic recordings.^{37 38} As a consequence, the RR interval is not fixed during apnea but shows a rather complex pattern in which both LF and very LF oscillations are evident. A new methodology for computing a time-varying spectrum analysis (Wigner-Ville transform) allowed us to study the pattern of change of these fluctuations during apnea. We found that the respiratory BP fluctuations were reduced, thus accounting for the decreases in RR interval fluctuations. In fact, RSA was markedly reduced yet did not disappear completely, and it was at a rhythm slower than that of respiration before apnea. This suggests that RSA is mainly due to the peripheral activity of respiration and its influence on BP, but even in the absence of respiration a small amount of RSA persists; this persistence favors the presence of a central oscillator.⁸ Other fluctuations, although reduced, were evident during apnea, and occasionally, the slow (0.1-Hz) rhythm increased toward the end of apnea (Figs 1 and 2): this may be related to the increase in sympathetic traffic.^{16 37 38} An important baroreflex contribution of this 0.1-Hz fluctuation has been proposed by De Boer et al¹⁴ from a mathematical model and has more recently been demonstrated in our laboratory by damped persisting oscillations in BP.³⁹ However, since these oscillations were more obvious at the end than at the beginning of apnea, they might be evidence of an additional coexisting central neuronal oscillator.^{33 40}

If RSA were due to baroreceptor-sensed respiratory changes in BP, then a respiration-like modulation of the carotid sinus should result in "RSA" despite apnea. The neck-suction technique is an ideal method to stimulate the carotid baroreceptors selectively, without any respiratory or hemodynamic effect other than reflex. Although in most applications the stimulation has been given as a sudden decrease in neck pressure, and the response has been evaluated simply in terms of reflex bradycardia,²⁴ the technique can be made far more flexible. We have built a servomechanism able to continuously vary the pressure within the neck chamber and that can be modulated by any predetermined signal. A sinusoidal signal was used at the same frequency as normal breathing, ie, 15 breaths/min, or 0.25 Hz. We found that if the subject suddenly stopped breathing during such neck suction, RSA continued unchanged, so that respiration (and other possible related mechanisms, such as cardiopulmonary reflexes) appeared unnecessary for the generation of RSA, provided that the carotid baroreceptors were sensing an oscillation of similar frequency and magnitude to that of the BP effects of respiration. Similar results were obtained whether the neck suction became operative only during the period of apnea and stopped thereafter or whether it was continuously cycled before, during, and after apnea. This experiment suggests that the arterial baroreceptors may contribute to the generation of RSA (Figs 3 and 4). We cannot assume from this experiment alone that the baroreflex is the only mechanism responsible for RSA, since we cannot exclude the possibility that neck suction might have entrained a central nervous oscillator normally entrained by respiratory afferent stimuli.

The persistence of the HF fluctuations, although attenuated, in the BP signals during apnea with neck suction supports the theory of a mechanical origin of these respiratory oscillations.

Potentiation or Suppression of RSA by Changing the Phase of Neck Suction
We next argued that if a central oscillator did play an important role in generating RSA, then this oscillator should be entrained by the neck suction regardless of the phase relationship between neck suction and respiration. Thus, if this hypothesis were correct, by summing up the effect of the centrally entraining neck suction and that of respiratory entrainment, we should have obtained a potentiation of RSA. Conversely, if RSA were the result of a baroreflex response to respiration-induced changes in BP, then the effects of these BP fluctuations on carotid distension and hence baroreceptor firing could have been counteracted by specifically timed counterpressure induced by neck suction that was out of phase with the respiration-induced BP fluctuations. So by finding a suitable phase shift between respiration and neck suction we might observe either a reduction or a potentiation of RSA.

Our experimental data indicate that in all the subjects RSA could be reduced by at least 50% by neck suction timed at the same frequency as respiration but with an appropriate phase shift. This delay was consistent and specific in timing in the same subject when tested 1 month or 2 years later but was different from subject to subject, probably because of differences in the time necessary for the increased venous return generated by inspiration to pass from the right heart to the systemic arteries, and hence to the carotid baroreceptors. It is possible also that differences in pulmonary damping and capacitance and differences in circulatory delay through the pulmonary and left heart vessels could explain the interindividual variability. We excluded a nonspecific effect of suction (not mediated by the baroreflex) by suctioning a nonbaroreflex area (thigh or leg), which did not counteract RSA but did produce some nonsignificant changes in the PS.

The neck pressure swing able to obtain the maximum RSA suppression ranged from 8 to 15 mm Hg, ie, a plausible and physiological respiratory pressure swing. Complete suppression of RSA was not expected, since it was not possible with this technique to exactly counterbalance the respiratory BP fluctuations in the neck arteries, nor was it possible to suppress afferent influences from other noncarotid receptors in the aorta or heart (Fig 5). It seems unlikely that the specific (for each individual) phase shift we found to optimally suppress RSA was caused by a central nervous "gating" mechanism. In fact, the phase shift necessary was not the same in each subject, ie, did not bear a fixed relation to respiration, as might be expected for a central nervous "gating" mechanism.⁴¹ Furthermore, the phase shift of neck suction was related more closely to BP than to the respiration phase. The most simple explanation for these results is that arterial pressure fluctuations in the neck region play a major role in the generation of RSA in conscious humans through the action of the carotid baroreceptors.

Comparison Between Pure Baroreflex-Induced and `Natural' RSA
In the above two experiments the neck suction and respiration were set at the same frequency, so the effect of pure baroreceptor stimulation was mixed with that of respiration. We therefore examined separately the pure baroreceptor effect and that of the "natural" RSA (ie, including the baroreceptors together with all other possible causes of RSA) by setting the respiration and the neck suction at two close but distinct frequencies, ie, 0.25 and 0.20 Hz, respectively. Saul et al⁴² have convincingly shown that the activities of both the sympathetic and the vagus are frequency dependent; therefore, to avoid differences due to the different frequencies of stimulation, the two stimuli were set at the closest possible frequency. At 0.20 and 0.25 Hz, two distinct peaks could be easily observed in all the subjects studied. The power of the "baroreceptor" peak was lower than the normal respiratory peak if the neck suction swing was 7 mm Hg, was in the same range of magnitude with a 15–mm Hg swing, and was two to three times greater with 30 mm Hg. These results point to the importance of the arterial baroreceptors in generating RSA. Suction applied to the leg or thigh cycled at 0.20 Hz induced a small effect, suggesting that other afferents could exert a central effect, but much less than neck suction and without any specific phase where the effect was greater or less.

Taylor and Eckberg⁴³ have studied the origin of RSA in humans after elimination of RR interval fluctuations by atrial pacing: arterial pressure oscillations were increased in the 40° tilt position but decreased in the supine position. The authors concluded that RSA seemed to buffer the BP fluctuations in the upright but not the supine position. To override the sinus rhythm, Taylor and Eckberg needed to pace the heart at significantly higher rates than the control SA, with consequent reductions in stroke volume. This and the probable discomfort from esophageal electrical pacing could have altered baroreflex afferent discharge and/or the central gain of the reflex arc when comparing the fixed (paced) rate with the normal sinus rhythm. A major role of body position in the relationship between HR and BP has been reported.⁴² Our results, obtained in sitting subjects, may be at least partially consistent with the hypothesis that RSA buffers respiratory arterial pressure oscillations in a partially upright position.

Study Limitations
Like all models, the model we used may be subjected to criticism. A system that normally works in closed-loop conditions is forced with an external input in such a way that what is analyzed becomes an input-output open-loop relationship: therefore baroreflex forcing should be as brief as possible. However, the sinusoidal stimuli used have the advantage of minimizing baroreceptor adaptation.^{10 22}

The RR interval response induced by baroreflex stimulation during apnea (SA) cannot really be described as natural RSA, but the similarity between the "natural" and the neck suction–induced responses suggests the possibility of a contribution of the carotid baroreceptors. Furthermore, animal experiments³² also suggest an important baroreceptor influence on RSA in normocapnic animals with intact vagus compared with vagotomized animals with increased central drive due to hypercapnia (when the "central" influence is dominant).

As this is a study of respiration, the measurement of tidal volume and/or end-tidal CO₂ could have provided interesting information, but in our laboratory preliminary tests have shown that the concomitant presence of the collar around the neck and the mouthpiece for measurement of respiratory gases was poorly tolerated.

However, during the first set of experiments we evaluated whether the neck collar had induced any changes in ventilation by calculating the modifications of the spectrum of the respiratory signal during controlled breathing (15 breaths/min) without and during continuous sinusoidal neck suction set at the same frequency as the respiration. Five subjects (50%) did not show any changes or even reduced their respiration, but the other five increased their ventilation. Therefore, at least in some cases, the neck suction may have affected the RSA, perhaps by stimulating the carotid chemoreceptors or baroreceptors, or as a consequence of an alerting stimulus. No significant changes, however, were observed between the different recordings with the neck suction.

In this study we did not try to examine all other possible causes of RSA, nor were the differential roles of the aortic with respect to the carotid baroreceptors assessed. By definition neck suction affects only the neck region, and thus a counterresponse by the aortic baroreceptors to the changes induced by the neck was to be expected. This, together with the relatively crude phase changes, might explain why carotid countersuction was unable to suppress RSA totally. Nevertheless, our results indicate that the carotid input was important.

Conclusions
The present investigation suggests a substantial role for the carotid baroreceptors in the origin of RSA in conscious humans. However, the observations that conscious animals with chronic sinoaortic deafferentation⁹ or during apnea⁸ show a clear RSA support the hypothesis of a multiple genesis of RSA under particular conditions. Our present finding does not exclude the presence of a central oscillator: the small RSA observed during undisturbed apnea actually suggests that a central oscillator is operative, although at a slower rate than either the normal or the controlled ventilation that we used (Fig 2). It is possible to envisage a more complex system for the generation of RSA that would involve both the baroreflex and central oscillator mechanisms (and perhaps other mechanisms). We have shown that during normal respiration, in healthy subjects, the baroreflex is the major system for generating both HF (RSA) and also slow oscillations of HR and BP³⁹ : this suggests that during normal respiration the less powerful central rhythm can be masked. On the other hand, when respiration ceases during apnea or when the baroreflex sensitivity is depressed (either in physiological, such as exercise, or pathological conditions), the contribution of the central and/or other oscillators to the generation of the RR oscillations, which we generally found to occur at a slower frequency than the normal RSA, can be observed.⁴⁰

	Selected Abbreviations and Acronyms

 

BP = blood pressure DBP = diastolic blood pressure HF = high frequency HR = heart rate LF = low frequency PS = power spectrum RSA = respiratory sinus arrhythmia SA = sinus arrhythmia SBP = systolic blood pressure

Received August 13, 1996; revision received November 12, 1996; accepted November 19, 1996.

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