Respiratory modulation of human autonomic function on Earth.

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The Journal of Physiology

Neuroscience

J Physiol 594.19 (2016) pp 5611–5627

Respiratory modulation of human autonomic function on Earth Dwain L. Eckberg1 , William H. Cooke2 , André Diedrich3 , Italo Biaggioni3 , Jay C. Buckey Jr4 , James A. Pawelczyk5 , Andrew C. Ertl3 , James F. Cox1 , Tom A. Kuusela6 , Kari U. O. Tahvanainen7 , Tadaaki Mano8 , Satoshi Iwase9 , Friedhelm J. Baisch10 , Benjamin D. Levine11,12 , Beverley Adams-Huet13 , David Robertson3 and C. Gunnar Blomqvist (Deceased)11 1

Departments of Medicine and Physiology, Hunter Holmes McGuire Department of Veterans Affairs Medical Center and Virginia Commonwealth University School of Medicine, Richmond, VA, USA 2 Department of Kinesiology, Health, and Nutrition, University of Texas at San Antonio, San Antonio, TX, USA 3 Department of Medicine, Division of Clinical Pharmacology, Autonomic Dysfunction Center, Vanderbilt University School of Medicine, Vanderbilt University, Nashville, TN, USA 4 Dartmouth Hitchcock Medical Center, Lebanon, NH, USA 5 Department of Physiology, Pennsylvania State University, University Park and Hershey, PA, USA 6 Department of Physics, University of Turku, Turku, Finland 7 Department of Clinical Physiology and Nuclear Medicine, South Karelia Central Hospital, Lappeenranta, Finland 8 Gifu University of Medical Science, 795-1 Nagamine Ichihiraga, Seki, Gifu, 501-3892, Japan 9 Department of Physiology, Aichi Medical University, Aichi, Japan 10 DLR-Institute for Aerospace Medicine, Cologne, Germany 11 Department of Medicine, University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA 12 Institute for Exercise and Environmental Medicine, Texas Health Presbyterian Hospital, Dallas, TX, USA 13 University of Texas Southwestern, Dallas, TX, USA

Key points

r We studied healthy supine astronauts on Earth with electrocardiogram, non-invasive arterial r r r

pressure, respiratory carbon dioxide concentrations, breathing depth and sympathetic nerve recordings. The null hypotheses were that heart beat interval fluctuations at usual breathing frequencies are baroreflex mediated, that they persist during apnoea, and that autonomic responses to apnoea result from changes of chemoreceptor, baroreceptor or lung stretch receptor inputs. R-R interval fluctuations at usual breathing frequencies are unlikely to be baroreflex mediated, and disappear during apnoea. The subjects’ responses to apnoea could not be attributed to changes of central chemoreceptor activity (hypocapnia prevailed); altered arterial baroreceptor input (vagal baroreflex gain declined and muscle sympathetic nerve burst areas, frequencies and probabilities increased, even as arterial pressure climbed to new levels); or altered pulmonary stretch receptor activity (major breathing frequency and tidal volume changes did not alter vagal tone or sympathetic activity). Apnoea responses of healthy subjects may result from changes of central respiratory motoneurone activity.

Abstract We studied eight healthy, supine astronauts on Earth, who followed a simple protocol: they breathed at fixed or random frequencies, hyperventilated and then stopped breathing, as a means to modulate and expose to view important, but obscure central neurophysiological mechanisms. Our recordings included the electrocardiogram, finger photoplethysmographic arterial pressure, tidal volume, respiratory carbon dioxide concentrations and peroneal nerve muscle sympathetic activity. Arterial pressure, vagal tone and muscle sympathetic outflow

Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

DOI: 10.1113/JP271654

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D. L. Eckberg and others

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were comparable during spontaneous and controlled-frequency breathing. Compared with spontaneous, 0.1 and 0.05 Hz breathing, however, breathing at usual frequencies (0.25 Hz) lowered arterial baroreflex gain, and provoked smaller arterial pressure and R-R interval fluctuations, which were separated by intervals that were likely to be too short and variable to be attributed to baroreflex physiology. R-R interval fluctuations at usual breathing frequencies disappear during apnoea, and thus cannot provide evidence for the existence of a central respiratory oscillation. Apnoea sets in motion a continuous and ever changing reorganization of the relations among stimulatory and inhibitory inputs and autonomic outputs, which, in our study, could not be attributed to altered chemoreceptor, baroreceptor, or pulmonary stretch receptor activity. We suggest that responses of healthy subjects to apnoea are driven importantly, and possibly prepotently, by changes of central respiratory motoneurone activity. The companion article extends these observations and asks the question, Might terrestrial responses to our 20 min breathing protocol find expression as long-term neuroplasticity in serial measurements made over 20 days during and following space travel? (Received 28 September 2015; accepted after revision 14 March 2016; first published online 29 March 2016) Corresponding author D. L. Eckberg: Ekholmen, 8728 Dick Woods Road, Afton, VA 22920, USA. [email protected]

Email:

Abbreviations pNN50, proportion of successive normal R-R intervals greater than 50 ms, divided by the total number of normal R-R intervals (%); RMSSD, root mean square of successive normal R-R intervals (ms).

Introduction

Methods

Space travel presents astronauts with physiological challenges that do not occur on Earth, and thus affords scientists unique, potentially rich opportunities to explore the effects of an utterly basic modulator of human physiology: gravity. In the first of two articles, we closely examine data recorded on Earth before the Neurolab Space Shuttle mission, to obtain potentially new physiological insights from the protocol we designed for space, and to better understand physiological changes set in motion by space travel. The terrestrial studies tested three variably contentious null hypotheses: (1) that R-R interval fluctuations occurring in parallel with systolic pressure fluctuations during normal respiration are mediated by vagal baroreflex mechanisms (Eckberg, 2009; Karemaker, 2009); (2) that haemodynamic and autonomic responses to apnoea can be explained simply on the basis of changes of chemoreceptor, baroreceptor, or pulmonary receptor activity (Kara et al. 2003); and (3) that the R-R interval fluctuations that occur during quiet breathing persist during apnoea, as evidence for the existence of a central respiratory oscillation (Cooper et al. 2003). The companion article (Eckberg et al. 2016) moves beyond the results of this terrestrial study and asks, Does space travel alter human central neurophysiological mechanisms? And if so, Might such changes reflect long-term neuroplasticity, triggered by exposure to microgravity?

Subjects

We studied six male astronauts and one male and one female backup astronaut whose mean age (and range) was 41 (38–46) years, and height and weight were 181 (159–193) cm and 82 (57–110) kg. All were healthy and none were taking medications. Although no subjects were competitive athletes, all were of average or above average fitness, and all performed some exercise regularly. The controlled breathing experiment was part of a larger series of autonomic studies conducted as part of the National Aeronautics and Space Administration (NASA) Neurolab Space Shuttle Mission (STS-90); astronaut subjects are designated by the same numbers used in the companion article and three earlier publications (Ertl et al. 2002; Fu et al. 2002; Levine et al. 2002). Ethical approval

This experiment was performed in accordance with guidelines established by the Declaration of Helsinki, and was approved by the Human Research Policies and Procedures Committee of NASA’s Johnson Space Center. The home institutions of the principal investigators (F.J.B., C.G.B., D.L.E. and D.R.) also approved the project. Astronauts and backup astronauts gave written informed consent.


Breathing, apnoea and circulatory control

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Measurements

Experimental sessions and protocol

We described measurements and equipment in the first of our articles on human autonomic responses to microgravity exposure (Cox et al. 2002). Briefly, we recorded the electrocardiogram with surface electrodes, respiratory carbon dioxide concentrations (air withdrawn from a mouthpiece into a mass spectrometer), tidal volume (VMM-2 turbine spirometer; Interface Associates, Laguna Niguel, CA, USA), beat-by-beat finger arterial pressure (Finapres, Ohmeda, Englewood, CO, USA; Imholz et al. 1998), and, in seven of the eight subjects, muscle sympathetic nerve activity (NASA Nerve Traffic Analyzer, Johnson Space Center, Houston, TX, USA). Nerve signals were amplified 70,000–160,000 times, filtered (700–2000 Hz) and integrated (time constant 0.1 s) to obtain mean voltage neurograms. Satisfactory recordings of muscle sympathetic nerve activity were defined as pulse synchronous bursts that increased during end expiratory apnoea or the release phase 4 of Valsalva straining and did not change during unexpected tactile or auditory stimulation.

We conducted all studies on Earth with subjects in the supine position. The fixed protocol is illustrated by Fig. 1, which shows data recorded from Astronaut No. 1 on Earth prior to his launch into space. Subjects breathed spontaneously (Fig. 1A, Spon) during an initial baseline period. Following this, they timed their inspirations and expirations to waveforms that scrolled across a laptop computer monitor, according to a random, ‘white noise’ signal, which was followed by 3 min periods of controlled frequency breathing at 0.25, 0.1 and 0.05 Hz (15, 6 and 3 breaths min–1 ). Subjects then hyperventilated (H) with room air for 15 s at a median rate of 1 (range: 0.98–1) Hz, held their breaths in end inspiration for as long as they could, and finally breathed again (Rec, recovery). The abrupt end-tidal carbon dioxide increase at the end of apnoea (Fig. 1A, extreme right) in this recording and virtually all others (97%) lasted only one breath. This figure documents synchronization of rhythms during slow breathing, a brief surge of muscle sympathetic nerve activity at the onset of apnoea (Fig. 1B, right, arrow),

Complete protocol, Astronaut No. 1, on Earth before launch Tidal carbon dioxide (%) 9 6

Spon

A

Random

0.25 Hz

0.1 Hz

0.05 Hz

H

Rec Apnoea

3 0 Muscle sympathetic nerve activity

B

Artefact

R-R interval (ms) 1.5 1.2 0.9 0.6

C Systolic pressure (mmHg)

180

D

150 120 100

300

500

700

800

1100

Time (s)

Figure 1. Recording showing entire protocol from one astronaut studied on Earth before launch into space The breathing mode is indicated in A, above the carbon dioxide tracing. Spon, initial period of uncontrolled, spontaneous breathing; H, hyperventilation; Rec, recovery, uncontrolled breathing after apnoea. Hypercapnia, at the beginning of recovery, lasted only one breath (A, extreme right). The onset of apnoea triggered a brief volley of sympathetic activity (B, right arrow). The right halves of tracings illustrate the synchronization and augmentation of rhythms by slow (0.1 and 0.05 Hz) breathing, and their dampening during apnoea. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

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which was followed by 10 s sympathetic silence, and a broad low frequency R-R interval change during apnoea (Fig. 1C, extreme right). Data acquisition

During preflight and postflight sessions, all analogue signals were filtered and integrated at a frequency range of up to 10 Hz, and then digitized at 500 Hz with Windaq software and hardware (DATAQ, Akron, OH, USA). All data were analysed with commercial software (WinCPRS, Absolute Aliens Ay, Turku, Finland). Muscle sympathetic nerve activity was detected automatically, as bursts occurring within a 1 s window centred on each subject’s R wave (one removed; Sundlȍf & Wallin, 1978). We identified sympathetic bursts in entire recordings iteratively, and after each iteration, adjusted the search window timing according to the burst latency measured during that iteration. When an iteration failed to detect additional bursts, burst selection was considered to be final. For comparisons among subjects and experimental sessions, we normalized all bursts according to the median burst area recorded during spontaneous breathing of each session; thus bursts with the same area as the median burst during spontaneous breathing were assigned a value of 100%. Sympathetic burst frequency was calculated from interburst intervals. Data analysis

We estimated tonic levels of vagal cardiac nerve activity with simple R-R intervals (Eckberg et al. 1988), and fluctuations of vagal cardiac nerve activity as the root mean square of successive normal R-R intervals (RMSSD) and the proportion of successive normal R-R intervals greater than 50 ms, divided by the total number of normal R-R intervals (pNN50) (Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology, 1996). Since we could not synchronize data across subjects on the onsets of breaths, time series represent 15 s averages, moved by 1 s steps. We estimated carbon dioxide levels during apnoea by linearly extrapolating between end-tidal measurements recorded during the last breath before apnoea and the first breath after the resumption of breathing. We estimated vagal baroreflex gain four ways. First, we calculated baroreflex sequence gain from the slopes of linear regression analyses of all parallel up-going and down-going systolic pressure–R-R interval pairs (Bertinieri et al. 1985; Fritsch et al. 1986). We paired each systolic pressure with the next R-R interval (Eckberg & Eckberg, 1982), and required that valid sequences comprise three or more pairs of data, each with systolic pressure changes 1 mmHg, R-R interval changes 5 ms,

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and linear regression coefficients 0.8 (Rothlisberger et al. 2003). (We also calculated up-going and down-going baroreflex sequences with the same R-R interval in which the systolic pressure occurred, and found no significant differences; P = 0.96 and P = 1). Second, we calculated vagal baroreflex transfer functions (Robbe et al. 1987), as the ratio between R-R interval and systolic pressure changes over low (0.04–0.15 Hz) and high (0.15–0.4 Hz) frequency ranges. Third, we integrated fast Fourier R-R interval and systolic pressure spectral power within the same frequency ranges, and calculated baroreflex gain as the square root of the ratio between R–R interval and systolic pressure integrated spectra, the alpha index (Pagani et al. 1988). This baroreflex index was considered valid when the squared coherence was > 0.50. To estimate changes of baroreflex gain over time, we iteratively calculated alpha index gain from 15 s windows, moved through the data by 1 s steps. Statistical analyses

Since many data sets were not distributed normally, we give some results as medians, 25th and 75th percentiles, and ranges. We compared results recorded during different breathing frequencies and breathing modes with a linear mixed effects model, with individual subjects modelled as random effects, to account for intrasubject correlations, and to specify the covariance structure for the longitudinally collected measurements (Singer & Willett, 2003; Littell et al. 2007). An autoregressive covariance structure was used for most analyses. Baroreflex responses were rank transformed prior to analysis. Statistical tests were two tailed, with P 0.05 considered to be significant. P values were not adjusted for multiple testing with this small sample size; however exact P values are given where practicable, to facilitate interpretation. Statistical analyses were performed with SAS Proc Mixed or Proc HPMixed version 9.4 (SAS Institute, Cary, NC, USA). Results Frequency controlled breathing

Median carbon dioxide spectral power was comparable for all four controlled frequency protocols (P = 0.84 (0.72 and 0.9)). The carbon dioxide spectral power and the frequency distribution during random breathing were positively skewed – the distribution was not rectangular, or ‘white noise’, as planned. As expected, carbon dioxide spectral power was much smaller at most individual frequencies during random breathing than during the other three breathing modes. The median (25th and 75th percentile) controlled breathing frequencies were random: 0.27 (0.26 and 0.29) Hz and 0.25 (0.25 and 0.25) Hz; 0.1


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(0.1 and 0.11) Hz and 0.05 (0.05 and 0.06) Hz. Tidal volume was significantly lower during 0.25 than during 0.1 and 0.05 Hz breathing, hyperventilation and recovery (all P < 0.0001). The median (25th and 75th percentile) frequencies of spontaneous breathing were 0.21 (0.16 and 0.25) Hz and 13 (10 and 15) breaths min–1 . The median end-tidal carbon dioxide concentration during spontaneous breathing was 5.5% (5.4 and 5.7%), and was significantly (P 0.001) higher than levels during random and 0.25 and 0.1 Hz breathing (all 4.5–4.6% (4.2–5.3%)). End-tidal carbon dioxide levels were significantly higher during 0.05 Hz than 0.25 and 0.1 Hz breathing (P = 0.01 and 0.03), but were comparable (P = 0.58) during 0.25 and 0.1 Hz breathing. As expected, median end-tidal carbon dioxide levels were significantly lower (P 0.003) during hyperventilation, 3% (2.3 and 3.7%), than during all other breathing modes. Carbon dioxide responses to apnoea are reported below. We present results in descriptive terms, figures and in an important Table 1, which lists mean, 95% confidence limits, and pairwise statistical comparisons. The low frequency alpha index vagal baroreflex gain was lower during 0.25 Hz breathing than during 0.1 and 0.05 Hz breathing. Baroreflex gain was comparable, however, during 0.1 and 0.05 Hz breathing (P = 0.07). The measurement considered to reflect vagal tone, R-R interval, was comparable over the three breathing frequencies (P = 0.41–0.74). The measurements considered to reflect oscillations of vagal firing, RMSSD and pNN50, were significantly higher during 0.1 than 0.25 Hz breathing (P = 0.05 and 0.02). Vagal baroreflexes

Figure 2 indicates that, as expected (since all spontaneous baroreflex estimates are based on correlations between R-R intervals and systolic pressures), median gains for all four baroreflex estimates varied during different breathing modes in similar ways. (We do not include high frequency baroreflex gains in this analysis, for reasons discussed below.) Although we calculate transfer function gains in this and the companion article (Eckberg et al. 2016), we rely on the well-validated (Diedrich et al. 2013) low frequency alpha baroreflex index over the other methods, in part on statistical grounds. Its values were less variable and closer to the median than the other three measurements; it had a log normal distribution (positively skewed) within each condition; and its coefficient of variation was more stable than the other metrics. The attributes of low frequency alpha baroreflex index estimates satisfied assumptions of the parametric modelling that we used. Figures 3 and 4 compare vagal baroreflex parameters during 0.25 and 0.1 Hz breathing. Figure 3 shows

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median and 25th and 75th percentile transfer functions, coherences and phases at low (0.04–0.15 Hz) and high (0.15–0.4 Hz) frequencies. At low frequencies (Fig. 3A), only one pairwise comparison of baroreflex gain, between 0.1 Hz breathing and late apnoea, was significant. At high frequencies (Fig. 3B), no pairwise comparisons were significant. Median gain was comparable at low and high frequencies: 12.3 (8.7 and 14) and 10.8 (10.1 and 14.5) ms mmHg–1 (P = 0.91). At low frequencies (Fig. 3C), coherence was higher during 0.1 Hz breathing, recovery and hyperventilation than during 0.25 Hz breathing. At high frequencies (Fig. 3D), 8 of 36 pairwise comparisons were significant, most notably between hyperventilation and all other controlled breathing modes. Across breathing modes, median coherence (Fig. 3C and D) was significantly higher at low than high frequencies: R = 0.85 (0.83–0.91) vs. 0.65 (0.47 − 0.7), P = 0.01. There were no significant pairwise differences of median phase at either low or high frequencies. Across breathing modes, the median phase angle (Fig. 3E and F) was significantly greater at low than high frequencies: −71 (−73 and −69) vs. −35 (−48 and −23) deg (P = 0.004). If negative values indicate that pressure changes precede R-R interval changes (as is likely), and if the 15 s hyperventilation values be excluded, median calculated latencies were 2 (2.1 and 2) vs. 0.4 (0.5 and 0.3) s for low and high frequency baroreflex estimates (P < 0.001). Notwithstanding comparable baroreflex gains (Fig. 3A and B), the coherence and phase data shown in Fig. 3C–F visually challenge the notion that frequency domain baroreflex estimates at low and high frequencies reflect the same physiology. We closely examined baroreflex gain at the two breathing frequencies, 0.1 and 0.25 Hz, because slow breathing (at 0.1 Hz) increases baroreflex gain (Bernardi et al. 2001; Lehrer et al. 2003; Table 1), and healthy supine subjects tend to breathe at 0.25 Hz (see below). Figure 4A and C shows systolic pressure (black) and R-R interval (grey) averages after threshold crossings set on early inspiration (vertical dashed lines). Note the differences in scales: both systolic pressure and R-R interval changes were much greater during 0.1 than 0.25 Hz breathing. Median latencies during 0.25 and 0.1 Hz breathing (Fig. 4B) were 0.5 (0.2 and 0.9) vs.1.9 (1.5 and 2.6) s, and all individual differences and the group difference were highly significant (P < 0.001, paired t test). Figure 4D shows median 15 s alpha index baroreflex gain, moved by 1 s steps through the two consecutive 180 s breathing periods. There was a striking, highly significant step increase of baroreflex gain at the transition between 0.25 and 0.1 Hz breathing frequencies, from 12.6 (10.5 and 12.6) to 15.7 (10.8 and 18.5) ms mmHg–1 (P < 0.0001).


97 (80, 114)

32 (25, 43)

58 (46, 69) F∗ , I

Burst area (% Spon.)

Bursts/min

Burst probability (%)

31 (26,39) G, H 54 (44, 64) F∗

97 (83, 111)

9 (1, 21) G∗

31 (21, 45) G∗

62 (55, 68) G 0.95 (0.84, 1.0) I 17 (13, 22) C∗ , F∗ , G∗ , H∗

29 (24, 36) G∗ , H∗ 54 (44, 64) F∗

94 (79, 109)

5 (0, 15)

26 (18, 39) G

62 (55, 68) G .96 (0.84, 1.08) I 13 (8, 18)

68 (62, 75)

130 (118, 142)

4.7 (4.3, 5.1)

0.25 Hz

C

31 (26, 40) G 57 (47, 67) F∗ , I

98 (83, 113)

11 (3, 25) C, E, F, G∗

35 (23, 51) C, F, G∗

63 (56, 69) G 0.99 (0.87, 1.1) F, I∗ 17 (12, 21) C∗ , F, G, H

67 (60, 73)

129 (117, 141)

4.8 (4.4, 5.2)

0.1 Hz

D

29 (24, 36) G∗ , H∗ 53 (42, 63) F

95 (80, 110)

5 (0, 16)

27 (18, 40) G

63 (56, 69) F, G∗ 0.94 (0.82, 1.05) I 16 (12, 21) C, F, G, H

68 (62, 75)

4.8 (4.1, 5.5) F∗ , G∗ 131 (119, 143)

0.05 Hz

E

28 (22, 37) G, H 42 (30, 55)

110 (92, 129) C, E

3 (0, 15)

32 (13, 35)

59 (48, 70) F∗ , I

119 (103, 136) A∗ , B∗ , C∗ , D∗ , E∗ 38 (29, 56)

2 (0, 10)

18 (12, 29)

12 (7,17)

0.89 (0.77, 1)

0.84 (0.72, 0.96) 11 (5, 17)

54 (47, 61)

72 (65, 78) F

126 (114, 138)

3.1 (2.6, 3.6)

Early apnoea

G

56 (49, 63)

64 (58, 71)

120 (108, 132)

3 (2.3, 3.7)

Hyperventilation

F

64 (53, 76) B∗ , C∗ , D, E∗ F∗ , I∗

141 (124, 158) A∗ , B∗ , C∗ , D∗ , E∗ , F∗ . G∗ , I∗ 38 (29, 55)

9 (1, 22) G

32 (21, 50) G∗

12 (7, 18)

5.2 (5, 5.5) F∗ , G∗ 148 (136, 160) A∗ , B∗ , C∗ , D∗ , E∗ , F∗ , G∗ 84 (78, 91) A∗ , B∗ , C∗ , D∗ , E∗ , F∗ , G∗ , I 63 (57, 70) F, G∗ 0.9 (0.78, 1.02)

Late apnoea

H

20 (15, 25) A, C∗ , E, F∗ , G∗ , H∗ 27 (18, 40) A∗ , B∗ , C∗ , D, E∗ , F∗ , G∗ , H 20 (7, 37) A, B∗ , C∗ , E∗ , F∗ , G∗ , H 120 (103, 137) A∗ , B∗ , C∗ , D∗ , E∗ , F∗ 26 (22, 33) B, D, G∗ , H∗ 48 (37, 60)

5.1 (4.4, 5.7) F∗ , G∗ 143 (131, 156) A, B∗ , C, D, E, F, G∗ 77 (71, 84) A∗ , B∗ ,C∗ , D∗ , E∗ , F∗ 65 (58, 72) F∗ , G∗ 0.81 (0.7, 0.93)

Recovery

I


Each column of measurements is designated by a letter. A letter in a different column indicates that that measurement is significantly larger than the value for same measurement. Asterisks indicate that P < 0.01. RMSSD, square root of mean squared differences of successive normal R-R intervals. pNN50 = proportion of successive normal R-R intervals greater than 50 ms, divided by the total number of normal R-R intervals. Burst probability = percentage of heartbeats with sympathetic bursts.

3 (1, 21) G

28 (18, 44) G

63 (57, 70) F, G∗ 0.98 (0.86, 1.1) F, I 15 (10, 21)

67 (60, 73)

128 (117, 140)

131 (119, 143)

68 (61, 74)

4.6 (4.3, 5)

5.5 (5, 5.9)

Random

Spontaneous breathing

pNN50 (%)

RMSSD (ms)

Baroreflex (ms/mmHg)

Pulse pressure (mmHg) R-R interval (s)

Diastolic pressure (mmHg)

End-tidal carbon dioxide (%) Systolic pressure (mmHg)

B

A

Table 1. Mean, 95 % confidence limits, and pairwise comparisons for all subjects and all breathing modes

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Hyperventilation, apnoea and recovery

n=8

15 10 5

oe a Re co ve ry

La

te a

pn

n

no e ap

rly

Ea

Hy

pe

rve

nti

lla

tio

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Hz

0.0

0.1

0.2

Ra n

5H z

0

a

Transfer Down Alpha index Up Median do m

Median vagal baroreflex gain (ms mmHg–1)

Since apnoea durations varied among subjects, we analysed the first and last 50 s of data separately. The

Breathing mode

Figure 2. Median (continuous black line and circles) vagal baroreflex estimates derived from four methods As expected, vagal baroreflex estimates tended to change in parallel with the different breathing modes. The highest median vagal gain was registered during 0.1 Hz breathing. (Transfer function and alpha index baroreflex gains rose to their highest levels during recovery from apnoea; extreme right.) Statistical analyses of changes of the alpha baroreflex estimate are listed in the Table 1.

median apnoea duration was 125 s (range: 86–220 s). (Three subjects had apnoea durations of less than 100 s, all 86 s; therefore, their last 50 s periods of apnoea included the terminal 14 s of their first 50 s periods.) For analyses of apnoea responses, we synchronized each subject’s data precisely on the beginnings and endings of apnoea, and calculated changes with 1 s time resolution. Figures 5 and 6, show data from the last 30 s of 0.05 Hz breathing, hyperventilation, the first and last 50 s of apnoea and recovery. (Note that since these data were derived from 15 s averages moved by 1 s steps (see Methods), the earliest change of each value began 7.5 s earlier than shown.) Figure 5 (top panels) shows end-tidal carbon dioxide levels and their linear extrapolations during apnoea. (The relation between lung carbon dioxide levels and apnoea duration is gently asymptotic (Mithoefer, 1959); therefore, although our linear projections are accurate at the beginning and end of apnoea, they modestly underestimate carbon dioxide levels between these points.) As expected, median carbon dioxide levels fell during hyperventilation (from 4.8% (4.1 and 5.5%) during 0.05 Hz breathing to 3.1% (2.3 and 3.7%)). Extrapolated carbon dioxide levels during early apnoea were less (P < 0.0003) than those measured during 0.05 Hz breathing, but extrapolated carbon dioxide levels during late apnoea (5.3% (4.4 and 5.7%)) and measured levels during recovery (5.1% (4.4 and 5.7%)) were comparable to those measured during 0.05 Hz breathing (P = 0.62 and 0.6).

Gain (ms/mmHg)

0.9

Phase (0)

Coherence

Median, 25th and 75 % transfer function baroreflex parameters (n = 8) 40

A

0.04 – 0.15 Hz

B

0.15 -- 0.6 Hz

20 0

0.6 0.3

C

D

0

E

F

–50

on ta

ne

ou s Ra nd om 0.2 5H z 0.1 Hz 0.0 Hy 5H pe rve z nti l a tio Ea n rly ap n oe La a te ap no ea Re co ve ry Sp on tan eo us Ra nd om 0.2 5H z 0.1 Hz 0.0 Hy 5H pe rve z nti lat Ea ion rly ap no La ea te ap no ea Re co ve ry

–100

Sp

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Breathing mode

Figure 3. Median, 25th and 75% (gray) vagal baroreflex parameters at low and high frequencies Baroreflex gain (A and B) was comparable at low and high frequencies. Coherence (C and D) was significantly higher, and the phase angle (E and F) was significantly greater at the low than the high frequency. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

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Systolic pressure (Fig. 5, second row) fell insignificantly during hyperventilation, rose significantly during late apnoea and remained elevated during early recovery. Pulse pressure (not shown) was higher during most breathing modes than during hyperventilation and early apnoea. R-R intervals did not change significantly during hyperventilation, apnoea and recovery (mean P = 0.28; range: 0.17–0.84)). (Note that bradycardia did not develop during apnoea.) One index of vagal fluctuations, RMSSD (not shown), was significantly higher during recovery than during most other breathing modes (including early apnoea). The other vagal fluctuation index, pNN50, was significantly higher during late than early apnoea. Baroreflex gain also was higher during random, 0.1 and 0.05 Hz breathing, and recovery than during hyperventilation and early and late apnoea. Figure 6 shows diastolic pressure and sympathetic metrics. Diastolic pressure rose steadily and substantially during apnoea, and returned to usual levels during recovery. Muscle sympathetic burst areas, were significantly greater during early and late apnoea and recovery than during most other breathing modes (Table 1). Sympathetic burst frequencies were significantly greater during early and late apnoea than during hyperventilation and recovery. The median percentage of heart beats with sympathetic bursts, burst probability, began to increase during hyperventilation, rose steadily during early apnoea, and never returned to levels measured

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during spontaneous (horizontal dashed line) or 0.05 Hz breathing. Burst probability was lower during hyperventilation, and was higher during late apnoea than during all controlled breathing modes. R-R interval oscillations

Figures 7 and 8 address the question, Do respiratory frequency R-R interval fluctuations persist during apnoea? Figure 7 shows individual R-R interval spectral powers during spontaneous breathing and the first and last 50 s of apnoea (note the widely different vertical scales). The frequencies of carbon dioxide spectral power (dotted lines), taken as indices of breathing frequency during spontaneous breathing, varied widely among our physically fit subjects. R-R interval spectral powers at spontaneous breathing frequencies (continuous grey lines) were small or not apparent. In all subjects, R-R interval spectral power was higher during early (black) than late (circles) apnoea, and in all but one subject (Subject 3) was higher during early apnoea than spontaneous breathing. Figure 8 depicts median and 25th and 75th percentile integrated R-R interval spectral powers in very low (0–0.04 Hz), low (0.04–0.15 Hz) and high frequency ranges (0.15–0.4 Hz), during spontaneous breathing and early and late apnoea. Subject 2 was excluded as an outlier for this analysis (see Fig. 7). Very low frequency power

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Figure 4. Responses to 0.1 and 0.25 Hz breathing: systolic pressures and R-R intervals signal averaged on early inspiration, latencies between the two measures, and moving vagal baroreflex gain Median systolic pressure and R-R interval changes triggered by inspiration (A and C), systolic pressure–R-R interval latencies (B), and the moving alpha index baroreflex gain (D) were all significantly greater during 0.1 than 0.25 Hz breathing. Symbols in B indicate median values. Gray areas in D indicates 25th and 75th%. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

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(Fig. 8A) dominated low and high frequency powers (Fig. 8B and C, repeated measures analysis of variance, P < 0.001). There were no significant differences between low and high frequency powers. Obviously, the validity of this analysis is challenged by the brief apnoea periods, and therefore, the confidence we place on our analysis of very low frequency rhythms is small. The 50 s periods yielded only 1 very low frequency cycle (apparent in Fig. 1C, extreme right), 6 low frequency cycles and 16 high frequency cycles. The power of high frequency oscillations (Fig. 8C, inset) is vanishingly small, notwithstanding their credibility as frequency domain data. We divided the results shown in Fig. 8 into 50 s early and late apnoea phases so that we could detect possible trends during apnoea. We also calculated spectra over entire apnoea periods for each subject. During entire apnoea periods, the median numbers of oscillations at very low, low and high frequencies were 2.5, 12.7 and 36, For the longer time series, spectral power at very low frequencies was significantly (P < 0.001) greater than power at low and

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high frequencies (which did not differ significantly from each other; P = 0.7). Median high frequency R-R interval spectral power was 0.4% of median very low frequency power. Discussion We studied haemodynamic and autonomic responses of astronauts to controlled frequency breathing and apnoea on Earth, for two reasons: first, to clarify important and contentious central autonomic mechanisms in a larger number of subjects than were involved with the space study (Eckberg et al. 2016, 8 vs. as few as 4), and second, to identify metrics for serial evaluation in astronauts. Our main conclusions are that R-R interval fluctuations at usual breathing frequencies are likely not to be baroreflex mediated, and are not present during apnoea; that vagal tone does not change during apnoea, but that vagal baroreflex gain declines significantly; that muscle sympathetic nerve burst areas, frequencies and Systolic pressure and vagal metrics

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Figure 5. Meidan, 25th and 75th% (gray) systolic pressure and vagal measurements during 0.05 Hz breathing, hyperventilation, the first and last 50 s of apnoea and the first 50 s of recovery End-tidal carbon dioxide concentrations were linearly extrapolated between the last measurement made during hyperventilation and the first measurement made during recovery. The vertical dashed lines, left panels, indicate the onsets of hyperventilation and apnoea, and the vertical dashed lines, right panels, indicate the end of apnoea.

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probabilities increase significantly during apnoea, even as arterial pressure climbs to new levels; and that the major haemodynamic and autonomic changes initiated by apnoea in healthy subjects are driven importantly, and possibly prepotently, by changes of respiratory motoneurone activity. Vagal baroreflex mechanisms

We revisited human vagal baroreflex physiology because of ongoing controversy over its underlying mechanisms (Eckberg, 2009; Karemaker, 2009). Our approach was non-pharmacological – simple breathing control – and, although some individual findings are not new (for example, Dornhorst et al., as long ago as 1952, showed that slow breathing augments arterial pressure fluctuations), the combination of our findings may be unique and as such, may provide refined insights into human baroreflex mechanisms (Dornhorst et al. 1952). Since arterial pressure fluctuates rhythmically with normal breathing (Hales, 1733), it was reasonable to suspect (de Boer et al. 1985; Elghozi et al. 1991; Scheffer et al. 1994) that each R-R interval change

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is caused by the preceding pressure change, and thus is baroreflex mediated. We tested this possibility by examining arterial pressure–R-R relations over different breathing frequencies, including what is considered ‘normal breathing’, which occupies a surprisingly narrow range: 0.22–0.28 Hz (Lenfant, 1967; Narkiewicz et al. 2006; Pinna et al. 2006; Wallin et al. 2010; Stankovski et al. 2013; Clemson PT, Hoag JB, Cooke WH, Stefanovska A & Eckberg DL, unpublished data). Our results are most unambiguously expressed by contrasts between the normal breathing frequency, 0.25 Hz, and slow breathing, 0.1 Hz. In a Point:Counterpoint debate, Eckberg (2009) argued that latencies between pressure and R-R interval changes during spontaneous breathing derived from time or frequency domain analyses (in two studies, 0.25 s; Eckberg, 1976; Koh et al. 1998) are too short to be mediated by baroreflexes. Karemaker (2009) responded that estimates of latencies based on frequency domain Subject

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Figure 7. Individual R-R interval spectral powers during spontaneous breathing and apnoea R-R interval spectral powers during spontaneous breathing are shown with continuous grey lines; carbon dioxide spectral powers (values exceeding 5% of maximum) are shown with dotted lines; and R-R interval spectral power during the first and last 50 s of apnoea are shown with continuous black lines and open circles. The carbon dioxide spectral powers indicate that during spontaneous breathing, subjects breathed at widely different frequencies; the R-R interval spectral powers at these frequencies (continuous grey lines) tended to be small, and during apnoea virtually absent. M: million.



analyses may differ from those derived from time domain analyses. Our new study documents a median time domain arterial pressure–R-R interval latency of 0.4 s (Fig. 4B). This reinforces the earlier conclusion that R-R interval changes during normal breathing occur too soon after inspiratory pressure changes to be baroreflex mediated. Conversely, the median time domain systolic pressure–R-R interval latencies of 1.6 s during 0.1 Hz breathing and 2 s across all breathing modes are fully compatible with baroreflex physiology (Eckberg, 1980; Diedrich et al. 2013). Although minor (< 30 ms) R-R interval prolongation can be provoked with latencies of 0.5 s, by intense, highly unphysiological stimuli (−60 mmHg neck suction with a rate of pressure change 3000 mmHg s–1 ; Eckberg, 1976) or electrical carotid sinus nerve stimulation (Borst & Karemaker, 1983), major R-R interval prolongation does not develop until > 1.5 s (Baskerville et al. 1979). We show also that over a range of breathing modes, arterial pressure and R-R interval fluctuations are more coherent and more consistently so, and latencies (phase angles) are greater and more consistently so at low than high breathing frequencies. Simple inspection of Fig. 3C–F challenges the notion that responses at low and high frequencies share a common physiological mechanism. We add two caveats: first (and obviously), baroreflex mediation becomes increasingly likely as breathing rate slows from 0.25 Hz, and second, baroreflex physiology could obtain in subjects breathing at 0.25 Hz, whose resting R-R intervals (and therefore, latencies between pressures and successive P waves) are extraordinarily long. Data recorded during fixed frequency breathing (Fig. 4) confirm the finding that slow breathing augments vagal

baroreflex gain (Bernardi et al. 2001; Lehrer et al. 2003). We extend this observation by identifying a clear step increase of alpha index baroreflex gain at the transition between 0.25 and 0.1 Hz breathing, which persisted during the entire 180 s of 0.1 Hz breathing (Fig. 4D). Moreover, we show that baroreflex augmentation is not limited to 0.1 Hz, but occurs also during 0.05 Hz breathing (Table 1). This confirms the finding of Stankovski et al. (2013) that baroreflex gain varies systematically over a wide range of breathing frequencies, including 0.1 Hz. Autonomic responses to hyperventilation, apnoea and recovery

Our experiment is relevant to sleep apnoea studies (Kara et al. 2003), which stress the importance of (1) chemoreceptor stimulation, (2) baroreflex opposition of sympathoexcitation, (3) elimination of sympathetic inhibition by pulmonary stretch receptor activity, and (4) reduction of sympathetic activity by breathing. Our results, obtained from healthy subjects – but see below – suggest that none of these mechanisms are necessary to explain autonomic responses to apnoea. First, chemoreceptor stimulation by hypercapnia cannot explain our findings: end-tidal carbon dioxide levels ranged from hypocapnia to normocapnia (Figs 1 and 5, and Table 1). It is unlikely that hypocapnia contributed to responses during early apnoea, because moderate end-tidal carbon dioxide reductions do not alter vagal baroreflex gain (Henry et al. 1998), and brief hyperventilation and hypocapnia do not alter muscle sympathetic nerve activity (St Croix et al. 1999; Shoemaker et al. 2002; Table 1).

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Figure 8. Meidan, 25th and 75th% (gray) integrated R-R interval spectral powers during spontaneous breathing and apnoea A very low frequency rhythm (A) dominated the three periods, and was particularly large during early apnoea. Neither low nor high frequency rhythms (B and C) were significantly different from each other during the three periods. The scales of low and high frequency spectral powers are expanded in the insets.

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Second, although we did not measure oxygen saturation (we could not add an additional recording channel, because other Neurolab protocols (Cox et al. 2002) used remaining channels), it is very unlikely that hypoxia contributed to our subjects’ responses. In one study (but see below), 20 s apnoeas preceded by room air breathing did not alter muscle sympathetic nerve activity (Leuenberger et al. 2005). In healthy subjects, reduction of oxygen saturation does not begin until the second minute of apnoea, and by that time has fallen only from 99% to 97% (Palada et al. 2008). Breathing 14% oxygen does not increase muscle sympathetic nerve activity (Somers et al. 1989), and breathing 12% oxygen does not increase sympathetic activity until after 11 min of exposure (Rowell et al. 1989). If hypoxia occurred in our subjects, it would not be expected to alter vagal (Bristow et al. 1971; Eckberg et al. 1982) or sympathetic (Halliwill & Minson, 2002) baroreflex gains. Moreover, apnoea alters physiology in ways that cannot be ascribed solely to hypoxia: sympathetic responses to identical levels of hypoxia are greater during apnoea than during breathing hypoxic gases (Somers et al. 1989; Smith et al. 1996; Steinback et al. 2010), and central respiratory motoneurone activity associated with individual breaths silences sympathetic motoneurones, and overrides sympathetic responses to hypoxia (Steinback et al. 2010). Finally, after resumption of breathing, muscle sympathetic nerve activity returns to normal too soon to be explained by reduction of chemoreceptor activity (Heusser et al. 2009). Third, our findings refine understanding of baroreflex function during apnoea. Muenter Swift et al. (2003) reported that neither vagal nor sympathetic baroreflex gain (experimentally altered by static neck suction and pressure) changes during apnoea. We found that spontaneous vagal baroreflex gain is significantly lower during early and late apnoea than during 0.1 and 0.05 Hz frequency breathing and recovery (Fig. 5 and Table 1). Although we did not estimate sympathetic baroreflex gain during our 50 s periods of analysis, we show (Fig. 6) that during and following apnoea, sympathetic burst frequencies move in parallel with diastolic pressure changes. Moreover, during early apnoea, burst probabilities increased, even as pressure was rising (Fig. 6) and the intervals between heart beats were shortening. These changes are opposite to what should have occurred if sympathetic baroreflexes had been functioning as usual (Sundlȍf & Wallin, 1978; Querido et al. 2011). Our results also speak against the possibility that increases of sympathetic activity result from absence of pulmonary stretch receptor activity. This notion is grounded in part on the prevalent concept that breathing rate influences the level of sympathetic activity. Although our finding that sympathetic activity was the same during

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0.25 Hz breathing and all other fixed-frequency breathing modes (Table 1) merely confirms those of earlier studies (Seals et al. 1990; Stankovski et al. 2013), its iteration may be important, given the view (Narkiewicz et al. 2006; Raupach et al. 2008; Roseng˚ard-Bärlund et al. 2011) that slow breathing reduces sympathetic activity. In our study, sympathetic burst areas and frequencies were similar when subjects breathed once every 4 s and once every 20 s (Table 1), despite the likelihood that increased tidal volumes during slow breathing augmented pulmonary stretch receptor activity (Molkov et al. 2014). Moreover, lung innervation is not necessary for sympathoexcitation to occur during apnoea (Khayat et al. 2004). Since our results point away from mechanisms thought to contribute to apnoeic responses of obstructive sleep apnoea patients, the question remains, What mechanisms underlie haemodynamic and autonomic responses of healthy subjects to apnoea? Abortive gasps during apnoea may help to answer this question. Small breaths that interrupt apnoea (Macefield & Wallin, 1995; Passino et al. 1997; Palada, 2008), breaths of 100% nitrogen, which do not reduce hypoxia (Seitz et al. 2013), and even abortive respiratory movements so small that they do not move air or change carbon dioxide levels can alter haemodynamic and autonomic function in major ways: Badra et al. (2001) documented abrupt R-R interval reductions as large as 250 ms, followed by increases as large as 400 ms during such tiny movements. We ask, Why is it that breathing as infrequently as once every 20 s does not increase sympathetic activity (Table 1), but cessation of breathing for only 7 s (Fritsch et al. 1991) does? Vagal and sympathetic baroreflex changes begin nearly instantaneously after the onset of apnoea, before any of the mechanisms discussed above could become operative: arterial pressure and muscle sympathetic nerve activity increase significantly after only 7 s of held expiration, and R-R intervals remain constant, notwithstanding rising arterial pressure. We propose an important role for changes of central respiratory motoneurone activity (Cohen, 1979; Richter & Spyer, 1990) in mediating apnoea responses. We cannot specify the precise nature of the central respiratory motoneurone changes that our protocol elicited: the autonomic responses we report during held inspiration, after a larger than usual breath, when expiratory motoneurone activity is likely to be absent, are reflected also during held expiration after a normal breath (Fritsch et al. 1991), when inspiratory activity is likely absent and expiratory activity is sustained (Sears, 1964). Our conclusions concern healthy subjects, and suggest that apnoea alone is sufficient to explain its major haemodynamic and autonomic consequences. We emphasize that this conclusion may not apply to sleep apnoea patients. Haemodynamic changes during voluntary held inspiration are different from those of


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patients who, in addition to apnoea, may have airway occlusion (Morgan et al. 1993), cumulative effects secondary to repetitive chemoreceptor stimulation (Xie et al. 2000, and alterations modulated by different sleep stages and arousal levels (Dempsey et al. 2010). R-R interval rhythms during apnoea

Human R-R interval fluctuations persist during apnoea at frequencies below 0.15 Hz (Agostoni, 1963; Hirsch & Bishop, 1981; Kollai & Mizsei, 1990; Trzebski & ´ Smietanowski, 1996; Piepoli et al. 1997; Badra et al. 2001). In 2003, Cooper and colleagues reported that R-R interval fluctuations during apnoea occur also at respiratory frequencies (Cooper et al. 2003). Since these are rhythms that by definition were present in the absence of breathing, Cooper considered them to be indirect evidence for the existence of a central respiratory oscillator. We examined Cooper’s provocative hypothesis by closely evaluating her data, and by collecting our own. Cooper et al. (2003) relied heavily on data from one subject (Fig. 1), whose resting breathing rate was 0.11 Hz. Although healthy research volunteers breathe spontaneously over a wide range of frequencies (Lenfant, 1967), 0.11 Hz is far below the average breathing frequencies of healthy resting subjects. Moreover, fully 70% of Cooper’s subjects breathed at rates below 0.22–0.28 Hz, the average range reported in the six studies cited above. Low and respiratory frequency cardiovascular oscillations have different underlying physiology (Koepchen, 1984); the relative contributions of low and respiratory mechanisms to R-R interval rhythms in subjects who breathe slowly cannot be known with certainty. We make two concessions regarding our own data, which do not support Cooper’s hypothesis. First, since the non-existence of a respiratory frequency rhythm during apnoea cannot be proven, our argument must be dealt with in quantitative terms: we report that if R-R intervals fluctuate at usual respiratory frequencies during apnoea, those fluctuations are small indeed (Fig. 8C, inset). Second, although increased sympathetic stimulation during apnoea must dampen vagal oscillations (Taylor et al. 2001), it seems unlikely that increased sympathetic opposition would extinguish them altogether. Limitations

Our protocol was designed for space research, and therefore, the number of subjects we studied is small (Pawelczyk, 2006). We cannot exclude the possibility that some negative statistical results might have been positive had we studied more subjects. We did not measure oxygen saturation, and we rely on earlier publications to support our conclusions. End-tidal carbon dioxide levels were significantly lower during controlled frequency than

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spontaneous breathing. On the basis of earlier research (Henry et al. 1998), however, we doubt that this influenced our results. A major limitation, which is inherent in all apnoea research, is that the ability of healthy subjects to hold their breaths is limited; as discussed, this limitation is particularly relevant when attempts are made to quantify very low frequency changes. Another limitation is that subjects may experience involuntary, apparently abortive respiratory movements during apnoea (Agostoni, 1963; Badra et al. 2001), which are beyond the control of the researcher. We discuss this above, and suggest that trivial movements so small that they do not move air or change chemoreceptor input may serendipitously provide unique information on central neurophysiological mechanisms, independent of the peripheral manifestations of actual breaths. Summary

Healthy human subjects bring great strength to physiological research: they can cooperate – in our case, by controlling their breathing. Our simple protocol permitted us to study important central mechanisms upon which contemporary physiologists disagree. First, our data speak against a baroreflex explanation for the rhythmic R-R interval fluctuations that occur at usual breathing rates. Second, we traced, with 1 s time resolution, haemodynamic and autonomic fluxes triggered by apnoea, and documented a continuous and ever changing reorganization of the relations between stimulatory and inhibitory inputs and autonomic outputs, which in our study could not be attributed to altered chemoreceptor, baroreceptor or pulmonary stretch receptor activity. Third, we make a case that responses to apnoea are driven importantly, and perhaps prepotently, by changes of central respiratory motoneurone activity. Fourth, we report that R-R interval fluctuations at usual respiratory rates do not persist during apnoea, and thus cannot be taken as indirect evidence for the existence of a central respiratory rhythm. References Agostoni E (1963). Diaphragm activity during breath holding: factors related to its onset. J Appl Physiol 18, 30–36. Badra LJ, Cooke WH, Hoag JB, Crossman AA, Kuusela TA, Tahvanainen KUO & Eckberg DL (2001). Respiratory modulation of human autonomic rhythms. Am J Physiol Heart Circ Physiol 280, H2674–H2688. Baskerville AL, Eckberg DL & Thompson MA (1979). Arterial pressure and pulse interval responses to repetitive carotid baroreceptor stimuli in man. J Physiol 297, 61–71. Bernardi L, Gabutti A, Porta C & Spicuzza L (2001). Slow breathing reduces chemoreflex response to hypoxia and hypercapnia, and increases baroreflex sensitivity. J Hyperten 19, 2221–2229.


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Bertinieri G, di Rienzo M, Cavallazzi A, Ferrari AU, Pedotti A & Mancia G (1985). A new approach to analysis of the arterial baroreflex. J Hyperten 3(Suppl. 3), S79–S81. Borst C & Karemaker JM (1983). Time delays in the human baroreceptor reflex. J Autonom Nerv Syst 9, 399–409. Bristow JD, Brown EB Jr, Cunningham DJC, Goode RC, Howson MG & Sleight P (1971). The effects of hypercapnia, hypoxia and ventilation on the baroreflex regulation of the pulse interval. J Physiol 216, 281–302. Cohen MI (1979). Neurogenesis of respiratory rhythm in the mammal. Physiol Rev 59, 1105–1173. Cooper HE, Parkes MJ & Clutton-Brock TH (2003). CO2 -dependent components of sinus arrhythmia from the start of breath holding in humans. Am J Physiol Heart Circ Physiol 285, H841–H848. Cox JF, Tahvanainen KUO, Kuusela TA, Levine BD, Cooke WH, Mano T, Iwase S, Saito M, Sugiyama Y, Ertl AC, Biaggioni I, Diedrich A, Robertson RM, Zuckerman JH, Lane LD, Ray CA, White RJ, Pawelczyk JA, Buckey JC Jr, Baisch FJ, Blomqvist CG, Robertson D & Eckberg DL (2002). Influence of microgravity on astronauts’ sympathetic and vagal responses to Valsalva’s manoeuvre. J Physiol 538, 309–320. de Boer RW, Karemaker JM & Strackee J (1985). Relationships between short-term blood-pressure fluctuations and heart-rate variability in resting subjects II: a simple model. Med Biol Eng Comput 23, 359–364. Dempsey JA, Veasey SC, Morgan BJ & O’Donnell CP (2010). Pathophysiology of sleep apnea. Physiol Rev 90, 47–112. Diedrich A, Crossman AA, Beightol LA, Tahvanainen KUO, Kuusela TA, Ertl AC & Eckberg DL (2013). Baroreflex physiology studied in healthy subjects with very infrequent muscle sympathetic bursts. J Appl Physiol 114, 203–210. Di Rienzo M, Castiglioni P, Iellamo F, Volterrani M, Pagani M, Mancia G, Karemaker JM & Parati G (2008). Dynamic adaptation of cardiac baroreflex sensitivity to prolonged exposure to microgravity: data from a 16-day spaceflight. J Appl Physiol 105, 1569–1575. Dornhorst AC, Howard P & Leathart GL (1952). Respiratory variations in blood pressure. Circulation 6, 553–558. Eckberg DL (1976). Temporal response patterns of the human sinus node to brief carotid baroreceptor stimuli. J Physiol 258, 769–782. Eckberg DL (1980). Nonlinearities of the human carotid baroreceptor-cardiac reflex. Circ Res 47, 208–216. Eckberg DL (2009). Point:Counterpoint: Respiratory sinus arrhythmia is due to a central mechanism vs. respiratory sinus arrhythmia is due to the baroreflex mechanism. J Appl Physiol 106, 1740–1742. Eckberg DL, Bastow H & Scruby AE (1982). Modulation of human sinus node function by systemic hypoxia. J Appl Physiol 52, 570–577. Eckberg DL, Diedrich A, Cooke WH, Biaggioni I, Buckey JC Jr, Pawelczyk JA, Ertl AC, Cox JF, Kuusela TA, Tahvanainen KUO, Mano T, Iwase S, Baisch FJ, Levine BD, Adams-Huet B, Robertson D & Gunnar Blomqvist C (2016). Respiratory modulation of human autonomic function: long-term neuroplasticity in space. J Physiol 594, 5629–5646.

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Additional information Competing interests None declared.

Author contributions D.L.E. conceived the research, obtained the funding, helped perform pilot studies, analysed the data, prepared the figures, and wrote the manuscript. W.H.C. helped develop the protocol, helped perform the terrestrial studies, and analysed the data. A.D. performed pilot studies, and formatted the data for analysis. I.B. coordinated the Neurolab protocols and contributed to their integration. Astronauts J.C.B. and J.A.P. performed studies in space. T.A.K. and K.U.O.T. contributed to protocol development and created the software used to analyse the data. A.C.E., J.F.C and T.M. and S.I. contributed to protocol development, and S.I. performed landing day studies. B.A.-H. performed statistical analyses. F.J.B., D.R., B.D.L. and C.G.B. all contributed to protocol development and integration. All authors have approved the final version of the manuscript and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. Funding This work was supported by National Aeronautics and Space Administration contracts NAS0-19541 and NAG2-408, and National Heart, Lung, and Blood Institute, UO1HL-56417. It was also supported by a grant from the Centennial Foundation of Helsingin Sanomat, Finland. Acknowledgements We thank the astronauts and backup astronauts who gave of themselves unstintingly to make this research successful. We also thank J. Philip Saul for contributing to the early development of the protocol, and the large number of scientists and engineers, including particularly Hasan Rahman and Suzanne McCollum, at Lyndon B. Johnson Space Center.


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Translational perspective The human central nervous system is in no way static or unchanging; central mechanisms change continuously in response to environmental changes, and also in response to changes that occur for no discernible reason. Human subjects can cooperate with research protocols, and thereby alter their central and peripheral physiological mechanisms. Our results cast further doubt on the notions that heart period fluctuations during quiet, normal breathing are mediated by vagal baroreflex mechanisms, and that they persist during apnoea, as peripheral evidence for the existence of a central respiratory rhythm. We systematically evaluated responses to apnoea and showed that explanations advanced to explain responses of sleep apnoea patients, including changes of chemoreceptor, baroreceptor and pulmonary receptor activity, are not necessary to explain apnoeic responses of healthy subjects.


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