Passive limb training modulates respiratory rhythmic bursts

Figures

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  Figure 1. A stable fictive respiration is spontaneously recorded from cervical segments of the entire in vitro CNS with hindlimbs attached. (A) Simultaneous extracellular recordings acquired 2 h from the beginning of surgical isolation. Traces correspond to first and fifth cervical (rC1; rC5) ventral roots (VR) on the right side of the cord, and to the left fifth and third cervical (lC3; lC5) VRs. (B) Time course of the average frequency calculated for 5-min bins (black dots) showing the stability of rhythm lasting for 140 min of continuous recordings (x-axis reports the time in minutes from the induction of anesthesia at the beginning of surgical procedures for tissue isolation; n = 6). (C) Time course of the average burst duration calculated for 5-min bins (black dots) showing the stability of burst duration lasting for 140 min of continuous recordings (x-axis reports the time in minutes from the induction of anesthesia at the beginning of surgical procedures for tissue isolation; n = 6). (D) An average event out of the 40 single events from the same preparation used in A (VRrC1) showing the shape of a single respiratory burst. Note that burst duration was measured as the time distance…

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  Figure 2. Respiratory rhythm is modulated by the progressive increase of passive pedaling speed. (A) 5-min traces from the right second cervical ventral root (VRlC2) in pre-BIKE control (ctrl), during passive leg mobilization at different speeds (BIKE 0.5 Hz, 1 Hz, 2 Hz, 3 Hz, 3.5 Hz) and immediately after switching off BIKE (rest). (B) Mean bursts of superimposed events from side-traces in (A). Shaded green rectangle corresponds to burst duration in control. (C) Time courses of inter-burst intervals, duration, and amplitude for the entire experiment related to traces in (A). Grey dots indicate when limbs are still, red dots correspond to steps of increasing pedaling speed and light blue dots point to the resting phase at the end of the experiment. (D) Whisker plot reports that respiratory frequency in control (grey box) increases during BIKE (red boxes) only at the highest speed of pedaling (3.5 Hz), recovering to baseline values right after termination of passive exercise (rest, light blue box; n = 10). (E) Whisker plot showing that average burst duration in control (grey box) is reduced for all BIKE speeds over 2 Hz (red boxes) without any recovery in the first post-BIKE rest (light…

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  Figure 3. A short and intense training session differently modulates the respiratory rhythm in relation to the basal breathing pace. (A) Scatter plot showing on the y-axis the modulation of respiratory rhythm frequency during 5 min of BIKE at 3.5 Hz, reported as a percentage of the breathing pace in pre-BIKE control (ctrl). On the x-axis the main respiratory frequency in control is expressed in Hz. Each dot corresponds to single BIKE sessions (n = 33 preparations). The green vertical dotted line is set at a 0.05 Hz value to split the dataset in two main fields based on the speed of the respiratory rhythm in pre-BIKE control (< 0.05 Hz, indicated as “slower breathers and > 0.05 Hz, as “faster breathers”). The horizontal green dotted line traces the 0% modulation of frequency, with values above corresponding to an increase in bursting frequency and values below stating a reduction. The rhythm of slower breathers speeds up during BIKE, while faster breathers are mostly unaffected. Red line traces the regression curve [y = 316 (± 167) exp(− x/0.02 ± 0.01) + 83 (± 22)]. (B) Scatter plot illustrating that the consistent reduction of burst duration during 5 min BIKE (3.5 Hz) does not depend upon…

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  Figure 4. BIKE slows down the fast respiratory rhythm induced by high potassium. (A) Sample trace recorded from the first right ventral root (VRrC1) showing a slow respiratory rhythm in control (ctrl, 0.06 Hz), which is accelerated by perfusion in high potassium Krebs solution (7.5 mM [K+], 0.12 Hz), slowed down by passive pedaling in the continuous presence of high potassium (7.5 mM [K+] + BIKE 3.5 Hz, 0.09 Hz) and remains unchanged during rest (rest in 7.5 mM [K+], 0.08 Hz). (B) Changes in frequency are accompanied by shorter respiratory bursts (superimposed averaged single events from corresponding traces in (A); color code as reported in the legend above). Whisker plots summarizing changes in frequency (C; n = 7), burst duration (D; n = 7) and amplitude (E; n = 7) in control (ctrl, grey boxes), high potassium (7.5 mM [K+], purple boxes); BIKE in high potassium (7.5 mM [K+] + BIKE 3.5 Hz, red boxes) and rest in high potassium (rest 7.5 mM [K+], cyan boxes). *, P < 0.05.

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  Figure 5. A longer BIKE session further affects the pace of bursting. (A) Sample traces from the right second cervical ventral root (VRrC2) recorded in pre-BIKE control (ctrl, 0.09 Hz), and during the early (BIKE 0–5 min) and late (20–25 min) phases of a long BIKE session (3.5 Hz; 25 min). (B) Average bursts of the corresponding traces in A showing a reduced burst duration during passive training. (C) Time course (bins = 5 min) tracing the dynamics of the mean rhythm frequency (Hz) from 5 preparations indicated as faster breathers in pre-BIKE control (0.07 ± 0.01). Passive training eventually slows down rhythm (BIKE 3.5 Hz, red bars), without recovering speed for the following 35 min of rest after the termination of pedaling. Whisker plots showing the effect of a long BIKE session as for rhythm frequency (D; *, P < 0.001; n = 5), burst duration (E; *, P < 0.001) and unchanged amplitude (F). Black boxes are pre-BIKE controls (ctrl), red boxes represent different phases of the BIKE training (BIKE 0–5, BIKE 20–25) and grey boxes indicate the first 5 min after BIKE termination (rest).

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  Figure 6. Effects of passive training on the respiratory rhythm rely on suprapontine structures. (A) (left). Cartoons with the ventral view of the neonatal rat brain illustrate the level of the pre-collicular transection for suprapontine decerebration (red dotted line) and the recording site from the left first cervical ventral root (VRlC1). For a slower breather preparation (A), stable fictive respiration in control (ctrl, upper trace) is slowed down after precollicular decerebration (middle trace), remaining unchanged following passive training (BIKE 3.5 Hz, 5 min). (B) Mean bursts from superimposed single events of the corresponding traces in A with the shaded green rectangle indicating burst duration in control. Similarly, for a faster breather preparation (C), stable fictive respiration was slowed down after precollicular decerebration and was unaffected by BIKE (3.5 Hz, 5 min). (D) Mean bursts from traces in C calibrated on burst duration in control (green rectangle). Whisker plots from seven experiments illustrating rhythm frequency (E) and burst duration (F; *, P = 0.001) in control (ctrl, grey boxes), after pre-collicular decerebration (orange boxes) and during passive training on…