Acute intermittent hypoxia (AIH) elicits long‐term facilitation (LTF) of respiration. Although LTF is observed when CO2 is elevated during AIH in awake humans, the influence of CO2 on corticospinal respiratory motor plasticity is unknown. Thus, we tested the hypotheses that acute intermittent hypercapnic‐hypoxia (AIHH): (1) enhances cortico‐phrenic neurotransmission (reflecting volitional respiratory control); and (2) elicits ventilatory LTF (reflecting automatic respiratory control). Eighteen healthy adults completed four study visits. Day 1 consisted of anthropometry and pulmonary function testing. On Days 2, 3 and 4, in a balanced alternating sequence, participants received: AIHH, poikilocapnic AIH, and normocapnic‐normoxia (Sham). Protocols consisted of 15, 60 s exposures with 90 s normoxic intervals. Transcranial (TMS) and cervical (CMS) magnetic stimulation were used to induce diaphragmatic motor‐evoked potentials and compound muscle action potentials, respectively. Respiratory drive was assessed via mouth occlusion pressure (P0.1), and minute ventilation measured at rest. Dependent variables were assessed at baseline and 30–60 min after exposures. Increases in TMS‐evoked diaphragm potential amplitudes were observed following AIHH vs. Sham (+28 ± 41%, P = 0.003), but not after AIH. No changes were observed in CMS‐evoked diaphragm potential amplitudes. Mouth occlusion pressure also increased after AIHH (+21 ± 34%, P = 0.033), but not after AIH. Ventilatory LTF was not observed after any treatment. We demonstrate that AIHH elicits central neural mechanisms of respiratory motor plasticity and increases resting respiratory drive in awake humans. These findings may have important implications for neurorehabilitation after spinal cord injury and other neuromuscular disorders compromising breathing. Key points The occurrence of respiratory long‐term facilitation following acute exposure to intermittent hypoxia is believed to be dependent upon CO2 regulation – mechanisms governing the critical role of CO2 have seldom been explored. We tested the hypothesis that acute intermittent hypercapnic‐hypoxia (AIHH) enhances cortico‐phrenic neurotransmission in awake healthy humans. The amplitude of diaphragmatic motor‐evoked potentials induced by transcranial magnetic stimulation was increased after AIHH, but not the amplitude of compound muscle action potentials evoked by cervical magnetic stimulation. Mouth occlusion pressure (P0.1, an indicator of neural respiratory drive) was also increased after AIHH, but not tidal volume or minute ventilation. Thus, moderate AIHH elicits central neural mechanisms of respiratory motor plasticity, without measurable ventilatory long‐term facilitation in awake humans.

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The diaphragmatic motor evoked potential (MEP) induced by transcranial magnetic stimulation (TMS) permits electrophysiological assessment of the cortico-diaphragmatic pathway. Despite the value of TMS for investigating diaphragm motor integrity in health and disease, reliability of the technique has not been established. The study aim was to determine within- and between-session reproducibility of surface electromyogram recordings of TMS-evoked diaphragm potentials. Fifteen healthy young adults participated (6 females, age=29±7 years). Diaphragm activation was determined by gradually increasing the stimulus intensity from 60-100% of maximal stimulator output (MSO). A minimum of seven stimulations were performed at each intensity. A second block of stimuli was delivered 30 minutes later for within-day comparisons, and a third block performed on a separate day for between-day comparisons. Reliability of diaphragm MEPs was assessed at 100% MSO using intraclass correlation coefficients (ICC) and 95% limits of agreement (LOA). MEPlatency (ICC=0.984, p<0.001), duration (ICC=0.958, p<0.001), amplitude (ICC=0.950, p<0.001) and area (ICC=0.956, p<0.001) were highly reproducible within-day. Between-day reproducibility was good to excellent for all MEP characteristics (latency ICC=0.953, p<0.001; duration ICC=0.879, p=0.002; amplitude ICC=0.789, p=0.019; area ICC=0.815, p=0.012). Data revealed less precision between-day versus within-day, as evidenced by wider LOA for all MEP characteristics. Large within- and between-subject variability in MEP amplitude and area was observed. In conclusion, TMS is a reliable means of inducing diaphragm potentials in most healthy individuals.

Intermittent hypoxia (IH) elicits respiratory motor plasticity (known as ventilatory long‐term facilitation [LTF]) in humans and other mammals. However, awake humans exhibit ventilatory LTF only when CO2 levels are raised slightly above eupneic levels in a sustained or intermittent fashion during the IH protocol. In this ongoing study, we seek to determine the effect of CO2 on IH‐induced respiratory motor plasticity by examining multiple, distinct mechanisms contributing to the ventilatory response during and following acute intermittent hypercapnic hypoxia (IHH). We hypothesized that IHH elicits a prolonged increase in cortico‐diaphragmatic conduction, resting neural drive to breathe, and respiratory motor output versus poikilocapnic IH and a normoxic, normocapnic control (CTRL). Five healthy young adults (age = 32 ± 5 years; 3 female) completed four study visits. Day 1 consisted of general subject characterization. On Days 2, 3 and 4 (separated by ≥72 h), subjects were randomly assigned to receive either: IHH (PETO2 = 55 ± 4 Torr, PETCO2 = 44 ± 3 Torr), IH (PETO2 = 55 ± 3 Torr, PETCO2 = 37 ± 2 Torr), or CTRL (PETO2 = 107 ± 3 Torr, PETCO2 = 36 ± 2 Torr). Protocols consisted of 15 episodes of 60 s exposure with 90 s breathing ambient room air. Cardiovascular responses (heart rate, blood pressure and oxygen saturation) were monitored throughout. Cortico‐diaphragmatic conduction was quantified by recording motor evoked potentials (MEPs) using transcranial magnetic stimulation and surface EMG electrodes. Resting neural drive to breathe was assessed using the mouth occlusion pressure technique (i.e. P0.1). Respiratory motor output was measured as minute ventilation () during resting isocapnic conditions (PETCO2 maintained within 1 mmHg of baseline) and adjusted based on the rate of metabolic CO2 production (/). Dependent variables were assessed at baseline, and 30‐60 min post. Following IHH, IH and CTRL, the mean change in MEP amplitude was +34 ± 48%, +9 ± 21%, and +2 ± 22%; the mean change in P0.1 was +26 ± 17%, +15 ± 16%, and +7 ± 16%; and the mean change in was +0.3 ± 1.0 L·min‐1, +0.3 ± 1.1 L·min‐1, and +0.3 ± 0.8 L·min‐1. When adjusted for , the mean change in / was ‐0.5 ± 1.0, +0.4 ± 1.8, and ‐0.2 ± 1.1. Based on these preliminary data in 5 subjects, we suggest that IHH elicits greater increases in cortico‐diaphragmatic conduction and resting neural drive to breathe vs. poikilocapnic IH and CTRL. Conversely, our IHH protocol does not elicit ventilatory LTF. We reason that measures such as MEP amplitude and P0.1 are less sensitive to behavioral influences vs. , and may provide unique insights into mechanisms of IH‐induced respiratory motor plasticity in awake healthy humans.

Episodic carotid body activation via acute intermittent hypoxia (AIH) elicits respiratory motor plasticity (known as long‐term facilitation [LTF]) in humans and other mammals. In rodents, AIH‐induced LTF requires serotonergic neuron activation in the medullary raphe nuclei and episodic serotonin release within respiratory motor nuclei, thereby strengthening descending synaptic inputs to spinal respiratory motor neurons. Humans exhibit ventilatory LTF under certain circumstances, although the details of its underlying mechanism are less clear. Recent evidence demonstrates that AIH enhances corticospinal motor evoked potentials (MEP) in a non‐respiratory motor pool innervating the first dorsal interosseous muscle of the hand. It is not known if AIH has similar effects on the phrenic/diaphragm system in humans. Thus, we tested the hypothesis that AIH augments diaphragm MEP amplitude. Twelve healthy young adults were recruited (age = 30 ± 7 years; 5 female). On Day 1, diaphragm MEPs and compound muscle action potentials (CMAPs) were assessed using transcranial (TMS) and cervical (CMS) magnetic stimulation, respectively. The diaphragm electromyogram was recorded using surface electrodes on the chest wall. Reliability of TMS‐ and CMS‐evoked potentials were determined prior to testing. Following baseline measures, subjects were exposed to AIH (15, 1‐minute episodes of 9% O2, with 1‐minute normoxic intervals) via a facemask attached to a hypoxic generator. Cardiovascular responses (heart rate, blood pressure and oxygen saturation) were monitored throughout. Stimulus‐response curves were determined for each subject by delivering stimuli prior to and 30 minutes post AIH. On Day 2 (at least 7 days later), 6 subjects returned for a normoxic (sham) trial. Subjects were blinded to the gas composition delivered on each visit. No significant AIH effects were observed in MEP (change = −1 ± 7%, p= 0.96) or CMAP amplitude (+1 ± 5%, p= 0.97) across the stimulus‐response curve. At an intensity equivalent to 50% of baseline peak amplitude (V50), the mean changes in MEP and CMAP amplitude were +4 ± 14% (p= 0.81) and −5 ± 6% (p= 0.75), respectively. Further, no effect of normoxic sham exposures was found during the control visit (MEP = −8 ± 9%, p= 0.97; CMAP = +7 ± 31%, p= 0.79). Between‐block coefficients of variation for TMS‐ and CMS‐evoked potentials were 22.9% and 6.9%, respectively. Within‐session reproducibility of evoked responses to TMS and CMS were good to excellent (ICC > 0.8). In conclusion, AIH alone does not influence cortical evoked potentials in the diaphragm as it does in a non‐respiratory motor system. We speculate that repetitive daily AIH and/or concomitant hypercapnia during AIH may reveal plasticity in diaphragm cortical evoked potentials in humans. Support or Funding Information This work was supported by the UF McKnight Brain Institute, DoD (SCIRP), NIH R01 HL147554 & OT2OD023854, Brooks Rehabilitation, and the Brooks PHHP Research Collaboration.