Neuroscan 4

Each electroencephalogram (EEG) was recorded using a SynAmps system (NeuroScan, CompuMedics, Charlotte, NC, USA) and collected through 32 Ag/AgCl electrodes embedded in an elastic cap (ElectroCap International, Inc., Eaton, OH, USA) according to the 10/20 international system and referenced to the right earlobe (A2). The left earlobe was recorded independently. The bandwidth of the amplifiers was set to 0.1–100 Hz, and the signal was digitized at a 500-Hz sampling rate. Impedances were kept below 5 kΩ. Two electrodes placed on the external canthus and superciliary arch of the left eye were used to record the electrooculogram.
The EEG recordings were re-referenced offline using the average of the earlobe signals (A1-A2). The continuous EEG recording was epoched from 200 ms pre-stimulus to 1000 ms post-stimulus. The ERP waveform was baseline corrected, and drift was removed using the linear detrend tool of the NeuroScan 4.5 software (CompuMedics; Charlotte, NC, USA). Only segments corresponding to correct answers were analyzed. All EEG epochs were visually inspected, and manual rejection of segments was performed. The artifact-free segments per the experimental condition of each participant were averaged. The same number of segments was used to build the ERPs’ average across the two groups and the two conditions.

Continuous EEG was acquired with 30 Ag–AgCl scalp electrodes mounted in an electrode cap (Quick-Cap, Compumedics Neuroscan 4.5) in accordance with the standard 10/20 system. The ground electrode was positioned between FPz and Fz. Electrical activity from both the mastoids was recorded, with the left mastoid (M1) as the reference. Two electrodes positioned above and below the left eye measured the vertical eye movement (VEOG), and another electrode pair placed on the outer canthi of each eye measured the horizontal eye movements (HEOG). The impedance at each electrode was maintained below 5 kΩ. The signal from the scalp electrodes was amplified 20,000 times (SynAmps 2 amplifier, Compumedics), sampled at 1000 Hz, and low pass filtered at 100 Hz online.

Behavioral data were recorded using a custom program written in LabVIEW (version 8.5, National Instruments, Austin, TX, USA). Participants applied force to the pressure-sensitive bulb that caused air to move through a rubber tube in a closed system, leading to a pressure change that was measured by a pressure sensor and converted to a voltage. There was a linear relationship between the pressure measurement and the voltage produced. EEG data were recorded from 32 electrode sites (32 channel Quik-Cap, Neuroscan, Compumedics, NC, USA) following the international 10–20 system for electrode placement and referenced to the linked mastoids. Impedance was maintained less than 5 kΩ. EEG data were collected with a DC—100 Hz filter and digitized at 500 Hz (Neuroscan 4.5, SynAmps2, Compumedics, NC, USA). Data were then saved for subsequent analysis.

An electrode cap based on the International 10–20 system was positioned on the subject's head by the experimenter. Figure 1 shows a schematic representation of the electrode sites of the cap. For a detailed review on the EEG setup, please refer to Chan and Davenport's (2008 (link)) review. Before the recording, the subjects were familiarized with the sensations of the paired inspiratory occlusions. Two inspiratory occlusions of 150 ms each, with a 500 ms inter-stimulus-interval, were provided randomly every 2–4 breaths. The onset of occlusion was identified as the start of mouth pressure change (Labchart V7, ADInstruments Inc., Bella Vista, Australia). Approximately 100 paired occlusions were provided during each of the two trials. The trigger box was sending parallel markers to the EEG recording software (Neuroscan 4.5, Compumedics Neuroscan Inc., Charlotte, NC, USA). The continuous EEG was sampled at 1 kHz with a 40-channel EEG system (NuAmps, Compumedics Neuroscan Inc., Charlotte, NC, USA), bandpass filtered from DC to 50 Hz and referenced to the bilateral mastoids. Individual electrode impedance was set below 5 kΩ. After each trial, the subjects were rating their subjective feeling of breathlessness with a visual analog scale (VAS) (0 = not at all breathlessness, and 100 = maximal level of breathlessness).

For the details of the RREP method, refer to the previous methodology paper [31 (link)]. Briefly, while breathing through the breathing circuit, the participant wore a 40-channel electrode cap (referenced to bilateral mastoids) connecting to an EEG system (NuAmps, Compumedics Neuroscan Inc., Charlotte, NC, USA). The impedance was set below 5 kΩ for every electrode. The EEG signal was sampled at 1 kHz and filtered from DC to 50 Hz. For the experiment, at least 100 paired inspiratory occlusions (150 ms each) with 500-ms ISI were provided randomly every 2 to 4 breaths. The paired stimuli were manually presented at the onset of inspiration by the experimenter triggering the occlusion valve closure via the trigger box. Parallel markers from the trigger box were sent to the Neuroscan recording software (Neuroscan 4.5, Compumedics Neuroscan Inc., Charlotte, NC, USA). During recordings, participants were watching a video (with sound) on a screen in order to be distracted from the stimuli.