Participants
Twenty male volunteers with ages ranging from 21 to 38 years (group M ± S.D., 27.0 ± 4.9 years) participated in the study. They were residing at a yoga center (Swami Vivekananda Yoga Research Foundation, Bangalore, India). Participants were recruited through flyers distributed in the center. The inclusion criteria were: (i) the participants had experience of alternate nostril yoga breathing for more than 3 months (mean experience ± S.D., 30.2 ± 24.4 months), (ii) male participants alone were studied as auditory evoked responses have been shown to vary with the phases of the menstrual cycle [13] and the P300 (evoked by visual stimuli) also varied with gender [14], and (iii) all of them were in normal health based on a routine clinical examination. Exclusion criteria included: (i) a history of smoking, (ii) respiratory ailments including nasopharyngeal abnormalities, (iii) taking medication or using other wellness strategies, and (iv) any impairment affecting attention. The variables to be recorded and the study design were described to the participants and their signed consent to participate in the study was obtained. The study was approved by the ethics committee of Patanjali Research Foundation.
Design of the study
Participants were assessed in two separate sessions namely, alternate nostril yoga breathing and breath awareness. For half the participants the alternate nostril yoga breathing session took place on the first day with breath awareness the next day. The remaining participants had the order of the sessions reversed. They were alternately allocated to either schedule to prevent the order of the sessions influencing the outcome. The participants were unaware about the hypothesis of the study. The assessments were done before and after each session which lasted for 40 minutes. The design has been shown schematically in Figure 1.
Recording conditions
The peak latencies and peak amplitudes of P300 were recorded using Nicolet Bravo System (U.S.A.). The P300 component was elicited with a simple discrimination task known as the ‘oddball’ paradigm, in which two auditory stimuli are presented in a random series so that one of them occurs infrequently i.e., considered the oddball [7]. In our experience yoga practitioners found auditory stimuli less distracting than visual or somatosensory stimuli. For assessments participants were seated in a sound attenuated and dimly lit cabin and were monitored on a closed circuit television with instructions being given through an intercom, so that participants could remain undisturbed during a session.
Electrode positions
Ag/AgCl disk electrodes were fixed with electrode gel (10–20 conductive EEG paste, D.O. Weaver, U.S.A.) at the Fz, Cz, and Pz scalp sites, with reference electrodes on linked earlobes and with the ground electrode on the forehead (FPz) according to the International 10–20 system [15]. The electro-ocular activity was recorded as an electro-occulogram (EOG), with a bipolar derivation from two electrodes placed 1 cm above and 1 cm below the outer canthus of the right eye to record the vertical EOG. The electrode impendence was kept below 5 kΩ at all sites.
Amplifier settings
The electroencephalographic (EEG) activity was amplified with a sensitivity of 100 μV. The low cut filter was at 0.01 Hz and the high cut filter was at 30 Hz. P300 Event Related Potentials (ERPs) were computer averaged in 300 trial sweeps in the 0 to 750 ms range. The pre-stimulus delay was 75 ms and the level of artifact rejection was set at 90 percent.
Stimulus characteristics
Binaural tone stimuli of alternating polarity delivered at 0.9 ms with a frequency of 1 KHz (50 cycles for the plateau, 10 cycles for the ramp) for the standard stimuli and 2 KHz (10 cycles for the plateau, 20 cycles for the ramp) for the target stimuli were used to trigger online averaging of the EEG. The percentage of standard stimuli was set at 80 and for the target stimuli at 20. The stimulus intensity was kept at 70 dB Sound Pressure Level (SPL). The inter stimulus interval was 1.1 ms.
Recording procedure
Participants were asked to avoid substances which influence cognitive performance (particularly tea and coffee for the caffeine content) on the day preceding and on the day of the recording. Where this was unavoidable the session was taken on another day. The P300 evoked potentials were recorded with eyes closed and participants seated comfortably. The ‘standard’ and ‘target’ auditory stimuli were delivered through close fitting earphones (TDH-39, Amplivox, UK). Participants were asked to distinguish between the two tones and mentally count the ‘target’ stimuli. The equipment gives the number of target stimuli delivered. Only those sessions in which the participants achieved 95 percent accuracy in counting target stimuli were included. None of the sessions had to be excluded for this reason. The P300 responses were recorded before and immediately after the intervention.
Interventions
Alternate nostril yoga breathing
Alternate nostril yoga breathing (ANYB) practice involves breathing through left and right nostrils alternately [16]. In this nostril manipulating pranayama the thumb and the ring finger of the right hand were used to manipulate or occlude the nostrils. This is a characteristic yoga gesture (nasika mudra in Sanskrit) prescribed during pranayama practice to manipulate the nostrils with ease [2]. Throughout this practice the awareness is directed to the breath and breathing.
Breath awareness
During breath awareness (BAW), the participants maintained awareness of the breath without manipulation of the nostrils. During these practices the participants’ attention was directed to the movement of air into and out of their nostrils. They also attempted to be aware of the air as it moved through the nasal passage.
Data extraction
The peak amplitude and the peak latency of the P300 were measured at three electrode sites; i.e., Fz, Cz and Pz. The peak amplitude (in μV) was defined as the voltage difference between baseline at stimulus delivery and the largest positive-going peak of the ERP waveform within 250–500 ms latency [7]. The peak latency (ms) was defined as the time from stimulus onset to the point of maximum positive amplitude within the latency window (i.e., 250-500 ms).
Data analysis
Statistical analysis was done using SPSS (Version 10.0). Data were analyzed using the repeated measures analyses of variance (ANOVA). There were separate ANOVAs for the peak amplitudes at the three sites (Fz, Cz and Pz) and for the peak latencies at the three sites (Fz, Cz and Pz). Hence there were six ANOVAs. Each ANOVA had two Within subjects factors. These were Sessions (ANYB, BAW) and States (Before, After).
Post-hoc tests with Bonferroni adjustment for multiple comparisons were used to detect significant differences between mean values, for pre-post comparisons.