The mode of breathing affects awareness-related brain potentials: Oral breathing shapes awareness-related brain potentials differently than nasal breathing

The mode of breathing affects awareness-related brain potentials: Oral breathing shapes awareness-related brain potentials differently than nasal breathing | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The mode of breathing affects awareness-related brain potentials: Oral breathing shapes awareness-related brain potentials differently than nasal breathing Viviana Leupin, Juliane Britz This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5655503/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Aug, 2025 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Cyclic variation in bodily signals can influence the conscious perception of sensory stimuli. We have previously shown that the respiratory phase affects the sensory processing of visual stimuli during nasal breathing: the P1 component was modulated by awareness only during inhalation. Breathing can affect brain activity both directly through the entrainment of rhythmic brain activity via mechanical stimulation of the olfactory bulb (OB) and indirectly through fluctuations in baroreceptor (BR) activity across the respiratory cycle. We here aim to differentiate the relative contribution of OB stimulation and BR activity by during oral breathing when the OB is not stimulated and show that the early correlates of awareness do not vary with the respiratory phase but vary with the cardiac phase, albeit with somewhat delayed timing. Furthermore, the P3a component was modulated by awareness only when BR activity was low (inhalation, diastole). Our findings clarify the interplay between OB stimulation and BR activity for the conscious processing of a visual stimulus: the fluctuations in BR activity alone cannot explain how early sensory processes affect the perceptual outcome. Only when both OB stimulation is present and BR activity is low, is the P1 the earliest ERP component modulated by awareness. Biological sciences/Neuroscience/Cognitive neuroscience/Consciousness Biological sciences/Neuroscience/Cognitive neuroscience/Perception Biological sciences/Neuroscience Biological sciences/Neuroscience/Cognitive neuroscience Consciousness brain–body interaction EEG respiratory phase oral breathing Figures Figure 1 Figure 2 Figure 3 Introduction Certain conditions allow to dissociate perception from sensation such that identical physical stimuli give rise to different perceptual outcomes. Stimuli at the sensory threshold are equally likely perceived or missed, and awareness fluctuates apparently randomly from one trial to another. Such stimuli are powerful tools to study the neural correlates of consciousness: because trial-by-trial fluctuations in awareness cannot arise from the stimulus itself, they can be related either to different responses of the brain to the same stimulus or to fluctuations in the pre-stimulus brain state. Both stimulus-evoked and state-dependent differences in brain activity make complimentary contributions to our understanding of how conscious awareness can arise. Investigations of evoked brain responses focus either on the brain areas involved in or the timing underlying conscious vs. unconscious processing of the same stimulus. Activity in sensory cortex is necessary but not sufficient for awareness 1 , which requires the dynamic interplay between sensory cortex and higher order brain areas in parietal and frontal cortex 2–4 . Both activity in a posterior occipito-temporo-parietal hot zone 5 and interactions between parietal and frontal cortex in a Global Neuronal Workspace (GNW) 6,7 have been proposed to be necessary for conscious awareness. Frontal cortex is predominantly recruited when subjects must report their percept, but not in no-report paradigms 8 . This suggests that the frontal cortex activation is more related to processes like selective attention or response selection, rather than reflecting awareness itself. These accounts make different predictions about whether activity in occipito-temporal and parietal areas reflect differences in awareness 5 or not 6,7 . Contrasting event-related potentials (ERPs) evoked by a stimulus when it is perceived or missed can elucidate the time-course underlying the emergence of awareness. Numerous studies claim different markers as the locus of awareness resolution: it remains debated whether sensory processing (reflected by the P1 component, ~80 – 120 ms after stimulus onset) differs between aware and unaware perception 9,10 or not 11–13 . Both perceptual and post-perceptual processes reflected by the mid-latency Visual Awareness Negativity (VAN) and the P3b/LPC component are reliably modulated by awareness and are more strongly pronounced for aware vs. unaware perception 6,13–15 , suggesting that awareness is only resolved after the initial sensory processing. Whether the VAN 16–18 or the P3b/LPC 6,13,14 are the earliest marker of awareness remains likewise a matter of debate. Similar to the frontal vs. posterior debate, when task-relevance is controlled 12 and no explicit report of awareness is required 8 , the P3b/LPC component is greatly reduced which implies that this component reflects post-perceptual processes rather than awareness itself 19 . Apparently random fluctuations in awareness have been reliably attributed to trial-by-trial variations in spontaneous brain activity. Both local and global EEG measures can predict whether an upcoming visual stimulus is perceived or missed. Local pre-stimulus occipital alpha power over occipital areas, an indicator of cortical excitability is lower for perceived than missed stimuli 20–22 , and global pre-stimulus microstates can predict differences in awareness 23–25 . The brain obviously plays a crucial role for conscious awareness; however, it is inextricably connected with the rest of the body, and it continuously integrates signals from both outside and inside the body. It is hence not surprising that awareness not only fluctuates the with the state of the brain state but also with cyclic fluctuations in bodily rhythms. The cardiac rhythm is the first pacemaker of the organism 26 , and its cycle is subdivided into the systolic (contraction /ejection of blood) and diastolic (relaxation /influx of blood) phase. Visual 27–30 , auditory 31 and somatosensory 32,33 stimuli are detected less likely and identified more slowly during the systole. These effects are likely generated by the rise in activity of pressure sensors (baroreceptors (BRs)) located in the aortic arch and the carotid sinus during the systole, when BRs relay information about the increased blood pressure to the brain. The processing of this interoceptive afferent visceral signal decreases cortical excitability 34 which can be described in the framework of gain control as the slope of the sigmoid relationship that maps input and output (I/O) strength. During high gain (low BR activity) the slope of the I/O function is steeper which amplifies relevant (sensory) stimuli and attenuates irrelevant (interoceptive) signals. Conversely, when the gain is low (high BR activity) the sensitivity of I/O function is reduced, affecting the ability of the brain to distinguish between interoceptive irrelevant signals and exteroceptive stimuli 35 . Respiration is another fundamental bodily rhythm whose primary function is gas exchange. It provides a constant rhythmic input into the organism, and awareness also fluctuates across the respiratory phase. Subjects perform better in a visuo-spatial task and detect visual stimuli more easily during inhalation 36,37 . Moreover, visual stimuli presented during inhalation elicit larger ERPs 37 , and subjects synchronize their breathing with task demands 37,38 . However, alternative accounts reported improved detection rates for exhalation both in visual 39 and somatosensory 38 modalities. Respiration can affect awareness through a direct and an indirect way. It directly drives both broad-band resting-state 36 and task-related oscillatory brain activity, both in rodents 40–43 and humans 44,45 : LFP amplitudes and power in structures of the limbic system crucial for memory consolidation are coupled to the respiratory cycle 45 and vary with the respiratory frequency of the organism 41,42,46 . Moreover, mechanical stimulation of the olfactory bulb (OB) during respiration drives phase-amplitude coupling (PAC, amplitude increase in a faster frequency band coupled to the phase of a slower frequency band) in these regions between the theta phase and gamma amplitude both in humans 45 and in rodents 41 . Breathing through the mouth 45 or OB ablation 41,42 bypassing OB stimulation abolishes the coupling between gamma amplitude and theta phase, making respiration an oscillatory scaffold underlying the coordination of activity between brain areas 42 . Respiration can affect awareness indirectly via cardio-respiratory coupling (respiratory sinus arrythmia (RSA)), i.e. the acceleration of the heart rate during inhalation and deceleration during exhalation 47 . The purpose of RSA is to adapt dynamically to fluctuations in metabolic demands through the dynamic interplay between the cardiac and the respiratory systems 48 ; RSA is partially generated by both the balance between the sympathetic and parasympathetic nervous system 49 and it is mediated by BR activity: during exhalation, both BR activity and vagal (parasympathetic) output increase, which triggers the baroreflex that decelerates the heart rate; during inhalation, low BR activation and higher sympathetic tone accelerate the heart rate 47 . It remains unexplored whether the effects of respiration on awareness can be better explained by the direct (entrainment of neural activity by OB stimulation) or the indirect (RSA, mediated by the balance between the sympathetic and parasympathetic nervous system and BR activity) mechanisms. We have previously shown that cyclic fluctuations of BR activity across the cardiac and respiratory cycle shape awareness-related brain activity for visual threshold stimuli in two ways: they affect 1) the earliest electrophysiological marker (P1 for low (diastole/inhalation), VAN for high (systole/exhalation) BR activity) and 2) the brain areas activated (frontal cortex for low and parietal cortex for high BR activity) when subjects become aware of a stimulus 50 . This provides a new answer to the debates of the earliest electrophysiological marker of awareness and the role of frontal and parietal cortex for awareness: both the earliest electrophysiological marker and the recruitment of frontal vs. parietal cortex depend on whether the visual threshold stimulus is presented along with signals from the body or not. In other words, cyclic fluctuations of gain control mediated by BR activity affect both the sensory processing and the trajectory of brain activity when subjects became aware of a threshold stimulus. It remains unknown whether this systemic gain control is mediated by rhythmic stimulation of the OB and concomitant entrainment of brain activity or not. That study could clearly show how the cardiac and respiratory phase shape awareness-related brain responses, but it could not determine whether respiration affected awareness-related ERPs in a direct or an indirect way because subjects breathed nasally. During nasal breathing, respiration can affect awareness both directly through the entrainment of oscillatory brain activity by mechanical OB stimulation and indirectly through BR mediated RSA. We here aim to further elucidate the mechanisms underlying the effect of the respiratory phase on awareness-related brain activity: the same subjects as in Leupin and Britz 50 performed the same task, albeit during oral respiration (which preserves the mechanism of BR mediated RSA but suppresses the effects of OB stimulation) to elucidate whether the entrainment of brain activity by mechanical OB stimulation is necessary for BR modulated gain control. This allows us to discern to what degree the direct and indirect effects of breathing are specific or general, i.e., whether they selectively affect specific ERP components or whether they affect all ERP components similarly. If the mode of breathing is specific and selectively affects BR modulated gain control, the sensory ERP components should be modulated differently for oral vs. nasal breathing. If the effects of the mode of breathing are more general, different ERP components should be modulated as a function of awareness during oral and nasal breathing. We contrast ERPs elicited by visual stimuli presented at the discrimination threshold when they were correctly classified with and without awareness (Fig. 1) as a function of both the respiratory (inhalation, exhalation) and cardiac (systole, diastole) phase. Cardiac phase was used as a control condition, because fluctuations in awareness-related processes during the cardiac cycle are primarily driven by fluctuations in BR activity and should be less affected by the mode of breathing. === insert Fig 1 about here === Results Behavioral results Subjects classified stimuli correctly in 87.1% (SD=6.3%) of trials with a mean ratio of correct aware/unaware responses of 52.6/47.4% (SD=11.1%); only correct trials were retained for further analyses. Supplementary Fig. S1 displays the reaction times as a function of awareness and respiratory (Supplementary Fig. S1 a ) and cardiac phase (Supplementary Fig. S1 b ). Reaction times were on average faster in the aware (736± 155 ms) compared to the unaware (935± 353 ms) condition. Because RT data were not normally distributed, we applied a Generalized Linear Mixed Model (GLMM) which revealed a main effect of awareness (estimate = 129.28, SE= 3.22, p = 10 -16 ). Neither the respiratory (estimate = - 0.98, SE= 2.92, p = 0.74 nor the cardiac (estimate = 1.25, SE=3.16, p = 0.69 ) phase significantly affected reaction times and neither the respiratory (estimate = - 7.78, SE= 4.02, p = 0.053) nor the cardiac phase (estimate = 0.71, SE = 3.92, p = 0.86 ) interacted with awareness. Stimulus-evoked potentials Overall, 13% of trials were discarded due to errors along with 26% of trials falling into the early phase of the diastole and the remainder was removed due to artifacts. An average of 257 trials were kept in the aware condition (123/133 in inhalation/exhalation, 128 in both systole and diastole) and 231 trials in the unaware condition (109/122 in inhalation/exhalation, 117/114 in the systole/diastole). The stimulus was only presented for 16 ms but it elicited clear canonical visual evoked potentials (VEPs). Awareness modulates ERPs Fig. 2 a and b display four time-windows in which awareness significantly modulated the ERPs (after FDR correction 51 for multiple comparisons across all 150 time-points and 128 electrodes). First, in the time window between 140 and 160 ms after stimulus onset ERP amplitudes over posterior electrodes were significantly more positive in the aware compared to the unaware condition. Next, in the period between 240 and 320 ms, the VAN component was significantly more negative in the aware condition over posterior electrodes. Next, both the P3a and P3b/LPC components were modulated by awareness: ERPs were significantly more positive over frontal electrodes for the P3a (320 to 360 ms) and over centro-parietal electrodes for the P3b/LPC (400 to 500 ms) in the aware compared to the unaware condition. === insert Fig 2 about here === Respiratory phase selectively modulates awareness-related ERPs Fig. 3 shows the effects of the respiratory phase on awareness-related ERPs. Unlike during nasal respiration 50 , the sensory ERP components were not modulated by awareness, and the VAN was the earliest component modulated by awareness between 240 and 320 ms, and it was equally so during inhalation and exhalation (Fig. 3 a , b , c ). Unlike for nasal respiration 50 , the P3a component (320-360 ms) was modulated by awareness, albeit only during inhalation, i.e. when the BRs are silent (Fig. 3 b , c ) but not during exhalation when they are active (Fig. 3 a , b ) with more positive-going ERPs in the aware than unaware condition over fronto-central electrodes. Cardiac phase selectively modulates awareness-related ERPs Fig. 3 depicts the ERP components modulated by awareness selectively averaged for the systole (Fig. 3 d , e ) and diastole (Fig. 3 e , f ). Unlike the respiratory phase, the cardiac phase selectively modulated sensory awareness-related ERPs: only in the diastole (silent BRs), but not the systole the P2 was more positive over posterior electrodes in the time-window between 140 and 160 ms when the stimulus was seen than when it was not (Fig. 3 d , e , f ). Like for the respiratory phase, the P3a was modulated by awareness during the diastole with more positive-going ERPs over fronto-central electrodes in the aware than the unaware condition from 320 to 360 ms (Fig. 3 e , f ). In the systole (active BRs), awareness neither modulated the P2 nor the P3a components (Fig. 3 d , e ). === insert Fig 3 about here === Discussion We show here that the mode of respiration shapes awareness-related brain potentials: during oral breathing, ERPs are modulated differently than during nasal breathing 50 . Respiration can influence awareness in two ways: directly, by mechanically stimulating of the OB to entrain oscillatory brain activity, and indirectly, by modulating cardiac frequency through RSA. This indirect influence is mediated by increased BR activity during exhalation and the interplay between the sympathetic and parasympathetic nervous systems. We here sought to identify the relative contributions of the direct and indirect influences of respiratory phase on awareness. In a previous study where the same subjects breathed nasally we have shown that cyclic fluctuations of BR activity across the cardiac and respiratory phase selectively modulate awareness-related ERPs such that the P1 was the earliest marker of awareness when BR activity was low (diastole, inhalation) and the VAN when it was high (systole, exhalation) 50 . During nasal respiration, both neuronal synchronization through OB stimulation and fluctuations of BR activity can account for the effects of the respiratory phase on awareness. We here isolate the effect of BR activity on awareness by omitting the effect of OB stimulation and concomitant entrainment of brain activity and retested the same subjects in the same paradigm during oral respiration. When breathing through the mouth, the early correlates of awareness do not vary with the respiratory phase, i.e. none of the sensory ERP components were modulated by awareness. This is in contrast to nasal breathing where the P1 was the earliest marker of awareness when BRs are silent during inhalation and the diastole 50 . Our findings clarify the interplay between OB stimulation and BR activity in the conscious processing of a visual stimulus: the fluctuations in BR activity alone cannot explain how early sensory processes affect the perceptual outcome. Only when both OB stimulation is present and BRA is low, the P1 is the earliest ERP component modulated by awareness, which suggests that the BR modulated gain control during inhalation appears to require additional entrainment of brain activity mediated by mechanical stimulation of the OB, supporting the notion of respiration as an oscillatory scaffold underlying the coordination of activity between brain areas 42 . When contrasting aware vs. unaware perception as a function of the cardiac phase, we found a significant effect of awareness only during the diastole in the P2 time window. Cyclic variations in cardiac activity affected the early sensory component of awareness even in the absence of OB stimulation, albeit at a slightly later latency. This suggests that the neural mechanisms which underlie long term parasympathetic tone during exhalation are distinct from the shorter-term phasic activation in the diastole. Overall, oral breathing reduces the influence of the respiratory phase over early sensory correlates of consciousness, and it affects the effects of the cardiac phase to a smaller degree. The following VAN, P3a, and P3b/LPC components were generally modulated by awareness. Both the VAN and P3b/LPC were observed during nasal respiration and consistently differentiate between aware and unaware conditions in visual discrimination paradigms 10,52 . In contrast, the P3a component was obtained exclusively in the oral modality, and, to our knowledge, it has not been reported to be modulated by awareness at the sensory threshold. This might indicate that the P3a is exclusively modulated by awareness during oral breathing. Both changes in autonomic activity and in oxygen metabolism and can explain these effects. The P3a is sensitive to novelty 53,54 , varies with task difficulty 55 , decreases with habituation 56 and varies with the skin conductance response (SCR) 57,58 . These characteristics indicate that this component reflects the orienting response towards motivationally relevant stimuli, which is mediated by the sympathetic (SNS) division of the autonomic nervous system (ANS) 59 . Here we show that the P3a component is modulated by awareness selectively in the diastole and inhalation when BR activity is reduced. This specific variation can be attributed to fluctuations in the autonomic nervous system (ANS) across the cardiac and respiratory cycles, which ultimately affect the P3a 60 . The SNS and parasympathetic nervous systems (PNS) interact in a complex antagonistic relationship to ensure optimal functioning of the organism. The ANS plays a crucial role in generating RSA: both central and peripheral generators contribute to enhancing SNS and PNS activity, respectively during inhalation and exhalation 47,61 . In particular, during inhalation the parasympathetically mediated chemoreflex and baroreflex are inhibited to increase the heart rate through sympathetic effectors 47 . Because the P3a component is sensitive to SNS activity, it is more pronounced in the diastole and inhalation when BR activity, and consequently PNS influence, is weaker and the SNS is more activated. The P3a is observed in fronto-central electrodes and originates from prefrontal cortices (PFC) 53,62 where oxygen consumption decreases during oral breathing 63 ; this depleted state can explain why we observe this component exclusively in the oral modality: because the processing demands required to become aware of the stimulus increase, the resulting P3a is stronger, potentially indicative of increased task difficulty 55 . PFC involvement might also be affected by changes in the ANS: in our previous study 50 we showed that the involvement of PFC in the processing of a stimulus varies with BR activity: activity spreads from the anterior insula to the PFC (the source of the P3a) in phases of weaker BR activity, while when BR activity is stronger activity spreads from the visceral posterior insula to parietal and temporal regions. Future studies should address whether phasic changes in P3a response during oral breathing are associated with differences in the skin conductance response to clarify the role of the SNS in these variations. The impact of oral breathing the autonomic system remains ambiguous. Breathing through the mouth decreases the efficiency of gas exchange 64 resulting in hypoxia and sleep apnea 65 , as well as decreases in the strength of the respiratory muscles 66 and in exercise capacity 67 . Overall, oral breathing increases stress on the organism which might affect the autonomic balance. Only few studies have explored this hypothesis albeit using indirect measurements of sympathetic activity derived from heart rate variability, and found that the respiratory route affected sympathetic activity differently based on the gender of the subject 68,69 . Clarifying the relationship between the breathing route and the global sympathetic state of the system might contribute to understand how bodily cycles interact with conscious processes. In our previous study 50 we hypothesized that increases in BR activity during the systole and exhalation would decrease cortical gain and consequently reduce the contribution of early sensory component to awareness. The absence of awareness-related modulations of the P1 component suggests that breathing through the mouth and thus the absence OB stimulation might generally decrease cortical gain of sensory cortices. However, we do find a modulation of the P2 exclusively in the diastole when BRs are silent. This effect suggests that OB stimulation interacts more strongly with mechanisms affecting the electrophysiological markers of awareness for the respiratory than the cardiac phase, providing further evidence that the modulation of awareness-related early sensory components across the cardiac cycle are mainly driven by BR activity. Taken together, we show how the mode of respiration modulates awareness-related brain potentials. Respiration can affect brain activity both directly by OB stimulation modulated entrainment of brain activity and indirectly through BR modulated RSA. We could previously show that BR modulated gain control can account for sensory processing differences across both the cardiac and respiratory phase when subjects breathe though their nose, and here we show that this effect is abolished for the respiratory phase and delayed for the cardiac phase when the same subjects breathe though their nose. To our knowledge, this is the first time that awareness-related ERPs have been found to vary with the mode of breathing. This is in line with and expands findings from intracranial recordings in humans and invasive recordings in rodents which show that oscillatory coordination between brain through PAC is present during nasal and absent during oral respiration. This suggests that to account for differences in gain control, BR activity fluctuations themselves are not enough but they need to be accompanied by entrainment of brain activity modulated by mechanical OB stimulation. Our results support the notion of breathing as an oscillatory scaffold that can modulate brain activity both during oral and nasal breathing 42 . The effects of OB stimulation on awareness-related ERPs are both specific to gain control and general: they specifically affect sensory ERP components and generally affect post-perceptual ERPs when the BRs are silent. Taken together, recording cardiac and respiratory signals along with brain activity provides rich information to open a new view on the intricate brain-body connection that encompasses the interaction between the bodily signals and both central and peripheral nervous system. Methods Participants Thirty-nine healthy right-handed subjects (25 female, age: 24.8 ± 5.1 years, range 18-42) without history of neurological, psychiatric, cardiological and respiratory disorders were recruited for the EEG study. They were the same subjects as in Leupin and Britz 50 , and the experimental sessions were two weeks apart with the order counterbalanced between subjects. The discrimination threshold could not be determined in five subjects, two subjects were excluded from the analysis due to poor signal quality. To maintain consistency in a repeated measure design across both nasal and oral conditions, we further excluded two participants who did not meet the criteria in the nasal condition. The data of 30 subjects (17 female, age 25.23 ± 5.42 years, range 18-42) was retained for analyses. Participants gave written informed consent received either with monetary compensation (20 CHF/hour) or course credits. The Ethics Committee of the University of Fribourg approved the full study protocol, and the study was conducted in accordance with the Declaration of Helsinki. Stimuli and procedure Target stimuli were Gabor gratings oriented either to the left (135°) or right (45°) and subtending a visual angle of 5° with 3 cpd of visual angle embedded in grayscale random dot noise. Psychopy3 was used to generate and display stimuli on a grey background on a ViewPixx Screen (1920 × 1080 pixel resolution, 120 Hz), and subjects viewed the stimuli in a dimly lit room from a chin-rest 70 cm from the screen. Participants were instructed to breathe through their mouth while having a piece of surgical tape placed over the nostrils to refrain from nasal breathing. They performed a threshold determination task followed by the main EEG experiment. The trial structure was the same for the threshold determination and EEG tasks. A white fixation cross (700-500 ms) was followed by a blank screen (100-300 ms) and then by the target stimulus (16 ms). After each trial the subject indicated the orientation of grating by pressing the “F”, (left index) or “J” (right index) key on a keyboard to indicate a left or right orientation, respectively, and then whether they saw (“J”) the stimulus or not (“F”). This measured respectively the objective accuracy and the subjective awareness of the stimulus. Before participating in the EEG experiment each subject had to perform a threshold determination procedure to determine the threshold stimuli that accounted for both performance and subjective awareness. Objective performance and awareness are usually confounded since subjective awareness is not required to correctly perform in a task 70 . To avoid this confound we kept performance constant and close to ceiling (> 75%) while maintaining the same proportion of correct aware and correct unaware trials for each stimulus orientation. When adopting standard adaptive staircase procedures 71 only one variable can be adapted at a time. For this reason, we used a two-step behavioral pre-test in which all stimuli close to the desired range were presented. In the first step, the approximate contrast level was determined by adjusting the contrast of the random dot noise mask in 20 linear steps from 30% to 100%, following the method implemented by Samaha 72 In case the difficulty of the task needed to be adjusted, this procedure was repeated by adjusting the opacity of the stimulus. In the second step, we redefined 20 stimuli for each orientation by centering (+/- 20%) the Michelson contrast around the stimuli which yielded values closest to desired threshold. These stimuli were pseudo-randomized and presented for a total 400 trials subdivided in 5 blocks (10 repetitions for each stimulus). This procedure was performed for each stimulus orientation to exclude that awareness was confounded with stimulus orientation, i.e. subjects consistently identified one but not the other orientation. For the EEG task, we retained the contrast levels that yielded the correct identification for more than 75% of the trials, and produced similar identification rates with and without awareness. Participants were presented with a total of 960 stimuli divided into 12 blocks, with each block consisting of 80 trials. To maintain the stability of the threshold, the noise contrast was readjusted throughout the task if needed. Electrophysiological recordings data processing The EEG was continuously recorded from 128 active Ag/AgCl electrodes (BioSemi®) referenced to the CMS-DRL ground. The ECG and respiration were simultaneously recorded with the EEG at 1024Hz/16 bit as external bipolar channels. ECG electrodes were placed on the right clavicle and lower left rib and a respiratory belt (SleepSense®) was placed on the lower abdomen. The cardiac and respiratory signals were preprocessed using the Python Neurokit2 toolbox 73 . The R-peak and the end of the T-wave were marked to indicate the start of systole and diastole. Similarly, the inhalation peak and exhalation trough marked the beginning of the inhalation and exhalation. Trials were then classified according to the cardiac and respiratory phase in which they occurred. We equalized the number of trials across the cardiac cycle to account for the fact that diastole can be twice as long as systole: only trials where the stimulus occurred during the period at the end of diastole, corresponding to the duration of systole within that cardiac cycle, were included 32 , as a result 26% of the trials were rejected. Respiratory cycles that deviated by 2.5 standard deviations faster or 1.5 standard deviations slower than the mean were excluded from further analyses. Only correct trials with (aware) and without awareness (unaware) were included in the analysis. All EEG analyses, including the pre-processing, averaging ,statistical analyses and the creation of images, were performed using the MNE-python toolbox version 1.0.3 74 . The EEG signal was initially re-referenced to the common average reference, filtered between 0.5 and 40 Hz using a FIR filter with a transition window of 10 Hz, and down sampled to 256 Hz. Independent component analysis (ICA) was applied to remove ocular, myogenic artifacts and cardiac field artifacts, and trials contaminated by ocular artifacts within 300 ms before and after stimulus onset were rejected. To correct the remaining ocular and myogenic artifacts the respective ICs were removed. The data was then segmented into epochs ranging from -200 ms to 1000 ms around stimulus onset, and artifact rejection and channel interpolation was performed using the Autoreject procedure 75 implemented in MNE. Analysis of behavioral data Behavioral analysis included only correct responses in the aware and unaware conditions. Reaction times (RTs) exceeding the 97.5th percentile or falling below the 2.5th percentile were excluded from the analysis. The effects of awareness, cardiac phase (systole/diastole), and respiratory phase (inhalation/exhalation) on RTs were assessed using two separate General Linear Mixed Effects Models (GLMMs), one for each physiological rhythm. Subjects was included as a random factor in the models, which employed an identity link function with an inverse distribution to account for deviations from the normal distribution typical of RTs 76 . GLMMs were implemented using R Statistical Software (v 4.3.3) 77 . Analysis of stimulus-evoked potentials For each subject and for each condition, the epoched data were separately averaged. We contrasted the aware and unaware condition as a function of the cardiac (systole, diastole) and the respiratory phase (inhalation, exhalation). Statistical differences in ERP amplitudes were assessed with mass univariate t-tests in the time window of -100 to 500 ms around stimulus onset. We had no a-priori assumptions about localization or timing of effects and applied False Discovery Rate (FDR) 51 to compensate for multiple comparisons across time and space. Declarations Acknowledgments This research was funded by Swiss National Science Foundation grant 10001C_189408 to J.B. We thank Roberto Caldara for providing the lab infrastructure and Alen Jelusic, Dunja Vulliemin, Amira El Hachimi, Jade Ueberschaer, Samuel Müller, Fania Maffeis and David Elmiger for help with data collection. Author contributions V.L. and J.B. designed research, V.L. performed research, V.L. analyzed data, V.L. and J.B. wrote manuscript. Data and Code Availability The code developed during the current study is available in the oral_24 repository 78 , https://zenodo.org/records/14499167. The consent forms signed by participants do not allow us to give free access to data but require us to check that data are shared with members of the scientific community. Therefore, data are not shared publicly but can be made available upon request to researchers. Please contact the corresponding author Juliane Britz ( [email protected] ). Competing interests The authors declare no conflict of interest References Tong, F. Primary visual cortex and visual awareness. Nat. Rev. Neurosci. 4 , 219–229 (2003). Dehaene, S., Changeux, J.-P., Naccache, L., Sackur, J. & Sergent, C. Conscious, preconscious, and subliminal processing: a testable taxonomy. Trends Cogn. Sci. 10 , 204–211 (2006). Lamme, V. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5655503","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":407548039,"identity":"7dd2ab6a-31b9-418c-a699-f2357b1cdbb0","order_by":0,"name":"Viviana Leupin","email":"","orcid":"","institution":"University of Fribourg, University of Fribourg","correspondingAuthor":false,"prefix":"","firstName":"Viviana","middleName":"","lastName":"Leupin","suffix":""},{"id":407548040,"identity":"2e1c4f79-1a41-43de-980a-117594675a5c","order_by":1,"name":"Juliane Britz","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIie3PMQrCMBSA4RcKcWndRdQr1ElE8SwGQTcXFweH1+W5WGcF0VuIY0rAqYeoB3Bws4NoTEFwMDo65F/ySPhIAuBy/WEMPZTPIdQjwBSAexLM/JmwF4kQUk14305ep6HhpKdvxFuJSOaHLrSqan7Kt2pc5kEG+cFyx0pgEqdDaC9FhJW9mnBeClmc2okMSEGY6r8090pQg8AzL7TdcqN7QcRGE86/ExWQLEiCv5DFCVWNBn57waI1HkeGJLGFNOcDdTlTr97yS9nlOuuIHXGW5TaCxeq/7crPAKBhO3S5XC6X6QGo5VXHtrEWawAAAABJRU5ErkJggg==","orcid":"","institution":"University of Fribourg, University of Fribourg","correspondingAuthor":true,"prefix":"","firstName":"Juliane","middleName":"","lastName":"Britz","suffix":""}],"badges":[],"createdAt":"2024-12-16 16:23:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5655503/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5655503/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-12518-1","type":"published","date":"2025-08-11T15:57:13+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":75059739,"identity":"63b1b496-d833-4c07-b539-06849af2ca9b","added_by":"auto","created_at":"2025-01-30 03:49:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":808022,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental procedure. A Gabor grating oriented either to the left or to the right was presented for 16 ms. The orientation of the stimulus was first (objective measure of accuracy) and followed by whether the stimulus was perceived or guessed (subjective measure of awareness).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5655503/v1/e54d6f837d366b69f8ce8049.png"},{"id":75059743,"identity":"96a92c3e-8c29-46da-8d0f-0ce3bf12a322","added_by":"auto","created_at":"2025-01-30 03:49:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3477544,"visible":true,"origin":"","legend":"\u003cp\u003eMass- univariate t-tests contrasting the aware and unaware conditions(\u003cstrong\u003ea\u003c/strong\u003e) and relative topographic maps (\u003cstrong\u003eb\u003c/strong\u003e) for the P2 (140- to 160 ms), VAN (240-320 ms), P3a (320-360 ms) and P3b/LPC (400-500 ms) components. Significant values are FDR corrected, blue t-values indicate a negative potential difference and red ones a positive potential difference between CA and CU conditions; significant electrodes for at least half the time window after FDR correction are indicated by a white dot on the topographic maps.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5655503/v1/0b6bdda5b6056f1a035145e8.png"},{"id":75059741,"identity":"4f5efb01-0e22-4678-92da-b5c691aab73b","added_by":"auto","created_at":"2025-01-30 03:49:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2841869,"visible":true,"origin":"","legend":"\u003cp\u003eModulation of the P2, VAN and P3a component as a function of awareness and of the respiratory and cardiac phase. Time course and location of the t -values denoting significant differences between the aware and unaware conditions (\u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003ef\u003c/strong\u003e) in exhalation (\u003cstrong\u003ea\u003c/strong\u003e), inhalation (\u003cstrong\u003ec\u003c/strong\u003e), systole (\u003cstrong\u003ed\u003c/strong\u003e) and diastole (\u003cstrong\u003ef\u003c/strong\u003e). The colored boxes highlight the significant period in which the P2 (140-160 ms), VAN (240-320 ms) and P3a (320 – 360 ms) components extend. \u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ee\u003c/strong\u003e) topo-maps illustrating the distribution of the effects of awareness for exhalation (\u003cstrong\u003eb\u003c/strong\u003e, top), inhalation (\u003cstrong\u003eb\u003c/strong\u003e, bottom), systole (\u003cstrong\u003ee\u003c/strong\u003e, top) and diastole (\u003cstrong\u003ee\u003c/strong\u003e, bottom). Significant electrodes for at least half of the time-window after FDR correction (applied over all 128 electrodes and all time-points from -100 to 500 ms) are shown with white dots, blue t-values indicate negative potential while red t-values indicate positive potential.\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5655503/v1/452be3ac4f0d1362c3def004.png"},{"id":89310529,"identity":"34dffca7-409d-46d4-8a8d-799028412b3e","added_by":"auto","created_at":"2025-08-18 16:07:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5296491,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5655503/v1/a43a35b4-e97a-467a-894a-aec4c6740e4f.pdf"},{"id":75059744,"identity":"02227c18-a422-4059-9f05-8540bf9e977b","added_by":"auto","created_at":"2025-01-30 03:49:26","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":980503,"visible":true,"origin":"","legend":"","description":"","filename":"oravepsubmissionSI.docx","url":"https://assets-eu.researchsquare.com/files/rs-5655503/v1/3898b4effc76b8ec87213e86.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The mode of breathing affects awareness-related brain potentials: Oral breathing shapes awareness-related brain potentials differently than nasal breathing","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCertain conditions allow to dissociate perception from sensation such that identical physical stimuli give rise to different perceptual outcomes. Stimuli at the sensory threshold are equally likely perceived or missed, and awareness fluctuates apparently randomly from one trial to another. Such stimuli are powerful tools to study the neural correlates of consciousness: because trial-by-trial fluctuations in awareness cannot arise from the stimulus itself, they can be related either to different responses of the brain to the same stimulus or to fluctuations in the pre-stimulus brain state. Both stimulus-evoked and state-dependent differences in brain activity make complimentary contributions to our understanding of how conscious awareness can arise.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInvestigations of evoked brain responses focus either on the brain areas involved in or the timing underlying conscious vs. unconscious processing of the same stimulus. Activity in sensory cortex is necessary but not sufficient for awareness\u0026nbsp;\u003csup\u003e1\u003c/sup\u003e, which requires the dynamic interplay between sensory cortex and higher order brain areas in parietal and frontal cortex\u003csup\u003e2\u0026ndash;4\u003c/sup\u003e. Both activity in a posterior occipito-temporo-parietal hot zone\u0026nbsp;\u003csup\u003e5\u003c/sup\u003e and interactions between parietal and frontal cortex in a Global Neuronal Workspace (GNW)\u0026nbsp;\u003csup\u003e6,7\u003c/sup\u003e have been proposed to be necessary for conscious awareness. Frontal cortex is predominantly recruited when subjects must report their percept, but not in no-report paradigms\u0026nbsp;\u003csup\u003e8\u003c/sup\u003e. This suggests that the frontal cortex activation is more related to processes like selective attention or response selection, rather than reflecting awareness itself. These accounts make different predictions about whether activity in occipito-temporal and parietal areas reflect differences in awareness\u0026nbsp;\u003csup\u003e5\u003c/sup\u003e or not\u0026nbsp;\u003csup\u003e6,7\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eContrasting event-related potentials (ERPs) evoked by a stimulus when it is perceived or missed can elucidate the time-course underlying the emergence of awareness. Numerous studies claim different markers as the locus of awareness resolution: it remains debated whether sensory processing (reflected by the P1 component, ~80 \u0026ndash; 120 ms after stimulus onset) differs between aware and unaware perception\u0026nbsp;\u003csup\u003e9,10\u003c/sup\u003e or not\u0026nbsp;\u003csup\u003e11\u0026ndash;13\u003c/sup\u003e. Both perceptual and post-perceptual processes reflected by the mid-latency Visual Awareness Negativity (VAN) and the P3b/LPC component are reliably modulated by awareness and are more strongly pronounced for aware vs. unaware perception\u0026nbsp;\u003csup\u003e6,13\u0026ndash;15\u003c/sup\u003e, suggesting that awareness is only resolved after the initial sensory processing. Whether the VAN\u0026nbsp;\u003csup\u003e16\u0026ndash;18\u003c/sup\u003e or the P3b/LPC\u0026nbsp;\u003csup\u003e6,13,14\u003c/sup\u003e are the earliest marker of awareness remains likewise a matter of debate. Similar to the frontal vs. posterior debate, when task-relevance is controlled\u0026nbsp;\u003csup\u003e12\u003c/sup\u003e and no explicit report of awareness is required\u0026nbsp;\u003csup\u003e8\u003c/sup\u003e, the P3b/LPC component is greatly reduced which implies that this component reflects post-perceptual processes rather than awareness itself\u0026nbsp;\u003csup\u003e19\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eApparently random fluctuations in awareness have been reliably attributed to trial-by-trial variations in spontaneous brain activity. Both local and global EEG measures can predict whether an upcoming visual stimulus is perceived or missed. Local pre-stimulus occipital alpha power over occipital areas, an indicator of cortical excitability is lower for perceived than missed stimuli\u0026nbsp;\u003csup\u003e20\u0026ndash;22\u003c/sup\u003e, and global pre-stimulus microstates can predict differences in awareness\u0026nbsp;\u003csup\u003e23\u0026ndash;25\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe brain obviously plays a crucial role for conscious awareness; however, it is inextricably connected with the rest of the body, and it continuously integrates signals from both outside and inside the body. It is hence not surprising that\u0026nbsp;awareness not only fluctuates the with the state of the brain state but also with cyclic fluctuations in bodily rhythms. The cardiac rhythm is the first pacemaker of the organism \u003csup\u003e26\u003c/sup\u003e, and its cycle is subdivided into the systolic (contraction /ejection of blood) and diastolic (relaxation /influx of blood) phase. Visual \u003csup\u003e27\u0026ndash;30\u003c/sup\u003e, auditory\u0026nbsp;\u003csup\u003e31\u003c/sup\u003e and somatosensory \u003csup\u003e32,33\u003c/sup\u003e stimuli are detected less likely and identified more slowly during the systole. These effects are likely generated by the rise in activity of pressure sensors (baroreceptors (BRs)) located in the aortic arch and the carotid sinus during the systole, when BRs relay information about the increased blood pressure to the brain. The processing of this interoceptive afferent visceral signal decreases cortical excitability \u003csup\u003e34\u003c/sup\u003e which can be described in the framework of gain control as the slope of the sigmoid relationship that maps input and output (I/O) strength. During high gain (low BR activity) the slope of the I/O function is steeper which amplifies relevant (sensory) stimuli and attenuates irrelevant (interoceptive) signals. Conversely, when the gain is low (high BR activity) the sensitivity of I/O function is reduced, affecting the ability of the brain to distinguish between interoceptive irrelevant signals and exteroceptive stimuli \u003csup\u003e35\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRespiration is another fundamental bodily rhythm whose primary function is gas exchange. It provides a constant rhythmic input into the organism, and awareness also fluctuates across the respiratory phase. Subjects perform better in a visuo-spatial task and detect visual stimuli more easily during inhalation \u003csup\u003e36,37\u003c/sup\u003e. Moreover, visual stimuli presented during inhalation elicit larger ERPs\u0026nbsp;\u003csup\u003e37\u003c/sup\u003e, and subjects synchronize their breathing with task demands\u0026nbsp;\u003csup\u003e37,38\u003c/sup\u003e. However, alternative accounts reported improved detection rates for exhalation both in visual\u0026nbsp;\u003csup\u003e39\u003c/sup\u003e and somatosensory\u0026nbsp;\u003csup\u003e38\u003c/sup\u003e modalities. Respiration can affect awareness through a direct and an indirect way.\u0026nbsp;It\u0026nbsp;directly drives both broad-band resting-state\u0026nbsp;\u003csup\u003e36\u003c/sup\u003e and task-related oscillatory brain activity, both in\u0026nbsp;rodents \u003csup\u003e40\u0026ndash;43\u003c/sup\u003e and humans \u003csup\u003e44,45\u003c/sup\u003e: LFP amplitudes and power in structures of the limbic system crucial for memory consolidation are coupled to the respiratory cycle\u0026nbsp;\u003csup\u003e45\u003c/sup\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eand vary with the respiratory frequency of the organism \u003csup\u003e41,42,46\u003c/sup\u003e. Moreover, mechanical stimulation of the olfactory bulb (OB) during respiration drives phase-amplitude coupling (PAC, amplitude increase in a faster frequency band coupled to the phase of a slower frequency band) in these regions between the theta phase and gamma amplitude both in humans \u003csup\u003e45\u003c/sup\u003e and in rodents \u003csup\u003e41\u003c/sup\u003e. Breathing through the mouth \u003csup\u003e45\u003c/sup\u003e or OB ablation \u003csup\u003e41,42\u003c/sup\u003e bypassing OB stimulation abolishes the coupling between gamma amplitude and theta phase, making respiration an oscillatory scaffold underlying the coordination of activity between brain areas \u003csup\u003e42\u003c/sup\u003e. Respiration can affect awareness indirectly via cardio-respiratory coupling (respiratory sinus arrythmia (RSA)), i.e. the acceleration of the heart rate during inhalation and deceleration during exhalation \u003csup\u003e47\u003c/sup\u003e. The purpose of RSA is to adapt dynamically to fluctuations in metabolic demands through the dynamic interplay between the cardiac and the respiratory systems\u0026nbsp;\u003csup\u003e48\u003c/sup\u003e; RSA is partially generated by both the balance between the sympathetic and parasympathetic nervous system\u0026nbsp;\u003csup\u003e49\u003c/sup\u003e and it is mediated by BR activity: during exhalation, both BR activity and vagal (parasympathetic) output increase, which triggers the baroreflex that decelerates the heart rate; during inhalation, low BR activation and higher sympathetic tone accelerate the heart rate\u0026nbsp;\u003csup\u003e47\u003c/sup\u003e. It remains unexplored whether the effects of respiration on awareness can be better explained by the direct (entrainment of neural activity by OB stimulation) or the indirect (RSA, mediated by the balance between the sympathetic and parasympathetic nervous system and BR activity) mechanisms.\u003c/p\u003e\n\u003cp\u003eWe have previously shown that cyclic fluctuations of BR activity across the cardiac and respiratory cycle\u0026nbsp;shape awareness-related brain activity for visual threshold stimuli in two ways: they affect 1) the earliest electrophysiological marker (P1 for low (diastole/inhalation), VAN for high (systole/exhalation) BR activity) and 2) the brain areas activated (frontal cortex for low and parietal cortex for high BR activity) when subjects become aware of a stimulus\u0026nbsp;\u003csup\u003e50\u003c/sup\u003e. This provides a new answer to the debates of the earliest electrophysiological marker of awareness and the role of frontal and parietal cortex for awareness: both the earliest electrophysiological marker and the recruitment of frontal vs. parietal cortex depend on whether the visual threshold stimulus is presented along with signals from the body or not. In other words, cyclic fluctuations of gain control mediated by BR activity affect both the sensory processing and the trajectory of brain activity when subjects became aware of a threshold stimulus. It remains unknown whether this systemic gain control is mediated by rhythmic stimulation of the OB and concomitant entrainment of brain activity or not.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThat study could clearly show how the cardiac and respiratory phase shape awareness-related brain responses, but it could not determine whether respiration affected awareness-related ERPs in a direct or an indirect way because subjects breathed nasally.\u0026nbsp;During nasal breathing, respiration can affect awareness both directly through the entrainment of oscillatory brain activity by mechanical OB stimulation and indirectly through BR mediated RSA.\u0026nbsp;We here aim to further elucidate the mechanisms underlying the effect of the respiratory phase on awareness-related brain activity: the same subjects as in Leupin and Britz\u0026nbsp;\u003csup\u003e50\u003c/sup\u003e performed the same task, albeit during oral respiration (which preserves the mechanism of BR mediated RSA but suppresses the effects of OB stimulation) to elucidate whether the entrainment of brain activity by mechanical OB stimulation is necessary for BR modulated gain control. This allows us to discern to what degree the direct and indirect effects of breathing are specific or general, i.e., whether they selectively affect specific ERP components or whether they affect all ERP components similarly.\u0026nbsp;If the mode of breathing is specific and selectively affects BR modulated gain control, the sensory ERP components should be modulated differently for oral vs. nasal breathing. If the effects of the mode of breathing are more general, different ERP components should be modulated as a function of awareness during oral and nasal breathing.\u003c/p\u003e\n\u003cp\u003eWe contrast ERPs elicited by visual stimuli presented at the discrimination threshold when they were correctly classified with and without awareness (Fig. 1) as a function of both the respiratory (inhalation, exhalation) and cardiac (systole, diastole) phase.\u0026nbsp;Cardiac phase was used as a control condition, because fluctuations in awareness-related processes during the cardiac cycle are primarily driven by fluctuations in BR activity and should be less affected by the mode of breathing.\u003c/p\u003e\n\u003cp\u003e=== insert Fig 1 about here ===\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eBehavioral results\u003c/h2\u003e\n\u003cp\u003eSubjects classified stimuli correctly in 87.1% (SD=6.3%) of trials with a mean ratio of correct aware/unaware responses of 52.6/47.4% (SD=11.1%); only correct trials were retained for further analyses. Supplementary Fig. S1 displays the reaction times as a function of awareness and respiratory (Supplementary Fig. S1 \u003cstrong\u003ea\u003c/strong\u003e) and cardiac phase (Supplementary Fig. S1 \u003cstrong\u003eb\u003c/strong\u003e). Reaction times were on average faster in the aware (736\u0026plusmn; 155 ms) compared to the unaware (935\u0026plusmn; 353 ms) condition. Because RT data were not normally distributed, we applied a Generalized Linear Mixed Model (GLMM) which revealed a main effect of awareness (estimate = 129.28, SE= 3.22, \u003cem\u003ep\u003c/em\u003e = 10\u003csup\u003e\u0026nbsp;-16\u003c/sup\u003e). Neither the respiratory (estimate = - 0.98, SE= 2.92, \u003cem\u003ep\u003c/em\u003e = 0.74 nor the cardiac (estimate = 1.25, SE=3.16, \u003cem\u003ep\u003c/em\u003e = 0.69 ) phase significantly affected reaction times and neither the respiratory (estimate = - 7.78, SE= 4.02, \u003cem\u003ep\u003c/em\u003e = 0.053) nor the cardiac phase (estimate = 0.71, SE = 3.92, \u003cem\u003ep\u003c/em\u003e = 0.86 ) interacted with awareness.\u003c/p\u003e\n\u003ch2\u003eStimulus-evoked potentials\u003c/h2\u003e\n\u003cp\u003eOverall, 13% of trials were discarded due to errors along with 26% of trials falling into the early phase of the diastole and the remainder was removed due to artifacts.\u0026nbsp;An average of 257 trials were kept in the aware condition (123/133 in inhalation/exhalation, 128 in both systole and diastole) and 231 trials in the unaware condition (109/122 in inhalation/exhalation, 117/114 in the systole/diastole). The stimulus was only presented for 16 ms but it elicited clear canonical visual evoked potentials (VEPs).\u003c/p\u003e\n\u003ch2\u003eAwareness modulates ERPs\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eFig. 2 \u003cstrong\u003ea\u003c/strong\u003e and \u003cstrong\u003eb\u003c/strong\u003e display four time-windows in which awareness significantly modulated the ERPs (after FDR correction \u003csup\u003e51\u003c/sup\u003e for multiple comparisons across all 150 time-points and 128 electrodes). First, in the time window between 140 and 160 ms after stimulus onset ERP amplitudes over posterior electrodes were significantly more positive in the aware compared to the unaware condition. Next, in the period between 240 and 320 ms, the VAN component was significantly more negative in the aware condition over posterior electrodes. Next, both the P3a and P3b/LPC components were modulated by awareness: ERPs were significantly more positive over frontal electrodes for the P3a (320 to 360 ms) and over centro-parietal electrodes for the P3b/LPC (400 to 500 ms) in the aware compared to the unaware condition.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e=== insert Fig 2 about here ===\u003c/p\u003e\n\u003ch2\u003eRespiratory phase selectively modulates awareness-related ERPs\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eFig. 3 shows the effects of the respiratory phase on awareness-related ERPs. Unlike during nasal respiration \u003csup\u003e50\u003c/sup\u003e, the sensory ERP components were not modulated by awareness, and the VAN was the earliest component modulated by awareness between 240 and 320 ms, and it was equally so during inhalation and exhalation (Fig. 3 \u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eUnlike for nasal respiration \u003csup\u003e50\u003c/sup\u003e, the P3a component (320-360 ms) was modulated by awareness, albeit only during inhalation, i.e. when the BRs are silent (Fig. 3 \u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e) but not during exhalation when they are active (Fig. 3 \u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003eb\u003c/strong\u003e) with more positive-going ERPs in the aware than unaware condition over fronto-central electrodes.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eCardiac phase selectively modulates awareness-related ERPs\u003c/h2\u003e\n\u003cp\u003eFig. 3 depicts the ERP components modulated by awareness selectively averaged for the systole (Fig. 3 \u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003ee\u003c/strong\u003e) and diastole (Fig. 3 \u003cstrong\u003ee\u003c/strong\u003e, \u003cstrong\u003ef\u003c/strong\u003e). Unlike the respiratory phase, the cardiac phase selectively modulated sensory awareness-related ERPs: only in the diastole (silent BRs), but not the systole the P2 was more positive over posterior electrodes in the time-window between 140 and 160 ms when the stimulus was seen than when it was not (Fig. 3 \u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003ee\u003c/strong\u003e, \u003cstrong\u003ef\u003c/strong\u003e). Like for the respiratory phase, the P3a was modulated by awareness during the diastole \u0026nbsp;with more positive-going ERPs over fronto-central electrodes in the aware than the unaware condition from 320 to 360 ms (Fig. 3 \u003cstrong\u003ee\u003c/strong\u003e, \u003cstrong\u003ef\u003c/strong\u003e). In the systole (active BRs), awareness neither modulated the P2 nor the P3a components (Fig. 3 \u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003ee\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e=== insert Fig 3 about here ===\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe show here that the mode of respiration shapes awareness-related brain potentials: during oral breathing, ERPs are modulated differently than during nasal breathing\u0026nbsp;\u003csup\u003e50\u003c/sup\u003e. Respiration can influence awareness in two ways: directly, by mechanically stimulating of the OB to entrain oscillatory brain activity, and indirectly, by modulating cardiac frequency through RSA. This indirect influence is mediated by increased BR activity during exhalation and the interplay between the sympathetic and parasympathetic nervous systems. We here sought to identify the relative contributions of the direct and indirect influences of respiratory phase on awareness. In a previous study where the same subjects breathed nasally we have shown that cyclic fluctuations of BR activity across the cardiac and respiratory phase selectively modulate awareness-related ERPs such that the P1 was the earliest marker of awareness when BR activity was low (diastole, inhalation) and the VAN when it was high (systole, exhalation)\u0026nbsp;\u003csup\u003e50\u003c/sup\u003e. During nasal respiration, both neuronal synchronization through OB stimulation and fluctuations of BR activity can account for the effects of the respiratory phase on awareness. We here isolate the effect of BR activity on awareness by omitting the effect of OB stimulation and concomitant entrainment of brain activity and retested the same subjects in the same paradigm during oral respiration.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhen breathing through the mouth, the early correlates of awareness do not vary with the respiratory phase, i.e. none of the sensory ERP components were modulated by awareness. This is in contrast to nasal breathing where the P1 was the earliest marker of awareness when BRs are silent during inhalation and the diastole\u0026nbsp;\u003csup\u003e50\u003c/sup\u003e. Our findings clarify the interplay between OB stimulation and BR activity in the conscious processing of a visual stimulus: the fluctuations in BR activity alone cannot explain how early sensory processes affect the perceptual outcome. Only when both OB stimulation is present and BRA is low, the P1 is the earliest ERP component modulated by awareness, which suggests that the BR modulated gain control during inhalation appears to require additional entrainment of brain activity mediated by mechanical stimulation of the OB, supporting the notion of respiration as an oscillatory scaffold underlying the coordination of activity between brain areas\u0026nbsp;\u003csup\u003e42\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eWhen contrasting aware vs. unaware perception as a function of the cardiac phase, we found a significant effect of awareness only during the diastole in the P2 time window. Cyclic variations in cardiac activity affected the early sensory component of awareness even in the absence of OB stimulation, albeit at a slightly later latency. This suggests that the neural mechanisms which underlie long term parasympathetic tone during exhalation are distinct from the shorter-term phasic activation in the diastole. Overall, oral breathing reduces the influence of the respiratory phase over early sensory correlates of consciousness, and it affects the effects of the cardiac phase to a smaller degree.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe following VAN, P3a, and P3b/LPC components were generally modulated by awareness. Both the VAN and P3b/LPC were observed during nasal respiration and consistently differentiate between aware and unaware conditions in visual discrimination paradigms\u0026nbsp;\u003csup\u003e10,52\u003c/sup\u003e. In contrast, the P3a component was obtained exclusively in the oral modality, and, to our knowledge, it has not been reported to be modulated by awareness at the sensory threshold. This might indicate that the P3a is exclusively modulated by awareness during oral breathing. Both changes in autonomic activity and in oxygen metabolism and can explain these effects.\u003c/p\u003e\n\u003cp\u003eThe P3a is sensitive to novelty\u0026nbsp;\u003csup\u003e53,54\u003c/sup\u003e, varies with task difficulty\u0026nbsp;\u003csup\u003e55\u003c/sup\u003e, decreases with habituation\u0026nbsp;\u003csup\u003e56\u003c/sup\u003e and varies with the skin conductance response (SCR)\u0026nbsp;\u003csup\u003e57,58\u003c/sup\u003e. These characteristics indicate that this component reflects the orienting response towards motivationally relevant stimuli, which is mediated by the sympathetic (SNS) division of the autonomic nervous system (ANS)\u0026nbsp;\u003csup\u003e59\u003c/sup\u003e. Here we show that the P3a component is modulated by awareness selectively in the diastole and inhalation when BR activity is reduced. This specific variation can be attributed to fluctuations in the autonomic nervous system (ANS) across the cardiac and respiratory cycles, which ultimately affect the P3a\u0026nbsp;\u003csup\u003e60\u003c/sup\u003e. The SNS and parasympathetic nervous systems (PNS) interact in a complex antagonistic relationship to ensure optimal functioning of the organism. The ANS plays a crucial role in generating RSA: both central and peripheral generators contribute to enhancing SNS and PNS activity, respectively during inhalation and exhalation\u0026nbsp;\u003csup\u003e47,61\u003c/sup\u003e. In particular, during inhalation the parasympathetically mediated chemoreflex and baroreflex are inhibited to increase the heart rate through sympathetic effectors\u0026nbsp;\u003csup\u003e47\u003c/sup\u003e. Because the P3a component is sensitive to SNS activity, it is more pronounced in the diastole and inhalation when BR activity, and consequently PNS influence, is weaker and the SNS is more activated.\u003c/p\u003e\n\u003cp\u003eThe P3a is observed in fronto-central electrodes and originates from prefrontal cortices (PFC)\u0026nbsp;\u003csup\u003e53,62\u003c/sup\u003e where oxygen consumption decreases during oral breathing\u0026nbsp;\u003csup\u003e63\u003c/sup\u003e; this depleted state can explain why we observe this component exclusively in the oral modality: because the processing demands required to become aware of the stimulus increase, the resulting P3a is stronger, potentially indicative of increased task difficulty\u0026nbsp;\u003csup\u003e55\u003c/sup\u003e. PFC involvement might also be affected by changes in the ANS: in our previous study\u0026nbsp;\u003csup\u003e50\u003c/sup\u003e we showed that the involvement of PFC in the processing of a stimulus varies with BR activity: activity spreads from the anterior insula to the PFC (the source of the P3a) in phases of weaker BR activity, while when BR activity is stronger activity spreads from the visceral posterior insula to parietal and temporal regions.\u003c/p\u003e\n\u003cp\u003eFuture studies should address whether phasic changes in P3a response during oral breathing are associated with differences in the skin conductance response to clarify the role of the SNS in these variations. The impact of oral breathing the autonomic system remains ambiguous. Breathing through the mouth decreases the efficiency of gas exchange\u0026nbsp;\u003csup\u003e64\u003c/sup\u003e resulting in hypoxia and sleep apnea\u0026nbsp;\u003csup\u003e65\u003c/sup\u003e, as well as decreases in the strength of the respiratory muscles\u0026nbsp;\u003csup\u003e66\u003c/sup\u003e and in exercise capacity\u0026nbsp;\u003csup\u003e67\u003c/sup\u003e. Overall, oral breathing increases stress on the organism which might affect the autonomic balance. Only few studies have explored this hypothesis albeit using indirect measurements of sympathetic activity derived from heart rate variability, and found that the respiratory route affected sympathetic activity differently based on the gender of the subject\u0026nbsp;\u003csup\u003e68,69\u003c/sup\u003e. Clarifying the relationship between the breathing route and the global sympathetic state of the system might contribute to understand how bodily cycles interact with conscious processes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn our previous study\u0026nbsp;\u003csup\u003e50\u003c/sup\u003e we hypothesized that increases in BR activity during the systole and exhalation would decrease cortical gain and consequently reduce the contribution of early sensory component to awareness. The absence of awareness-related modulations of the P1 component suggests that breathing through the mouth and thus the absence OB stimulation might generally decrease cortical gain of sensory cortices. However, we do find a modulation of the P2 exclusively in the diastole when BRs are silent.\u0026nbsp;This effect suggests that OB stimulation interacts more strongly with mechanisms affecting the electrophysiological markers of awareness for the respiratory than the cardiac phase, providing further evidence that the modulation of awareness-related early sensory components across the cardiac cycle are mainly driven by BR activity.\u003c/p\u003e\n\u003cp\u003eTaken together, we show how the mode of respiration modulates awareness-related brain potentials. Respiration can affect brain activity both directly by OB stimulation modulated entrainment of brain activity and indirectly through BR modulated RSA. We could previously show that BR modulated gain control can account for sensory processing differences across both the cardiac and respiratory phase when subjects breathe though their nose, and here we show that this effect is abolished for the respiratory phase and delayed for the cardiac phase when the same subjects breathe though their nose. To our knowledge, this is the first time that awareness-related ERPs have been found to vary with the mode of breathing. This is in line with and expands findings from intracranial recordings in humans and invasive recordings in rodents which show that oscillatory coordination between brain through PAC is present during nasal and absent during oral respiration. This suggests that to account for differences in gain control, BR activity fluctuations themselves are not enough but they need to be accompanied by entrainment of brain activity modulated by mechanical OB stimulation.\u003c/p\u003e\n\u003cp\u003eOur results support the notion of breathing as an oscillatory scaffold that can modulate brain activity both during oral and nasal breathing \u003csup\u003e42\u003c/sup\u003e. The effects of OB stimulation on awareness-related ERPs are both specific to gain control and general: they specifically affect sensory ERP components and generally affect post-perceptual ERPs when the BRs are silent. Taken together, recording cardiac and respiratory signals along with brain activity provides rich information to open a new view on the intricate brain-body connection that encompasses the interaction between the bodily signals and both central and peripheral nervous system.\u003c/p\u003e"},{"header":"Methods","content":"\u003ch2\u003eParticipants\u003c/h2\u003e\n\u003cp\u003eThirty-nine healthy right-handed subjects (25 female, age: 24.8 \u0026plusmn; 5.1 years, range 18-42) without history of neurological, psychiatric, cardiological and respiratory disorders were recruited for the EEG study. They were the same subjects as in Leupin and Britz\u0026nbsp;\u003csup\u003e50\u003c/sup\u003e, and the experimental sessions were two weeks apart with the order counterbalanced between subjects.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe discrimination threshold could not be determined in five subjects, two subjects were excluded from the analysis due to poor signal quality. To maintain consistency in a repeated measure design across both nasal and oral conditions, we further excluded two participants who did not meet the criteria in the nasal condition. The data of 30 subjects (17 female, age 25.23 \u0026plusmn; 5.42 years, range 18-42) was retained for analyses. Participants gave written informed consent received either with monetary compensation (20 CHF/hour) or course credits. The Ethics Committee of the University of Fribourg approved the full study protocol, and the study was conducted in accordance with the Declaration of Helsinki.\u003c/p\u003e\n\u003ch2\u003eStimuli and procedure\u003c/h2\u003e\n\u003cp\u003eTarget stimuli were Gabor gratings oriented either to the left (135\u0026deg;) or right (45\u0026deg;) and subtending a visual angle of 5\u0026deg; with 3 cpd of visual angle embedded in grayscale random dot noise. Psychopy3 was used to generate and display stimuli on a grey background on a ViewPixx Screen (1920 \u0026times; 1080 pixel resolution, 120 Hz), and subjects viewed the stimuli in a dimly lit room from a chin-rest 70 cm from the screen. Participants were instructed to breathe through their mouth while having a piece of surgical tape placed over the nostrils to refrain from nasal breathing. They performed a threshold determination task followed by the main EEG experiment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe trial structure was the same for the threshold determination and EEG tasks. A white fixation cross (700-500 ms) was followed by a blank screen (100-300 ms) and then by the target stimulus (16 ms). After each trial the subject indicated the orientation of grating by pressing the \u0026ldquo;F\u0026rdquo;, (left index) or \u0026ldquo;J\u0026rdquo; (right index) key on a keyboard to indicate a left or right orientation, respectively, and then whether they saw (\u0026ldquo;J\u0026rdquo;) the stimulus or not (\u0026ldquo;F\u0026rdquo;). This measured respectively the objective accuracy and the subjective awareness of the stimulus.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBefore participating in the EEG experiment each subject had to perform a threshold determination procedure to determine the threshold stimuli that accounted for both performance and subjective awareness. Objective performance and awareness are usually confounded since subjective awareness is not required to correctly perform in a task \u003csup\u003e70\u003c/sup\u003e. To avoid this confound we kept performance constant and close to ceiling (\u0026gt; 75%) while maintaining the same proportion of correct aware and correct unaware trials for each stimulus orientation. When adopting standard adaptive staircase procedures \u003csup\u003e71\u003c/sup\u003e only one variable can be adapted at a time. For this reason, we used a two-step behavioral pre-test in which all stimuli close to the desired range were presented. In the first step, the approximate contrast level was determined by adjusting the contrast of the random dot noise mask in 20 linear steps from 30% to 100%, following the method implemented by Samaha \u003csup\u003e72\u003c/sup\u003e In case the difficulty of the task needed to be adjusted, this procedure was repeated by adjusting the opacity of the stimulus. In the second step, we redefined 20 stimuli for each orientation by centering (+/- 20%) the Michelson contrast around the stimuli which yielded values closest to desired threshold. These stimuli were pseudo-randomized and presented for a total 400 trials subdivided in 5 blocks (10 repetitions for each stimulus). This procedure was performed for each stimulus orientation to exclude that awareness was confounded with stimulus orientation, i.e. subjects consistently identified one but not the other orientation.\u003c/p\u003e\n\u003cp\u003eFor the EEG task, we retained the contrast levels that yielded the correct identification for more than 75% of the trials, and produced similar identification rates with and without awareness. Participants were presented with a total of 960 stimuli divided into 12 blocks, with each block consisting of 80 trials. To maintain the stability of the threshold, the noise contrast was readjusted throughout the task if needed.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eElectrophysiological recordings data processing\u003c/h2\u003e\n\u003cp\u003eThe EEG was continuously recorded from 128 active Ag/AgCl electrodes (BioSemi\u0026reg;) referenced to the CMS-DRL ground. The ECG and respiration were simultaneously recorded with the EEG at 1024Hz/16 bit as external bipolar channels. ECG electrodes were placed on the right clavicle and lower left rib and a respiratory belt (SleepSense\u0026reg;) was placed on the lower abdomen.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe cardiac and respiratory signals were preprocessed using the Python Neurokit2 toolbox\u0026nbsp;\u003csup\u003e73\u003c/sup\u003e. The R-peak and the end of the T-wave were marked to indicate the start of systole and diastole. Similarly, the inhalation peak and exhalation trough marked the beginning of the inhalation and exhalation. Trials were then classified according to the cardiac and respiratory phase in which they occurred. We equalized the number of trials across the cardiac cycle to account for the fact that diastole can be twice as long as systole: only trials where the stimulus occurred during the period at the end of diastole, corresponding to the duration of systole within that cardiac cycle, were included\u0026nbsp;\u003csup\u003e32\u003c/sup\u003e, as a result 26% of the trials were rejected. Respiratory cycles that deviated by 2.5 standard deviations faster or 1.5 standard deviations slower than the mean were excluded from further analyses. Only correct trials with (aware) and without awareness (unaware) were included in the analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll EEG analyses, including the pre-processing, averaging ,statistical analyses and the creation of images, were performed using the MNE-python toolbox version 1.0.3\u0026nbsp;\u003csup\u003e74\u003c/sup\u003e. The EEG signal was initially re-referenced to the common average reference, filtered between 0.5 and 40 Hz using a FIR filter with a transition window of 10 Hz, and down sampled to 256 Hz. Independent component analysis (ICA) was applied to remove ocular, myogenic artifacts and cardiac field artifacts, and trials contaminated by ocular artifacts within 300 ms before and after stimulus onset were rejected. To correct the remaining ocular and myogenic artifacts the respective ICs were removed. The data was then segmented into epochs ranging from -200 ms to 1000 ms around stimulus onset, and artifact rejection and channel interpolation was performed using the Autoreject procedure\u0026nbsp;\u003csup\u003e75\u003c/sup\u003e implemented in MNE.\u003c/p\u003e\n\u003ch2\u003eAnalysis of behavioral data\u003c/h2\u003e\n\u003cp\u003eBehavioral analysis included only correct responses in the aware and unaware conditions. Reaction times (RTs) exceeding the 97.5th percentile or falling below the 2.5th percentile were excluded from the analysis. The effects of awareness, cardiac phase (systole/diastole), and respiratory phase (inhalation/exhalation) on RTs were assessed using two separate General Linear Mixed Effects Models (GLMMs), one for each physiological rhythm. Subjects was included as a random factor in the models, which employed an identity link function with an inverse distribution to account for deviations from the normal distribution typical of RTs\u003csup\u003e76\u003c/sup\u003e. GLMMs were implemented using R Statistical Software (v 4.3.3)\u003csup\u003e77\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAnalysis of stimulus-evoked potentials\u003c/h2\u003e\n\u003cp\u003eFor each subject and for each condition, the epoched data were separately averaged. We contrasted the aware and unaware condition as a function of the cardiac (systole, diastole) and the respiratory phase (inhalation, exhalation). Statistical differences in ERP amplitudes were assessed with mass univariate t-tests in the time window of -100 to 500 ms around stimulus onset. We had no a-priori assumptions about localization or timing of effects and applied False Discovery Rate (FDR)\u0026nbsp;\u003csup\u003e51\u003c/sup\u003e to compensate for multiple comparisons across time and space.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eThis research was funded by Swiss National Science Foundation grant 10001C_189408 to J.B. We thank Roberto Caldara for providing the lab infrastructure and Alen Jelusic, Dunja Vulliemin, Amira El Hachimi, Jade Ueberschaer, Samuel M\u0026uuml;ller, Fania Maffeis and David Elmiger for help with data collection.\u003c/p\u003e\n\u003ch2\u003eAuthor contributions\u003c/h2\u003e\n\u003cp\u003eV.L. and J.B. designed research, V.L. performed research, V.L. analyzed data, V.L. and J.B. wrote manuscript.\u003c/p\u003e\n\u003ch2\u003eData and Code Availability\u003c/h2\u003e\n\u003cp\u003eThe code developed during the current study is available in the \u003cem\u003eoral_24\u003c/em\u003e repository\u003csup\u003e78\u003c/sup\u003e, https://zenodo.org/records/14499167. The consent forms signed by participants do not allow us to give free access to data but require us to check that data are shared with members of the scientific community. Therefore, data are not shared publicly but can be made available upon request to researchers. Please contact the corresponding author Juliane Britz ([email protected]).\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare no conflict of interest\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTong, F. Primary visual cortex and visual awareness. \u003cem\u003eNat. Rev. Neurosci.\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 219\u0026ndash;229 (2003).\u003c/li\u003e\n\u003cli\u003eDehaene, S., Changeux, J.-P., Naccache, L., Sackur, J. \u0026amp; Sergent, C. Conscious, preconscious, and subliminal processing: a testable taxonomy. \u003cem\u003eTrends Cogn. Sci.\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 204\u0026ndash;211 (2006).\u003c/li\u003e\n\u003cli\u003eLamme, V. Towards a true neural stance on consciousness. \u003cem\u003eTrends Cogn. Sci.\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 494\u0026ndash;501 (2006).\u003c/li\u003e\n\u003cli\u003eLumer, E. 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Zenodo https://doi.org/10.5281/ZENODO.14446172 (2024).\u003cbr\u003e \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Consciousness, brain–body interaction, EEG, respiratory phase, oral breathing","lastPublishedDoi":"10.21203/rs.3.rs-5655503/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5655503/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCyclic variation in bodily signals can influence the conscious perception of sensory stimuli. We have previously shown that the respiratory phase affects the sensory processing of visual stimuli during nasal breathing: the P1 component was modulated by awareness only during inhalation. Breathing can affect brain activity both directly through the entrainment of rhythmic brain activity via mechanical stimulation of the olfactory bulb (OB) and indirectly through fluctuations in baroreceptor (BR) activity across the respiratory cycle. We here aim to differentiate the relative contribution of OB stimulation and BR activity by during oral breathing when the OB is not stimulated and show that the early correlates of awareness do not vary with the respiratory phase but vary with the cardiac phase, albeit with somewhat delayed timing. Furthermore, the P3a component was modulated by awareness only when BR activity was low (inhalation, diastole). Our findings clarify the interplay between OB stimulation and BR activity for the conscious processing of a visual stimulus: the fluctuations in BR activity alone cannot explain how early sensory processes affect the perceptual outcome. Only when both OB stimulation is present and BR activity is low, is the P1 the earliest ERP component modulated by awareness.\u003c/p\u003e","manuscriptTitle":"The mode of breathing affects awareness-related brain potentials: Oral breathing shapes awareness-related brain potentials differently than nasal breathing","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-30 03:49:21","doi":"10.21203/rs.3.rs-5655503/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-02-13T05:54:39+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-30T04:03:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-27T09:06:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"85616102178030105039789740311099148911","date":"2025-01-14T12:17:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"234307387264679489391876368000075639800","date":"2025-01-14T01:45:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"302959560074598055763566805154193037371","date":"2025-01-11T10:36:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"259766350394641398294526215135325016428","date":"2025-01-11T06:40:37+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-01-11T06:36:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-01-10T13:24:06+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-12-30T04:06:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-12-26T09:22:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-12-16T16:20:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8a302288-c00d-4c45-ab8e-1d492cc7b6db","owner":[],"postedDate":"January 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":43451631,"name":"Biological sciences/Neuroscience/Cognitive neuroscience/Consciousness"},{"id":43451632,"name":"Biological sciences/Neuroscience/Cognitive neuroscience/Perception"},{"id":43451633,"name":"Biological sciences/Neuroscience"},{"id":43451634,"name":"Biological sciences/Neuroscience/Cognitive neuroscience"}],"tags":[],"updatedAt":"2025-08-18T16:00:00+00:00","versionOfRecord":{"articleIdentity":"rs-5655503","link":"https://doi.org/10.1038/s41598-025-12518-1","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-08-11 15:57:13","publishedOnDateReadable":"August 11th, 2025"},"versionCreatedAt":"2025-01-30 03:49:21","video":"","vorDoi":"10.1038/s41598-025-12518-1","vorDoiUrl":"https://doi.org/10.1038/s41598-025-12518-1","workflowStages":[]},"version":"v1","identity":"rs-5655503","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5655503","identity":"rs-5655503","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}