Measurement of Cervical Neuronal Activity during Stress Challenge Using Novel Flexible Adhesive Surface Electrodes

This study introduces a flexible, adhesive-integrated electrode array that was developed to enable non-invasive monitoring of cervical nerve activity. The device uses silver-silver chloride as the electrode material of choice and combines it with a novel electrode array consisting of a customized biopotential data acquisition unit and integrated graphical user interface (GUI) for visualization of real-time monitoring. Preliminary testing demonstrated this novel electrode design can achieve a high signal to noise ratio during cervical neural recordings. To demonstrate the capability of the surface electrodes to detect changes in cervical neuronal activity, the cold-pressor test (CPT) and a timed respiratory challenge were employed as stressors to the autonomic nervous system. This sensor system recording, a new novel technique, was termed Cervical Electroneurography (CEN). By applying a custom spike sorting algorithm to the novel electrode measurements, neural activity was classified in two ways: 1) pre-to-post CPT, and 2) during a timed respiratory challenge. Unique to this work: 1) rostral to caudal channel position-specific (cephalad to caudal) firing patterns and 2) cross challenge biotype-specific change in average CEN firing, were observed with both CPT and the timed respiratory challenge. Future work is planned to develop an ambulatory CEN recording device that could provide immediate notification of autonomic nervous system activity changes that might indicate autonomic dysregulation in healthy subjects and clinical disease states.


Introduction
The autonomic nervous system (ANS) links the central nervous system (CNS; brain and spinal cord) with peripheral organ systems, including the integumentary (sweat glands), circulatory (heart, blood vessels), digestive (gastrointestinal tract glands and sphincters, kidney, liver, salivary glands), endocrine (adrenal glands), reproductive (uterus, genitals), respiratory (bronchiole smooth muscles), urinary (sphincters), and visual (pupil dilator and ciliary muscles) systems [1][2][3] . The autonomic nervous system is colloquially divided into two main divisions: the "sympathetic" and "parasympathetic" nervous systems. However, both branches continuously coordinate through concerted feedback mechanisms to carefully control peripheral organ systems 4,5 . A large body of empirical evidence suggests that autonomic nervous system imbalance is associated with various pathological conditions that can include heterogeneous disease states, such as diabetic autonomic neuropathy, hyperhidrosis, orthostatic intolerance/postural tachycardia syndrome, pure autonomic failure, autonomic dysreflexia, Takotsubo cardiomyopathy, and vasovagal syncope, and it also contributes to pathophysiology associated with autoimmune inflammatory disorders such as Rheumatoid Arthritis 6,7 . Moreover, mental health disorders (e.g., Post-traumatic Stress Disorder (PTSD) and Major Depression Disorder) regularly exhibit circadian autonomic dysregulation, with heightened sympathetic and concomitant low parasympathetic drive most commonly reported [8][9][10][11][12] .
The human cervical spine (neck) is the site of a confluence of autonomic neural structures that are in close proximity to each other, including the major parasympathetic neuronal output transmitted by the vagus nerve. The vagus nerve communicates directly to the visual, heart, respiratory, and digestive systems, and the major sympathetic neuronal output transmitted by the middle and superior cervical ganglion is located approximately 1-2 cm deep to the vagus nerve.
The sympathetic ganglion, carotid body, and the vagus nerve are all localized within the carotid artery sheath, and, potentially due to this close proximity, sympathetic fibers have been observed in vagus nerve fascicles (called hitch-hikers), which further indicates multi-sourced neuronal signaling at this cervical level 13 . The superior cervical ganglion and the thoracic sympathetic ganglion output directly to the integumentary, visual, circulatory, respiratory, and digestive organ systems. Given the immense peripheral organ system control generated from cervical autonomic neuronal structures found within the superficial cervical neck 14 , decoding these signals to understand the role they play in health and disease could have significant impact on a host of conditions. Prior preclinical work has recorded resting microelectrode vagus nerve action potentials during lipopolysaccharide or inflammatory cytokine injection [15][16][17] . Recent work has shown that vagus nerve action potentials uniformly synchronize with the respiratory cycle in porcine 18,19 and in one recent human microelectrode study 20 . Other preclinical work measured (via cuff electrode) superior cervical ganglion activity with hypertensive stress tests, i.e., injection of adrenaline 21,22 or during painful stimuli 23 . These studies uniformly demonstrate immediate (within seconds) change in cervical sympathetic neuronal (superior cervical ganglion) activity with each challenge [21][22][23] . However, the invasive implantation and the risk associated with acute and chronic surgical cuff electrode complications have likely precluded, to date, any reported human cervical vagus nerve, carotid body, or superior cervical ganglion recording with validated stress models.
To enable recording with human autonomic stress models, a noninvasive, adhesive-integrated and skin conformal silver-silver chloride electrode array was developed that is capable of conformal positioning over the human left superior anterior cervical area overlying multiple neural structures (i.e., the vagus nerve and its branches, trigeminal nerve branches, sympathetic chain and its branches, the hypoglossal and glossopharyngeal nerves, as well as muscle and dermal sympathetic nerves), and its ability to monitor cervical nerve activity was tested using two widely used and validated stress tests, the cold pressor stress test (CPT) and a timed respiratory challenge.
CPT is performed by immersing the hand into a container filled with ice water, which is known to trigger a sympathetic reaction that involves blood vessel constriction and, thus, an increase in blood pressure 24,25 . It also increases the reflexive modulatory vagal tone by activating multiple brain stem nuclei that coordinate afferent and efferent vagus nerve signaling 26,27 . The effect of CPT -namely pain -on heart rate is bimodal: subjects routinely demonstrated either an increase or a decrease 28,29 . Likewise, in timed respiratory challenge studies, muscle sympathetic nerve activity was bimodal; subjects either increased or decreased muscle sympathetic nerve firing 30 .
To date, there is a paucity of inter-challenge within subject physiological measures with CPT and timed respiratory challenge. To fill this gap, the newly developed flexible adhesive electrode array was deployed for non-invasive cervical electroneurography (CEN) during a sequential CPT and timed respiratory challenge. Multi-stress challenge CEN measures could help to further disambiguate human autonomic biotype amongst healthy and disease states in several ways: 1) by facilitating the development of biomarkers of response to pharmacologic and or neuromodulation therapies, 2) enabling the prediction of inflammatory response to pain, and 3) aiding differentiation of sterile vs. non-sterile inflammation.

Results
The state-of-the-art custom surface electrode array that was tested is adhesive integrated and flexible, so that it can be non-invasively attached to a subject's anterior cervical neck. The electrode array was placed lateral to the trachea and medial to the sternocleidomastoid for this study. Silversilver chloride was utilized as the material of choice for the custom electrodes (Figure 1) in tandem with a customized biopotential data acquisition unit (Figure 2c). The custom electrode array allowed subjects to freely move (lateral rotation as well as forward, reverse, and lateral flexion and extension) without distorting the adhesiveness and robustness of the physical structure of the electrode array (Figure 2a, 2b). Power line noise and its harmonics were minimized by the low impedance between the electrodes and skin.
Cervical electroneurography recordings were carried out with four electrode "channels" positioned rostral to caudal to evaluate cervical signal: 1) pre-to-post CPT and 2) during a timed respiratory challenge. All channels were run through a spike sorting algorithm to identify putative action potentials or nerve firing patterns associated with different nerve branches. Heart rate was derived from ECG recording by finding QRS peaks.
In a sample subject, the change in cervical neural firing was observed to coordinately increase with onset of CPT; the cervical neural firing was extrapolated from the spike sorting algorithm (Figure 3a). Simultaneous recordings from Channel #1 (rostral overlying nodose ganglion, C2/3 cervical dermatome, and auriculotemporal branch of the trigeminal nerve) and heart rate were compiled (Figure 3a). Amongst an array of clusters, responsive clusters were identified.
Responsive clusters were defined as clustered groups that significantly increased in firing (by greater than 2 standard deviations) during CPT (for at least 40% of the stress challenge) compared to pre-CPT baseline activity.  With the CPT challenge, approximately 20 unique spike clusters were recorded in each subject.
Spike cluster firing activity was evenly divided into 5 temporally separated intervals over the duration of the CPT (0-20%, 20-40%, 40-60%, 60-80%, and 80-100%) with the aim of meticulously measuring onset and acquiescence of neural firing. Percent duration was employed to allow for temporal normalization of measures during the CPT. Average firing change (normalized by percentage change) for each channel across all clusters was computed at each CPT time interval and compared as follows: 1) within channel to baseline firing and 2) between channels at each time interval (Figure 4). All four channels at both the 0-20% and the 20-40% intervals showed significant increased firing activity when compared to baseline (p < 0.05). During the 0-20% CPT interval, Channel #4 (lower carotid artery) demonstrated greater neural firing (p<.05) when compared to both Channel #1 (over nodose ganglion) and Channel 2 (over lower nodose ganglion and 3rd trigeminal nerve, i.e., the auriculotemporal branch) as shown in Figure   4. Furthermore, during the 20-100% intervals of CPT, the increase in overall firing was observed to be greater for Channel #2 than for Channel #1 (Figure 4 b-d). rostral Channel #1 recording post-spike sorting analysis for the cold-pressor test (CPT). The green vertical line is when the CPT started and the subject immersed their right hand into the ice-water bucket, while the red vertical line is when the CPT stopped. Channel #1 (blue vertical line = neural firing) captures the amplitude of the recorded neural potential data. The peak of each detected spike (blue line) that exceeds the threshold value was sorted by dots with different colors, representing the different clusters. Heart rate in beats per minute is plotted in magenta using the right y-axis. (b) Exemplar subject cervical electroneurography (CEN) neural firing change during the cold pressor test. Top left: Responsive clusters at the Caudal Channel #4 (located over the carotid artery, vagus nerve, glossopharyngeal nerve, sympathetic chain, and sensory C2/C3 dermatomal nerves) immediately increase in firing frequency with cold pressor test (CPT) onset (i.e., hand placed in ice water bath). Top right: Responsive clusters from the rostral channel #1 (located over nodose ganglion and in close approximation to the auriculotemporal nerve) demonstrate delayed firing onset (at 120-150 sec). Bottom panels: Both Caudal channel #3 (overlying the carotid artery, vagus nerve, glossopharyngeal nerve, sympathetic chain, and sensory C2/C3 dermatomal nerves) and Rostral channel #2 (located over nodose ganglion and in close approximation to the auriculotemporal nerve) increase in firing frequency at 90-120 sec post CPT initiation. In all panels, the transition from purple to orange lines indicates increases in cervical neural firing greater than 2 SD above baseline (i.e., prior to CPT start), and the green line denotes initiation of CPT, while cessation is denoted by the red line.
Prior work by Mourot and colleagues 29 categorized subjects into two separate biotype groups, For the first interval of 0-20%, channels #3 and #4 demonstrate highly significant increases in firing frequency (** = p<.001), while channels #1 and #2 also increased in firing frequency, but to a lesser extent (* = p<.05) when compared to baseline. Channel #4 (lower carotid artery) demonstrated significantly greater (brackets) firing than channels #1 and #2 (p<.05). Panel B: All channels remained at relatively increased activity (p<.05) when compared to baseline. An increase in firing in Channel #2 was observed when compared to channel #4 (p<.05). Panels C, D, E: All channels do not show increases in activity when compared to baseline. Channel 2 continued to demonstrate significantly greater firing than Channel 4 for the remaining CPT duration (p<.05). Channel #1 = overlying nodose ganglion; channel #2 = overlying lower nodose ganglion; Channel #3 = overlying carotid artery (upper segment); Channel #4 = overlying carotid artery (lower segment). X Axis: Channel 1-4; Y Axis: Percentage Increase in neural firing frequency with respect to baseline. activity.

Cold Pressor Test Increaser (CPTi) or Cold Pressor Test Decreaser (CPTd), based on their HR
responses under CPT (see methods). In this CPT experiment, similar group/biotypes to those described by Mourot and colleagues 29 were observed that were also equally distributed between CPTd (N=5) and CPTi (N=4). Building on Mourot's biotype categorization (i.e., groups separated based on CPTd or CPTi), we aimed to determine if nested biotypes could be observed during the CPT as well as during the timed respiratory challenge. During the CPT, greater neural firing (p<.05) was observed in the CPTi compared to the CPTd group for the first two intervals (0-20% and 20-40%) and across all channels (Supplemental Figure 1). The spline generalized estimating equations (GEE) showed that the change in firing frequency from 0 to 50 seconds was different between two CPT groups at Rostral Channel #2, Caudal Channel #3, and Caudal Channel #4 at the trend level (p<0.1) (Supplemental Table 1, Supplemental Figure 2). The models also show that the slope change in firing frequency from 0 -50 seconds to 50 -200 seconds was met a trend level in between CPT groups across all four channels (p<0.06) (Supplemental Table 1).
Moreover, in addition to the observed group effect on neural firing (both during CPT and timed respiratory challenge), there was a significant group effect (CPTd vs. CPTi) on heart rate during the timed respiratory challenge (Supplemental Figure 1a).

Discussion
In this study, a flexible, adhesive-integrated electrode array was presented for the non-invasive monitoring of neural activity at the anterior cervical area. The peel-and-stick device substituted the use of an invasive and cumbersome needle electrode. Because of the self-adhering and noninvasive design of the electrode array, the array enabled the measurement of electrical signals over a prolonged period. Overlying the electrode array at the left superior anterior cervical area spanning rostral to caudal in a diagonal fashion enabled measurement of biopotentials from the skin surface that could arise from the activation of multiple superficial and deep neural structures during CPT and a timed respiratory challenge. The nerve structures that could be measured by the electrode array include the vagus nerve and its branches, the sympathetic chain of interconnected ganglia, the hypoglossal nerve, and glossopharyngeal nerves, as well as muscle electric signals and the potential activity of dermal sympathetic nerves. These neural recordings provided a sufficient signal to noise ratio to enable an electrocardiographic-derived heart rate estimate and neuronal spike sorting throughout the CPT. Moreover, spike sorting of signals collected from the different channels of the electrode array revealed statistically significant temporal and spatial relationships. One basic requirement for spike sorting is the spatial relationship between channels.
In the presented electrode device, both the electrode size and associated impedance, as well as the electrode shape and its arrangement on specific patterns can be customized 31 . This versatile electrode shaping can aid in neural structure source identification. Accurate CEN potential sourcing of underlying nerval structures may provide a powerful tool to evaluate sympathetic versus parasympathetic activity and their correlated homeostatic processes. Once these signal sourcing challenges can be resolved, CEN may prove a valuable tool for assessing body homeostasis.
For the electrode design, Ag/AgCl was chosen as the sensor layer material due to high signalto-noise ratio, lower skin-to-electrode impedance, and the non-polarizable nature of Ag/AgCl electrodes, which allows the Clion to take part in free charge exchange, preventing charge buildup [33][34][35] . Thin film technology enabled a high-resolution interconnect array that allows us to keep the form factor of the connector cable with 0.50 mm width (standard width used for a zero insertion force (ZIF) connector interface). The sensors were built using standard microfabrication processes followed by screen printing of Ag/AgCl ink, which yielded a final product that combined the best properties of each fabrication method (thin film and thick film The electrode design, recording technique, and electrode placement enabled the measurement of multiple neuronal signals characteristic of different possible sources. As in prior work, dermal sympathetic nerve firing, also referred to as skin sympathetic nerve activity, was ultimately detected, which is known to immediately increase with CPT onset; specifically, dermal sympathetic sudomotor firing results in eccrine sweat gland excretion that increases skin conductance, a phenomenon also known as electrodermal activity (EDA) 32,36 . Other sources of signals likely were detected as well, including deeper muscular sympathetic neurons, carotid bodies, sympathetic chain, and ganglia spanning the carotid body, middle and superior cervical ganglion, in addition to action potentials emanating from the glossopharyngeal nerve, the vagus nerve and its rostral ganglia, the nodose and jugular ganglia.
Spike sorting the electrode array CPT recordings enabled identification of key temporal and spatial signal characteristics that could reflect different neural source responsivity to the CPT. As part of the spike sorting analysis, the detected spike waveforms were decomposed from each measurement into multiple clusters. After applying spike sorting to the measured signal, it was found that amongst all channels (Rostral to caudal Channel #1 = overlying nodose ganglion, Channel #2 = overlying lower nodose ganglion, Channel #3 = overlying carotid artery (upper segment), Channel #4 = overlying carotid artery (lower segment)), multiple neural clusters had significant increases in firing compared with baseline during CPT, followed by a gradual return to baseline after CPT termination (Figure 3b). Specifically, the neuronal firing was most significant (p < 0.05) during the first two (0-20% and 0-40% periods) of the five CPT time intervals ( Figure   4). Importantly, this detected temporal distribution of increased activity, demonstrating that the presented pipeline could detect (within 1 min) increases in cervical neuronal activity known to occur with CPT 24-28 , which is indicative of changes in autonomic nervous system activity.
Additionally, this activity pattern followed the expected effects of CPT on several neuronal structures within the coverage of the anterior CEN array, including the vagus nerve (increased vagal tone during CPT) 25,26 , muscle and skin sympathetic nerve activity or EDA (increased activity followed by return to baseline), and carotid bodies (responsive to CPT induced increases in blood pressure) 27,32 . Collectively, the presented electrode array enabled the non-invasive detection and cluster isolation of neuronal spikes corresponding to CPT that may emanate from an aggregation or component of the vagus nerve, dermal, skin, muscle, and carotid body sympathetic response, while other neural structures may also contribute.
Unique to this study, significant spatiotemporally distinct cluster responsivity was observed when comparing Channels #1 and #2 (overlying the rostral auriculotemporal nerve and nodose ganglion) to Channels #3 and #4 (overlying the caudal carotid artery and sympathetic chain ganglia). Specifically, during the first 0-20% interval of the CPT, the caudal carotid artery sensor (Channel #4) demonstrated significantly higher firing frequency than the rostral nodose ganglion sensors (Channels #1 and #2) (p < .05) (Figure 4). In contrast, during the last 3 intervals of the CPT, the rostral nodose ganglion sensor (Channel #2) showed significantly higher firing frequency when compared to that of the caudal carotid artery sensor (Channel #4). Moreover, both caudal carotid artery sensors (Channels #3 and #4) demonstrated highly significant (p < .001) increases in firing frequency during the first interval of 0-20%, while the rostral sensors (Channels #1 and #2) also captured increased firing, but to a lesser degree than the caudal sensors (Channels #3 and #4) (p < .05) (Figure 4).
Prior work provided a premise for the observed delayed activity at the rostral Channels #1 and #2 (positioned over the trigeminal auriculotemporal branch distribution) when compared to the caudal Channels #3 and #4 (positioned over the carotid artery, carotid body, and sympathetic chain showed relatively less activity at this interval. However, greater activity in Channel #2 was observed in the last half of the CPT. As sympathetic output to the forehead is controlled by the Trigeminal nerve, the relative delay in neural firing in Channels #1 and #2 (positioned over the auriculotemporal branch of the Trigeminal nerve and the nodose ganglion) coincides with previous reports that demonstrate a delayed increase in trigeminal nerve sympathetic output that is known to occur with vagal-mediated reflex stress responses, associated with onset of motion/cybersickness, and CPT [37][38][39] . Future work will measure EGG, magnetoencephalography (MEG), and cervical electroneurography (CEN) during CPT to record multi-sourced coordinate neural changes that may further identify high resolution temporally dependent autonomic reflexes and give insight into visceral-brain coupling recently observed between EGG and MEG recordings 58 .
Extraordinarily, biotype-specific change in average CEN firing during the CPT and timed respiratory challenges was consistently observed (Figure 5, Supplement Figure 1, 2). To our knowledge, this is the first cross challenge report that indicates a direct relation between timed respiratory challenge CEN and heart rate change during CPT. Prior work by our group demonstrated that pre-deployed warfighter dysautonomia is predictive of eventual development of post-traumatic stress disorder (PTSD) in servicemembers 66 . Because PTSD is known to have a baseline hyper-sympathetic drive, it is concordantly co-morbid with cardiac disease risk 67 .
Therefore, it was postulated that the hyper-sympathetic timed respiratory challenge response identified in the CPTi group may indicate a biotype at risk for mental health disorder and or hyperinflammatory response. In support of this construct, PTSD sympathetic neural firing is highly increased during CPT when compared to healthy controls 68 . Future work will determine if CENderived hyper-sympathetic biotypes can predict immune cell response in an in-vivo human inflammatory model (intravenous lipopolysaccharide injection), in healthy subjects and servicemembers with PTSD.
Although these findings are promising, the present study has several limitations. First, the data are cross-sectional with relatively small sample size. This pilot study was meant to demonstrate feasibility of the flexible, adhesive-integrated electrode array for CEN recording during validated stress challenges, i.e., CPT and timed respiratory challenge. These findings need to be validated in a larger study cohort of healthy subjects and patients with a mental health disorder, namely PTSD.
In addition, these findings would have more impact if the study were done in larger populations of both sexes, with the aim to identify sex effects. For instance, we did not control for menstrual cycle for all female participants, which could impact CEN recording during CPT 68 . Prior work also demonstrated that sympathetic responses are both longer in duration and larger in amplitude during afternoon testing 69 . Although we did not control for exact time of day for the timed respiratory challenge or CPT, all testing occurred within the window of 13:00 to 18:00 and not during nighttime hours.
In conclusion, this study revealed three major findings: First, a flexible, adhesive-  Heights, OH), which acted as a backing that eased handling of the device. The silicone bilayer was then cut into a rectangular shape, which yielded the final sensor array as shown in Figure 1.

Participants
A total of 10 mentally and physically healthy subjects (2 females and 8 males, ages 21.8 ± 2.1) were recruited and enrolled in the study. The study was approved by the University of California San Diego Institutional Review Board (IRB#171154) and all research was performed in accordance with guidelines and regulations. All subjects provided written informed consent prior to participation and agreed to disclose identifying images for open-access publication.

Electrode Placement
Conductive gel was applied to the electrode surface using a syringe to improve conductivity.
The device was then applied to the neck with the rostral electrode approximately at the skin above the Trigeminal nerve auriculotemporal branch and the nodose ganglion and the caudal electrode at the skin above the carotid artery. Once attached, the PET backing was peeled off (Figure 1a).  Figure 1b).

Timed Respiratory Challenge
Respiration vital sign change was measured by a respiration belt transducer (BIOPAC system Inc, Goleta, CA, USA). The respiration belt was modified to function as a potentiometer, soldered in series with a 5k ohm resistor that was powered by the 3.3V power pin on the Arduino board.
The voltage variation caused by the resistance change during each breath was measured by the HackEEG system as one of the biopotential channels. The respiration signal was filtered by a 5 Hz zero-phase low-pass filter in post-processing. 120 seconds after baseline recording, the subjects underwent a timed respiratory challenge.
The timed respiratory challenge consisted of regular breathing (5 s inhale and 5 s exhale) that was carried out for 10 cycles. Post timed respiratory challenge data was then recorded for an additional 120 seconds. One subject's respiratory data was excluded due to irrevocable data loss.

Cold Pressor Test
Five minutes after the timed respiratory challenge, subjects were asked to perform the CPT task. Subjects were required to fully immerse their right hand into an ice water container to determine the cold pressor tolerance. Chilled water and freezer fresh ice (3°C) were immediately added to the container with water temperature kept at 4-7°C. Cold pressor tolerance was carried out for each subject; subjects were asked to keep their right hand in the ice water container for at least 1 minute (5 minutes maximum) and only withdraw their hand if they could no longer tolerate the pain. Subjects were told to stay still as well as avoid talking and swallowing during the experiment to avoid EMG activity/artifacts.

Defined Cold Pressor Test decreaser (CPTd) and Cold Pressor Test increaser (CPTi)
In this work, two common groups were identified: Cold Pressor Test increasers (CPTi) and Cold Pressor Test decreasers (CPTd). As previously defined by Mourot and colleagues 29 , individuals in the CPTi group experienced increased HR when reacting to CPT, i.e., first an increase in HR was observed and then either a further increase in HR occurred, or the HR remained elevated until the end of the test. It should be noted that if HR decreased less than 5 beats, the HR was considered maintained, and the participant was categorized to the CPTi group. As previously defined by Mourot and colleagues 29 , CPTd individuals were observed to react to the test with an initial increase followed by a decrease in HR of more than 5 beats per min (mean over 10 s) compared to the peak HR achieved during the test.

Data Processing
A customized graphic user interface (GUI) installed on a Linux terminal was written in Python to monitor and display physiological signals in real time. In post processing, the cervical signal was filtered by a 20-1,000 Hz zero-phase bandpass filter. Powerline noise and harmonics were also removed by notch filters. In each cervical neural recording, a spike sorting algorithm was carried out to differentiate detected spikes into different clusters. First, spike detection was performed by setting a threshold as: Thr = 3σ, with σ = ( cluster spikes into groups. The clustered neural groups were characterized by different firing rate and amplitude behaviors. The firing rate was counted as the number of neural group activity per second, and the amplitude was derived by calculating the peak-to-peak difference of each detected waveform (Figure 1c).

Statistical Analysis
Two-tailed paired sample t-test was used to identify differences between the firing frequency change of each individual channel when compared to baseline (Pre-CPT) activity across 5 intervals for the entire CPT duration (Figure 4), as well as between each channel at each time interval (Figure 4). The same methodology was used to test pairwise pre-to-post difference in 1) channel firing during the timed respiratory challenge (Figure 5c), 2) channel firing over the duration of the CPT challenge (Supplementary Figure 1b), and 3) HR change during the CPT (Supplementary   Figure 1a), for all within and between CPTd and CPTi group comparisons.
Two tailed two-sample t-test was used to compare neuronal firing differences between the CPTd (N=5) and CPTi (N=4) groups during the timed respiratory challenge, i.e., average firing frequency difference between the first and last 60 seconds of measurement (Figure 5c) Table 1).
To further understand interactions between groups (CPTi vs. CPTd) neural firing during the timed respiratory challenge, a linear regression model with inference based on GEE was utilized.
Differences in groups (CPTi vs. CPTd) neural firing pre-to-post the timed respiratory challenge were determined with the generalized estimating equations (Supplemental Table 2).