Comsol Oxygenator Gas Exchange

1,6*

In the brain, increased neural activity is correlated with increases of cerebral blood flow and tissue oxygenation. However, how cerebral oxygen dynamics are controlled in the behaving animal remains unclear. We investigated to what extent cerebral oxygenation varies during locomotion. We measured oxygen levels in the cortex of awake, head-fixed mice during locomotion using polarography, spectroscopy, and two-photon phosphorescence lifetime measurements of oxygen sensors. We find that locomotion significantly and globally increases cerebral oxygenation, specifically in areas involved in locomotion, as well as in the frontal cortex and the olfactory bulb. The oxygenation increase persists when neural activity and functional hyperemia are blocked, occurred both in the tissue and in arteries feeding the brain, and is tightly correlated with respiration rate and the phase of respiration cycle. Thus, breathing rate is a key modulator of cerebral oxygenation and should be monitored during hemodynamic imaging, such as in BOLD fMRI.

Open

Recherche Médicale, U1128 Paris, France. 3 Laboratory of Neurophysiology and New Microscopies, Université Paris Descartes, Paris, France. 4 Graduate NATURE COMMUNICATIONS | (2019) 10:5515 | https://doi.org/10.1038/s41467-019-13523-5 | www.nature.com/naturecommunications

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n adequate oxygen supply is critical for proper brain function1, and deficiencies in tissue oxygen is a noted comorbidity in human diseases2 and aging3. For these reasons, there has been a great deal of interest in studying dynamics of cerebral oxygenation4–8. However, there is a gap in our understanding of how behavior, such as natural exercises like locomotion, affects cerebral oxygenation. In natural envir- onments, animals and humans have evolved to spend a sub- stantial portion of their waking hours locomoting9. As exercise is known to have a positive effect on brain health10, a better understanding of the basic brain physiology accompanying these behaviors can give insight into how exercise can improve brain function. During movement, neuromodulator release and neural activity in many brain regions is elevated11–14, and there is an increase in cardiac output and respiratory rate. How these changes in local and systemic factors interact to control cerebral oxygenation is a fundamental question in brain physiology but is not well understood. Most cerebral oxygenation studies are per- formed in anesthetized animals7,8,15,16 (but see refs. 4,17,18), or non-invasively in humans. Anesthesia causes large disruptions of brain metabolism and neural activity19, and non-invasive human studies are impeded by technical issues, making accurate deter- mination of any aspect of brain tissue oxygenation problematic.

In addition to the importance of understanding oxygen dynamics in the brain to basic physiology, a better grasp of natural oxygen dynamics in the brain will greatly aid in the interpretation of functional MRI signals20, which allow non- invasive imaging of neural activity. Previous work in awake pri- mates has shown that tissue oxygen signals correspond well with changes observed with blood-oxygen level dependent (BOLD) fMRI5. Neurally-driven BOLD signals are generated by vessel dilation21. However, in addition to the BOLD fMRI signals of a neural origin (i.e., those generated by neurovascular coupling), BOLD signals can arise from sources that are not directly linked to underlying neural activity, such as pure vascular effects, respiration22,23, cardiac pulse rate, and autonomous hemody- namic regulation. Resolving the nature of non-neural sources of BOLD contrast has been an area of active research23, and will be helpful in better spatially resolving the neurally-generated BOLD signals24.

Here we investigated how and by what mechanisms voluntary exercise impacts brain tissue oxygenation. We used intrinsic optical signal (IOS) imaging11,25, electrophysiology, Clark-type

Dye

measurement4,7,8,17,18 to elucidate how vasodilation, neural activity, and systemic factors combine to generate changes in brain oxygenation. All experiments were performed in awake mice that were head-fixed on a spherical treadmill11,12 or rotating disk4,18,26 that allowed them to voluntarily locomote.

We find that cerebral oxygenation rises during locomotion in cortical regions that do not experience vasodilation, as well as when vasodilation is blocked. Oxygen levels increase in the arteries that supply the cortex during exercise, consistent with an increase in systemic oxygenation. Finally, oxygen fluctuations are correlated with spontaneous and locomotion-evoked chan- ges in respiration rate, as well as the phase of the respiration cycle, also consistent with a dynamic regulation in systemic oxygenation.

Results

Locomotion drives localized cortical vasodilation. We first assessed the spatial extent of cortical hemodynamic responses and their relationship to voluntary locomotion using IOS imaging11,25 (Fig. 1a). Imaging was done through a thin-skull window over the right-hemisphere (Fig. 1b). When the brain is illuminated with 530 nm light, reflectance decreases report dilations of arteries, capillaries and veins, which correspond with increases in cerebral blood volume (CBV). During locomotion, we observed region- specific changes in reflectance. There was a pronounced decrease in reflectance (corresponding to an increase in CBV) in forelimb/ hindlimb representation of somatosensory cortex (FL/HL), while in frontal cortex (FC) there was no change, or a slight increase in reflectance (Fig. 1b). To better localize the area of decreased CBV, we used a smaller region of interest (ROI, 2–4 mm rostral and 0.5–2.5 mm lateral from bregma, ~4 mm2) more rostral in FC than in our previous study11. We also assayed cerebral blood flow (CBF) using laser Doppler flowmetry, which will evaluate flow changes in a ~1 mm2 area. The locomotion-evoked CBF showed similar spatial pattern of responses (Fig. 1c) as CBV. We quan- tified how locomotion affected both CBV and CBF in two com-

Plimentary

ways.

Locomotion-Triggered

average, generated by aligning the IOS or laser Doppler signals to the onset of locomotion (see “Methods”) using only locomotion events ≥5 s in duration (Fig. 1d). The locomotion-triggered average showed no significant change in CBF in FC (n = 5 mice, Wilcoxon signed-rank test, p = 0.22), and a large increase in FL/HL (n = 5 mice, Wilcoxon signed-rank test, p = 0.03). The locomotion-triggered average showed significant increase in optical intensity in FC (n = 11 mice, Wilcoxon signed-rank test, p < 0.001) while decrease in optical intensity in FL/HL (n = 11 mice, Wilcoxon signed-rank test, p = 0.0122). We also calculated the hemodynamic response function (HRF)25,27, which is the linear kernel relating locomotion events to observed changes in CBV and CBF (Supplementary Fig. 1), using all locomotion events. When we quantified the net CBF using the HRFs, loco- motion actually drove a significant decrease in flow in FC and increase in FL/HL (Supplementary Fig. 1). Using the HRFs to quantify the net CBV, we obtained same conclusions as derived from locomotion-triggered average. This shows that locomotion and the accompanying cardiovascular changes do not drive global increases in CBF/CBV, rather CBF/CBV increases are under local control. This lack of non-specific flow increase in the cortex during locomotion is likely because of autoregulation of the feeding arteries at the level of the circle of Willis and larger resistance arteries, as well as increased blood flow to the muscles28.

To assess neural activity during locomotion, we measured the local-field potential (LFP) and multi-unit activity (MUA) in a separate group of seven mice (six sites in FL/HL and four sites in FC) using multi-channel linear electrodes (Fig. 1e). We used electrophysiological measures of neural activity, as they are more sensitive than calcium indicators (which fail to detect about half of the spikes even under ideal conditions29), and do not disrupt normal neural activity as genetically encoded calcium indicators can do. Since gamma-band (40–100 Hz) LFP power has been observed to be the strongest neural correlate of hemodynamic signals in rodents25,30, primates5 and humans, and increases in gamma-band activity are also closely associated with the increases in metabolic demand31, we quantified how locomotion affects neural activity by generating locomotion-triggered averages of gamma-band LFP power and spiking (see “Methods”). We observed that both the gamma-band LFP power and spike rate increased during locomotion across all layers in both FL/HL (Fig. 1f, h, i) and FC (Fig. 1g–i). The slow rise in neural activity a few hundred milliseconds before the onset of locomotion is due to low-pass filtering of the MUA signal (5 Hz, see “Methods”) and windowing (1 s duration) required to estimate the LFP power21, as well as the ramping up of neural activity due to arousal changes seen before voluntary locomotion32. As optogenetic stimulation of fast spiking inhibitory neurons has been shown to induce large increases in blood oxygenation in somatosensory cortex33, we

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NATURE COMMUNICATIONS | (2019) 10:5515 | https://doi.org/10.1038/s41467-019-13523-5 | www.nature.com/naturecommunications sorted recorded spikes into fast spiking (FS, putatively inhibitory) and regular spiking (RS, putatively excitatory) spikes (see “Methods”). We found that FS and RS neurons exhibited a similar degree of rate increases during locomotion in both FL/HL and FC areas (Supplementary Fig. 2).

Taken together, our results show that a short bout of locomotion increases neural activity, which is followed by an increase in CBV and CBF in FL/HL, and a small decrease or no change in CBV and CBF in FC. Together with our previous work11,34, these results suggest that coupling between neural activity and hemodynamics are brain region-specific (Fig. 1j), as seen in many other neurovascular coupling studies in the cortex and other brain regions35. The lack of observed vasodilation in the FC is not due to a lack of sensitivity of our IOS imaging paradigm, as if the vasodilation in FC had the same relationship to neural activity as in FL/HL, we would expect to see a 2%

Power (A.U.)

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decrease in the reflectance (Fig. 1j), which is easily detectable with our IOS setup25. As tissue oxygenation reflects the balance between oxygen supply and utilization36, we would expect that in FL/HL, increased activity of the neurons will be more than matched by an increased blood supply, leading to an increase in tissue oxygenation. However, increased neural activity in FC during locomotion will not be matched by an increase in the blood supply and should lead to a decrease in oxygenation in FC.

Locomotion drives cortex wide increases in brain oxygenation. To test if the brain region-dependent differences in neurovascular coupling drove regional differences in brain oxygen dynamics during locomotion, we measured partial pressure of tissue oxygen (PtO2) in awake, behaving mice (n = 37 mice, 23 in FL/HL, and 14 in FC; 148.2 ± 28.3 min of recording for each mouse) using Clark-type polarographic electrodes (Fig. 2a). Signals from these electrodes are similar to those obtained with BOLD fMRI5, but with sub-second response time (Supplementary Fig. 3a, b), long- term stability (Supplementary Fig. 3c, d) and higher spatial resolution. We measured oxygen dynamics at different cortical depths by sequentially advancing the probe from the cortical surface into deeper layers. We observed a laminar-dependence of resting PtO2 in awake mice, with lower oxygenation in layer I than other layers in both FL/HL and FC (Supplementary Fig. 4a, b, see also refs. 4,17), though this is probably too thin a section of brain to be distinguishable with laminar fMRI. Resting PtO2 levels were similar at each cortical depth in both FL/HL and FC (Supplementary Fig. 4a, b). No difference in onset time or peak time was observed in the HRFs of PtO2 across layers, though the onset time was shorter in FC (Supplementary Fig. 4d, e), con- sistent with fMRI measurements using ultrashort stimuli37. Note that because we are measuring tissue oxygenation in this case, any oxygen dynamics in the vasculature will be temporally blurred by the diffusion dynamics of oxygen in the tissue. These results, together with the observation that resting PtO2 is similar in somatosensory cortex and olfactory bulb glomerular layer4, indicate that the spatial distribution of oxygen in the brain under normal (non-anesthetized) physiological condition is relatively homogenous. This quantification of laminar tissue oxygen dynamics should aid interpreting the complicated dynamics of laminar fMRI signals38.

Locomotion produces large, sustained dilation of arteries39 and increases in CBF and CBV11,34 in somatosensory cortex. These locomotion-induced dilations were not due to systemic effects, as they have been shown to be unaffected by drugs that do not cross the blood brain barrier (BBB) that increase or decrease the heart rate40 and are blocked by the suppression of local neural activity25. The locomotion-induced dilations are comparable in magnitude to those elicited by episodic whisker stimulation25 which is known to elevate oxygenation, so one would expect increases in tissue oxygenation in FL/HL during locomotion. As anticipated, we observed increases in PtO2 during locomotion in FL/HL in all layers (Fig. 2b–d). Because the supply of blood to FC does not increase, but neural activity does, one would expect a decline in tissue oxygenation during locomotion. Surprisingly, we also observed a very similar PtO2 increase in FC (Fig. 2b–d) to that observed in FL/HL, despite small decreases in CBV or CBF, and an increase in neural activity. The elevation of PtO2 in FC during locomotion suggests that other factors can increase oxygenation in the brain.

Polarographic probes provide measures of oxygen tension over a small region of brain tissue, and the response may be affected by vasculature type and density41,42 surrounding the probe. To distinguish compartment-specific oxygen tension in the tissue, arterial and venous blood spaces, we then mapped the spatial distribution of locomotion-evoked brain oxygenation response using optical imaging spectroscopy43 (Fig. 2e). Taking advantage of differences in the optical absorption spectra of oxyhemoglobin (HbO) and deoxyhemoglobin (HbR)43, we collected reflectance images during rapid alternating green (530 nm) and blue (470 nm) illumination. Note that spectroscopic measurements report oxygen concentrations in red blood cells, while polarography reports average oxygen concentration in tissue near the electrode.

The oxygen levels in the tissue will differ from that in the blood somewhat due to the constraints of oxygen diffusion from the blood into the tissue and ongoing metabolic processes in neurons and glial cells. Using cerebral oxygenation index (HbO-HbR)44, the spectroscopic measures of hemoglobin oxygenation were similar to measurements from the tissue using polarographic probes: both methods yielded an increase in oxygenation during locomotion in both FC and FL/HL (Fig. 2f, g). These oxygenation changes persisted even when the heart rate increase associated with locomotion was pharmacologically blocked or occluded (Supplementary Fig. 5b, d, e), indicating they were not driven by increased cardiac output during locomotion.

Moreover, locomotion-induced elevations in oxygenation were present in the parenchyma, arterial and venous blood (Fig. 2f). As oxygen levels in the brain strongly depends on the arterial oxygen content8, we made direct measurements of oxygen partial pressure in the center of pial arteries (PaO2) using two-photon phosphorescence lifetime microscopy (2PLM, Fig. 2h)4,7,8,17,18, with a new phosphorescent probe (Oxyphor 2P) which has a very high brightness, improving measurement speed and imaging depth45. We asked if the oxygen levels increased in the center of large pial arteries that supply blood to the brain. As blood in these arteries will have minimal time to exchange oxygen in their transit through the heart and carotid artery to the brain, oxygen levels in these arteries will track systemic oxygenation levels. We Fig. 1 Locomotion drives cortical region specific hemodynamic and neural responses. a Experimental setup for IOS imaging. b Example showing CBV change during voluntary locomotion. Top left, an image of thin-skull window and corresponding anatomical reconstruction; scale bar = 1 mm. Top right, reflectance map before (1 s), during (49 s) and after (94 s) a voluntary locomotion event. Bottom, percentage change in reflectance (ΔR/R0) during locomotion events for each brain region. Wh, vibrissae cortex; V1, visual cortex. c Example showing locomotion-evoked changes of CBF in FC (top) and FL/ HL (bottom) in the same animal. d Population average of locomotion-triggered average of CBV (n = 11 mice, left) and CBF (n = 5 mice, right) responses in both FL/HL (green) and FC (blue). Data are shown as mean ± SD. e Top, all laminar electrophysiology measurement sites in FC (n = 4 mice) and FL/HL (n = 6 mice). The squares indicate the measurement sites showing in (f) and (g). Bottom, layout of the electrodes and measurement depth. f Example trial showing the large increase in gamma-band LFP power (top), raw signal (middle), and spike raster (bottom) during locomotion from a site 800 µm below the pia in FL/HL. Shaded area indicates the time of locomotion. g As in (f) but for FC. h Group average of locomotion-evoked spike rate responses in both FC (top, n = 4 mice) and FL/HL (bottom, n = 6 mice). i As in (h) but for locomotion-evoked gamma-band LFP power responses. j Changes of ΔR/R0, 2–5 s after the onset of locomotion plotted against spike rate change 0–2 s after the onset of locomotion in FL/HL (green ellipse) and FC (blue ellipse). For each ellipse, the radius along the vertical axis is the SD of ΔR/R0 across all 11 mice; the radius along the horizontal axis is the SD of spike rate across all animals (n = 4 for FC and n = 6 for FL/HL). The black dot in the center of each ellipse represents the average value of ΔR/R0 and spike rate response. The diagonal line shows the prediction of linear coupling.

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NATURE COMMUNICATIONS | (2019) 10:5515 | https://doi.org/10.1038/s41467-019-13523-5 | www.nature.com/naturecommunications measured PaO2 in cortical and olfactory bulb arteries and found that PaO2 increased during locomotion (Fig. 2i). Taken together, these measurements are consistent with an increase in systemic blood oxygenation that leads to a brain-wide increase of oxygenation in the tissue and vascular compartments during locomotion. The increase in oxygenation accompanying the decrease in CBV and CBF in FC suggests that neurovascular coupling is not the only process controlling brain oxygenation46 during locomotion.

Cortical oxygen increases even when vasodilation is blocked. Our observation that locomotion induced localized blood flow/ volume increases, but cortical-wide increases in brain oxygena- tion, led us to hypothesize that activity-dependent vasodilation may not be necessary for an increase in oxygenation. To test this, we pharmacologically blocked glutamatergic and spiking activity by infusing/superfusing a cocktail of 6-cyano-7-nitroquinoxaline- 2,3-dione (CNQX, 0.6 mM), (2R)-amino-5-phosphonopentanoic acid (AP5, 2.5 mM) and muscimol (10 mM) to suppress local neural activity. We first infused a cocktail of CNQX/AP5/mus- cimol via a cannula into FL/HL25, while concurrently monitoring neural activity, CBV and blood oxygenation (n = 4 mice, Fig. 3a).

The cocktail infusion suppressed resting gamma-band LFP power by 80 ± 12% and spiking activity by 82 ± 3% relative to vehicle infusions. Similarly, the standard deviation (SD) of these neural activity measurements during resting periods, an indicator of spontaneous neural activity levels, was decreased by 75 ± 18% in the gamma-band LFP power and by 85 ± 6% in the MUA amplitude. To quantify CBV responses, we selected a semicircular ROI centered on the cannula and with a radius specified by the distance between the electrode and cannula (Fig. 3a), to ensure the ROI only included suppressed cortex25. Accompanying this neural activity blockade, baseline reflectance from the ROI increased (indicating decreased CBV), and the locomotion- evoked decrease in reflectance (vasodilation) was almost com- pletely suppressed (Fig. 3b–d), consistent with our previous study25 showing that intracerebral infusion of a cocktail of CNQX/AP5/muscimol suppressed sensory-evoked CBV increase.

However, the block of neural activity was less effective during locomotion (Fig. 3b), likely due to the large increases of neural

During

locomotion13,14. Nevertheless, this is conducive for testing our hypothesis, as a complete block of vasodilation and an incomplete block of neural activity increases should lead to a decrease in oxygenation. However, if there is no oxygenation decrease, or the oxygenation increases, this would indicate that the oxygenation of the inflowing blood is elevated during locomotion. When locomotion-induced vasodilation was blocked, the locomotion- evoked increase in HbO-HbR persisted, though the increase was smaller (Fig. 3b–d, Supplementary Fig. 5b, c, e). This increase was surprising, as we were able to completely block the locomotion-

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Fig. 2 Cortex-wide increases in oxygenation during locomotion. a Top, experimental setup. Bottom, measurement sites. b Example traces showing PtO2 responses to locomotion at sites 800 µm below brain surface in FL/HL (left) and FC (right). Top, black ticks denote binarized locomotion events; Middle, PtO2 responses to locomotion; Bottom, example of data showing spectrogram of LFP (white trace showing the gamma-band LFP power). c Locomotion- evoked cortical tissue oxygenation increases (ΔPtO2) at all measured depths in both FL/HL (left, n = 23 mice) and FC (right, n = 14 mice). Gray shaded area indicates locomotion. d HRF of tissue oxygenation at different depths in both FL/HL (top, n = 23 mice) and FC (bottom, n = 14 mice). Vertical black line showing the start of a brief impulse of locomotion. e Schematic showing the optical imaging spectroscopy setup. f Left, example data showing spatial distribution of locomotion-evoked response of ΔR/R0 and HbO-HbR in an example mouse. Right, locomotion-triggered average of ΔR/R0 and HbO-HbR for the same mouse in FC (blue) and FL/HL (green). g Group average of locomotion-evoked response of ΔR/R0 and HbO-HbR in FC (n = 4 mice) and FL/HL (n = 4 mice). h Schematic showing the measurement of PaO2 using 2PLM. i Locomotion induced PaO2 increases in five arteries (three in the cortex (green) and two in the olfactory bulb (purple)) from a total of four mice. Mean response of all arteries is shown as a black line. Solid lines and shaded area in (c), (d), (f) and (g) denote mean ± SEM, respectively.

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induced vasodilation, and there was still a small locomotion- induced increase in neural activity, which should result in a net decrease in oxygenation.

We further studied the effects of the suppressed vasodilation on oxygen responses in the tissue in a separate set of mice using polarographic electrodes (n = 9 mice, 5 in FC and 4 in FL/HL).

We topically applied a cocktail of CNQX/AP5/muscimol to the cortex, while measuring spontaneous and locomotion-evoked neural activity and PtO2 in the superficial cortical layers (100–200 µm below the pia). The efficacy of the cocktail in suppressing neural activity was monitored with two electrodes spanning the oxygen measurement site25,30 (Fig. 3e). Similar to intracortical infusions, superfusing the cocktail potently sup- pressed resting gamma-band LFP power by 89 ± 8% and the SD by 77 ± 21% (Fig. 3g). Resting PtO2 increased by ~70% following the suppression of neural activity and vasodilation (before: 23.09 ± 10.60 mmHg; after: 34.64 ± 11.11 mmHg; Fig. 3h), con- sistent with neural signaling being a major component of metabolic demand47. To quantitatively assay locomotion-evoked oxygen and neural responses, we calculated the linear kernels (i.e., the HRF) relating PtO2 and the gamma-band LFP power to locomotion. To ensure that vasodilation was blocked, we only analyzed those animals (n = 4 mice, 3 in FC and 1 in FL/HL) that showed >50% suppression of locomotion-evoked neural activity.

In these animals, application of CNQX/AP5/muscimol reduced peak amplitude of gamma-band LFP neural response function (NRF) by 81 ± 8% (Fig. 3i). If activity-dependent vasodilation is the only determinate of tissue oxygenation, we would expect the HRF of PtO2 shows profound reductions, since the vasodilation was blocked by the suppression of neural activity (Fig. 3b–d).

However, the peak amplitude of PtO2 HRF was not changed (82 ± 51% of before cocktail application, Fig. 3i). Taken together, these results show that suppressing vasodilation does not block the locomotion-evoked oxygen increases.

Respiration drives changes in tissue and blood oxygenation. One possible driver of the increases in cerebral oxygenation is the increase in respiration during locomotion. Changes in respiration affect blood oxygen levels in the carotid artery48,49 in anesthetized animals, and in humans, inhalation of 100% oxygen can elevate brain oxygen levels50. However, it is not known if normal fluc- tuations in respiration rate can impact cerebral oxygenation during normal behaviors. We tested whether respiration was correlated with oxygenation during locomotion by simulta- neously measuring cortical tissue oxygenation and respiration (Fig. 4a). Locomotion was accompanied by a robust increase in respiratory rate (Fig. 4a, Supplementary Fig. 6), and fluctuations in respiratory rate on the time scale of seconds were linked to fluctuations in PtO2 (Fig. 4a). We quantified how well the fluc- tuations of respiratory rate and gamma-band LFP power (which has been shown to be the LFP band most correlated with

N = 9

N = 9

Fig. 3 Locomotion-evoked cortical oxygenation increases persist when vasodilation is blocked. a Experimental setup for spectroscopy. b Locomotion- evoked gamma-band LFP power (top left), MUA power (top right), ΔR/R0 (bottom left) and HbO-HbR (bottom right) in one representative mouse following aCSF (black, 12 locomotion events) and CNQX/AP5/muscimol (red, 14 locomotion events) infusion. c Locomotion-evoked spatial distribution of ΔR/R0 (top) and HbO-HbR (bottom) for the same mouse shown in (a) and (b) following aCSF and CNQX/AP5/muscimol infusion. d Group average of locomotion-evoked ΔR/R0 (top, * paired t-test, t(3) = 7.4235, p = 0.0051) and HbO-HbR (bottom, * paired t-test, t(3) = 8.0007, p = 0.0041) after aCSF or CNQX/AP5/muscimol infusion in four mice. The orange circle denotes the mouse shown in (b) and (c). e Experimental setup for simultaneous PtO2 and LFP measurements. f Example of resting PtO2 fluctuations (top) and resting gamma-band LFP fluctuations (bottom) in FL/HL in a single mouse.

g Comparison of spontaneous LFP activity (left, * Wilcoxon signed-rank test, p = 0.0039) and fluctuations (SD, right, * paired t-test, t(8) = 5.0246, p = 0.0010) before (black) and after (red) application of CNQX/AP5/muscimol in FL/HL (4 mice, black circle) and FC (five mice, orange circle). h As (g) but for spontaneous PtO2 activity (left, * paired t-test, t(8) = 3.2712, p = 0.011) and fluctuations (SD, right, * paired t-test, t(8) = 0.7542, p = 0.4723). i Top left, NRF of gamma-band LFP power (n = 4 mice, 1 in FL/HL and 3 in FC) before and after application of CNQX/AP5/muscimol. Vertical black line indicates the start of a brief impulse of locomotion. Bottom left, as in top left but for HRF of PtO2. Top right, peak amplitude of NRF of gamma-band LFP power before and after application of CNQX/AP5/muscimol (*paired t-test, one sided, t(3) = 3.4299, p = 0.0208). Bottom right, as in top right but for peak amplitude of HRF of PtO2 (paired t-test, t(3) = 0.5861, p = 0.599). Solid lines and shaded area in (b) and (i) denote mean ± SEM, respectively. Data are shown as mean ± SD in all other graphs.

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by calculating the cross-correlation. During periods of rest, increases in gamma-band LFP power were correlated with decreased oxygenation (Fig. 4d, e), which was unexpected as gamma-band power increases during rest are correlated with vasodilation25,30,51. Because the decrease takes place with near zero time lag (Fig. 4d, e), it seems as though the dilation induced by spontaneous neural activity are insufficient relative to the metabolic demand. In contrast, respiration rate increases were correlated with increased oxygenation with a slight delay, con- sistent with the transit time of the blood from the lungs to the brain (Fig. 4b, c). When periods of locomotion were included, the correlation between gamma-band LFP power and oxygenation was positive, suggesting that the coupling depends on animal’s state51 (Fig. 4d, e). The coupling between other frequency bands of the LFP and oxygen increases was negative (Supplementary Fig. 7), consistent with previous reports showing decreases in the power of these bands during voluntary locomotion32 (see also Supplementary Fig. 8). Because cortical excitability and respira- tory rate are correlated during locomotion (likely due to the reciprocal connections between respiratory and modulatory regions52), we sought to disentangle their respective contributions to cerebral oxygenation using partial coherence analysis53. We found that the coherence between respiratory rate and PtO2 was not due to the co-varying neural component (Supplementary Fig. 9b), nor was the coherence between gamma-band power and PtO2 affected by removing the respiratory rate contribution (Supplementary Fig. 9c). Thus, the partial coherence analysis indicates that respiration and neural activity (and likely vasodi- lation) affect tissue oxygenation independent of each other.

The correlated fluctuations in respiratory rate and PtO2 suggests that the oxygen tension of arterial blood should also track the respiratory rate. To test this, we simultaneously monitored respiration and PaO2 in the pial arteries using 2PLM. In mice with irregular respiration, where respiratory rate transients of a few seconds occurred without locomotion, PaO2 followed respiration rate fluctuations (Fig. 4f), showing that changes in respiration rate can alter the oxygenation of the arterial blood entering the cortex.

We then asked if PaO2 tracked the phase of respiration, that is, whether the concentration of oxygen in the blood entering the brain fluctuated in phase with the inspiration-expiration cycle.

This requires measuring PaO2 at rates high enough (>5 Hz) to capture fluctuation in PaO2 due to respiration (nominally 2.5 Hz).

*

Fig. 4 Respiration drives changes in cerebral tissue and arterial blood oxygenation. a Measuring respiration using a thermocouple. b Cross-correlation between PtO2 and respiratory rate during rest (top) and periods including rest and locomotion (bottom). c Peak amplitude (top, Wilcoxon signed-rank test, p = 0.3125) and peak time delay (bottom, Wilcoxon signed-rank test, p = 0.7422) of cross-correlation between PtO2 and respiratory rate during rest (black) and periods including rest and locomotion (red). d As (b) but for correlation between PtO2 and gamma-band LFP power. e As (c) but for peak time (* paired t-test, t(7) = 6.1918, p < 0.001) and peak time delay (* Wilcoxon signed-rank test, p = 0.0234) of cross-correlation between PtO2 and gamma- band LFP power. f Example data showing the temporal relationship between respiratory rate (black) and PaO2 (blue) in the center of one artery (white arrow) in somatosensory cortex during rest. g Schematic showing measurement of PaO2 fluctuations driven by respiration cycle. h Top, PaO2 change in one artery during the respiratory cycle at rest. Each filled circle denotes averaged PaO2 over a 20 ms window aligned to a specific phase of the respiration cycle and averaged over 15 recordings. The solid curve shows filtered data (first-order binomial filter, five repetitions). Tmin and Tmax denote the time period (40 ms) PaO2 reaches minimum and maximum, respectively. Bottom, power spectrum of PaO2 (red) and 95% confidence interval (CI, black) given by randomizing the phase of the PaO2 signal. The PaO2 power at the respiratory frequency (~2.5 Hz) is significantly greater than the 95% CI level. i PaO2 at Tmax and Tmin for the 15 recordings from the artery shown in (h). ** Wilcoxon signed-rank test, p < 0.01. j Normalized PaO2 at Tmin and Tmax for six vessels (three in cortex, and three in olfactory bulb, n = 5 mice) with statistically significant PaO2 power spectrum peaks at the respiratory frequency. * Wilcoxon signed-rank test, p = 0.0313. Data are shown as mean ± SEM in (b) and (d), and ± SD in all other graphs. Blue shaded region in (b) and (d) shows 95% CI of cross-correlation.

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