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Weak correlations between hemodynamic signals and ongoing neural activity during the resting state

An Author Correction to this article was published on 30 May 2022

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Abstract

Spontaneous fluctuations in hemodynamic signals in the absence of a task or overt stimulation are used to infer neural activity. We tested this coupling by simultaneously measuring neural activity and changes in cerebral blood volume (CBV) in the somatosensory cortex of awake, head-fixed mice during periods of true rest and during whisker stimulation and volitional whisking. We found that neurovascular coupling was similar across states and that large, spontaneous CBV changes in the absence of sensory input were driven by volitional whisker and body movements. Hemodynamic signals during periods of rest were weakly correlated with neural activity. Spontaneous fluctuations in CBV and vessel diameter persisted when local neural spiking and glutamatergic input were blocked, as well as during blockade of noradrenergic receptors, suggesting a non-neuronal origin for spontaneous CBV fluctuations. Spontaneous hemodynamic signals reflect a combination of behavior, local neural activity, and putatively non-neural processes.

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Fig. 1: Behavior drives CBV fluctuations.
Fig. 2: Neurovascular coupling was consistent across behavioral states.
Fig. 3: Predicting CBV changes from neural activity using HRFs.
Fig. 4: Variance of residual signal is similar across behavioral states.
Fig. 5: Spontaneous CBV fluctuations continue in the absence of neural activity and noradrenergic input.

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Acknowledgements

We thank L. Abbott, M. Adams, P. Blinder, D. Feldman, and N. Zhang for comments on the manuscript, and J. Berwick, D. Kleinfeld, and C. Mateo for discussions. This work was supported a Scholar Award from the McKnight Endowment Fund for Neuroscience, and grants R01NS078168, R01EB021703, and R01NS079737 from the NIH to P.J.D.

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A.T.W. and P.J.D. designed the experiments, A.T.W., C.E., and Q.Z. performed experiments and analyzed the data, A.T.W. and P.J.D. wrote the paper.

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Correspondence to Patrick J. Drew.

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Integrated Supplementary Information

Supplementary Figure 1 Details of the experimental model.

a: Photo of the experimental setup showing an awake, head-fixed mouse. The stereotrode and cannula are visible on the head. b: An example of the automated detection of whisker angle. Images of the whiskers at various times are indicated by colored boxes and arrows. The light blue box shows the whiskers at rest. The purple and orange boxes show the whiskers at various positions during volitional movement. c: An example of neural data during sensory stimulation (vertical gray bar) and the subsequent whisking (orange tick marks) showing wideband neural activity (0–10 kHz), multi-unit activity (300–3000 Hz), and local field potentials (0–150 Hz). Individual spikes (black tick marks, >5 standard deviations threshold shown with horizontal dashed lines) were detected in all animals. d: An example of the resting CBV signal (blue) compared to the reflectance obtained from a piece of clay placed over the cranial window during an experimental session (black). This shows the instrumentation noise is minuscule compared to the CBV signal. e: A comparison of the resting variance to the variance measured from the occluded cranial window which served as an illumination control surface. The variance of the instrumentation noise was 0.16% of the mean resting variance across all animals (n = 12). f: A comparison of the mean CBV amplitude changes following contralateral whisker stimulation between animals with only implanted electrodes (n = 12) or cannula and electrodes (n = 9) and animals with no implants (n = 3). The circles show individual animals. Bars show the mean of each group. The sensory-evoked CBV response was not affected by implantation of stereotrodes or cannulas (One-way ANOVA, n = {3,12,9}, p = 0.40, F(2,23) = 0.95). g: A reconstruction of the position and depth of implanted stereotrodes (colored circles) and the position of cannulas (black squares) for all animals. Stereotrode depths are designated by color. Scale bar = 1 mm. h: Whisking and CBV response to auditory stimuli for a representative animal. Auditory stimuli elicited volitional whisking behaviors, which then drove a hemodynamic response. Top: 28 examples of the detected whisker movements following auditory stimulation. Bottom: the average measured reflectance following auditory stimuli and compared to the response following volitional whisking without any stimulus. See Figure 1e, for population comparison of behavioral CBV variance. i: Averaged reflectance changes relative to baseline (top left) for a representative animal in response to a contralateral whisker stimulation (middle row) and during volitional whisking (bottom row). Brightness shows the mean normalized reflectance change from baseline. The light blue circular regions show the positions of individual macro-vibrissae barrel reconstructed from layer IV cytochrome oxidase staining. The locations of the implanted stereotrode (blue filled circle) and sensory regions are shown (top right). UJ: upper jaw, LJ: lower jaw, N: nose, FP: forepaw, FL: forelimb, HP: hindpaw, HL: hindlimb, TR: trunk, DZ: dysgranular zone. Scale bars = 1 mm.

Supplementary Figure 2 Heart-rate fluctuations are poorly correlated with sensory-evoked and spontaneous resting CBV changes.

a: Example of heart-rate fluctuations from a ~35-second-long period of rest. b: A scatterplot showing the root mean squared amplitude of heart rate fluctuations at rest for all animals (n = 12). c: The stimulus-triggered average heart rate among animals shows a small heart rate increase following contralateral (n = 9, mean±std:2.2±1.3%, blue, shaded region indicates standard deviation) and auditory (mean±std:0.4±1.6%, gray) stimulation. The peak heart rate for each animal (circles) is plotted to the right. Black circles indicate the mean of all animals. The heart rate increase following contralateral stimulation was not significantly larger than increases following an auditory stimulus (paired t-test, p = 0.92, t(8) = 0.11, n = 9). d: A comparison of the average stimulus-triggered reflectance changes driven by contralateral and auditory stimuli (solid blue and gray traces, respectively, shaded region shows the standard deviation). The contralateral puff elicited a larger CBV response than the auditory stimulus (blue, gray circles, paired t-test, p = 1×10−3, t(8) = −5.06). e: There was no significant linear relationship between CBV and heart rate increases following whisker and auditory stimuli in the data shown in c and d (n = 9, iteratively reweighted least squares, bisquare weighting, MATLAB function: robustfit, slope 0.002±0.002(mean±standard error), p = 0.35, t(7) = 0.99). Each animal is represented by a circle, fitted line shown as solid gray line, standard error is shown by shaded region and includes the zero slope. f: The power spectral density of the heart rate during periods of rest, averaged over all animals (n = 12). g: The mean cross-correlation between heart rate and CBV. Shaded region shows the standard deviation across animals (n = 12). The dashed red lines represent the 95% confidence interval based on reshuffled data (bootstrap, MATLAB function: bootci, 1000 resamples). h: A pooled scatter plot of the mean heart rate and root mean squared (R.M.S.) CBV during individual periods of rest (colored points) for all animals. Black line shows the linear fit using linear mixed-effects regression. There was a significant relationship between heart rate and the amplitude of the resting CBV variance (p = 0.004, t(532) = −2.9) but this relationship only explained 12.5% of differences in resting CBV variance (R 2 = 0.125).

Supplementary Figure 3 Neurovascular coupling in facial-nerve-transected animals.

a: A comparison of the mean stimulus triggered average CBV between animals with facial nerve transections (red, n = 3) and intact animals (black, n = 12) (left). Statistical comparison of the responses shows no significant difference between the response amplitudes (middle left, unpaired t-test, p = 0.52, t(13) = 0.66), full-width at half-max (middle right, unpaired t-test, Bonferroni corrected, p = 0.12, t(13) = 2.33), and the time to peak response (right, unpaired t-test, p = 0.24, t(13) = 1.24). Circles show results from individual animals. b: The mean cross-correlation between gamma-band neural power and ΔR/R during non-sensory evoked behavior for animals with facial nerve transections (red, n = 3) and intact animals (black, n = 12) (left). There were no significant difference in the maximum correlation coefficients between gamma-band power and normalized reflectance (middle left, unpaired t-test, p = 0.41, t(13) = −0.86), width of the cross correlation (middle right, unpaired t-test, p = 0.08, t(13) = −1.92), or the lag at maximum correlation (right, unpaired t-test, p = 0.81, t(13) = 0.24) compared between mice with intact facial nerves (black circles) and mice with transected facial nerves (red circles). c: same as b, but for the correlation between MUA and ∆R/R. (Unpaired t-test, Max correlation coefficient: p = 0.88, t(13) = −0.16; FWHM: p = 0.07, t(13) = −1.95; Time to peak: p = 0.31, t(13) = 1.05).

Supplementary Figure 4 Spontaneous cross-correlation between the LFP and reflectance in the barrel cortex in the absence of sensory stimulation.

Top: cross-correlation between MUA power and ∆R/R during all periods without whisker stimulation. The shaded region shows the population standard deviation (n = 12). Bottom: cross-correlation between ΔR/R and various frequency bands of the LFP. Color bar shows the magnitude of the correlations.

Supplementary Figure 5 Attributes of gamma-band power and MUA HRFs.

a: The amplitude, time to peak (TTP) and full-width at half-max (FWHM) of the gamma-band derived HRFs. Each circle represents the attribute from a single animal (n = 12) normalized by the sensory-evoked HRF attribute. Horizontal bars and shaded boxes show the median and interquartile range, respectively (amplitude comparison: Wilcoxon signed rank, Bonferroni corrected, stimulation vs. whisking: p = 0.64, z = −0.47, stimulation vs. rest: p = 0.06, z = −1.88. Time to peak comparison: paired t-test, stimulation vs. whisking: p = 0.23, t(11) = −1.28; stimulation vs. rest: p = 0.06, t(11) = 2.09. Full-width-half-maximum comparison: paired t-test, Bonferroni corrected, stimulation vs whisking: p = 0.11, t(11) = −1.74, Stimulation vs rest: p = 0.95, t(11) = −0.07). b: Same as a but for HRFs calculated from MUA power. (amplitude comparison: Wilcoxon signed rank, Bonferroni corrected, stimulation vs. whisking: p = 0.05, z = −2.27, stimulation vs. rest: p = 0.07, z = −2.12. Time to peak: Paired t-test, stimulation vs. whisking: p = 0.41, t(11) = −0.86; stimulation vs. rest: p = 0.80, t(11) = −0.26. Full-width-half-maximum comparison: paired t-test, stimulation vs. whisking: p = 0.97, t(11) = 0.04; stimulation vs. rest: p = 0.47, t(11) = 0.75). c: The attributes of the gamma-band derived HRF categorized according to stereotrode depth. Colored circles denote the depth of the implanted stereotrode obtained from histological reconstructions of each animal (Supragranular: n = 6, Granular: n = 3, Infragranular: n = 3). There was no detectable dependency of HRF attributes on the cortical depth of the recording site(HRF amplitude: Mann-Whitney U Test, Supragranular vs. granular: p = 0.07, z = 1.83, supragranular vs. infragranular: p = 0.74, z = 0.33, granular vs. infragranular: p = 0.30, z = −1.04; HRF time to peak: unpaired t-test, supragranular vs. granular: p = 0.81, t(16) = −0.38, supragranular vs. infragranular: p = 0.53, t(16) = −0.64, granular vs. infragranular: p = 0.51, t(10) = 0.69; HRF FWHM: Mann-Whitney U-test, supragranular vs. granular: p = 0.21, z = −1.26, supragranular vs. infragranular: p = 0.15, z = 1.45; granular vs. infragranular: p = 0.13, z = −1.52). d: Same as c, but for MUA derived HRF attributes (HRF amplitude: Mann-Whitney U-test, supragranular vs. granular: p = 0.81, z = 0.23, supragranular vs. infragranular: p = 0.21, z = 1.26, granular vs. infragranular: p = 0.30, z = −1.04; HRF TTP: unpaired t-test, supragranular vs. granular: p = 0.39, t(16) = −0.87, supragranular vs. infragranular: p = 0.39, t(16) = −0.88, granular vs. infragranular: p = 0.10, t(10) = −1.83; HRF FWHM: Mann-Whitney U-test, supragranular vs. granular: p = 0.07, z = 1.83; supragranular vs. infragranular: p = 0.07, z = 1.83, granular vs. infragranular: p = 0.30, z = 1.04).

Supplementary Figure 6 HRFs calculated from lower frequencies of the LFP.

a: Mean stimulus (top) and whisking (bottom) triggered neural power in the LFP at frequencies below 5 Hz (n = 12). b: A comparison of the mean HRFs derived from neural power in the 10–30 Hz band. Each HRF was calculated from a single behavioral state: stimulation (dark blue), volitional whisking (orange), and rest (light blue) and averaged across animals (n = 12). c: Same as b, but calculated from neural power in the 0.1–8 Hz band. d: Population summary of the goodness of fit (R2) of the CBV predictions based on neural power in the 10–30 Hz band. Circles represent median R2 for each animal. R2 calculated on predictions of the average behavior-triggered CBV are outlined in black, filled black circles show the population medians (Rest: R2 = 0.015). Circles without black outlines show medians of predictions from individual trials. e: Same as d, but for HRFs calculated from neural power in the 0.1–8 Hz. The median R2 of predictions using the 0.1–8 Hz band of the LFP of resting CBV was 0.018.

Supplementary Figure 7 Additional analyses of the CBV signal.

a: Population summary of the correlation between gamma-band power derived CBV predictions and measured CBV, separated according to behavior. Circles represent median correlation coefficient (R) for each animal (n = 12). R values calculated using predictions of the average behavior-triggered CBV are outlined in black (Friedman test, p = 1×10−4, χ2(2,22)  =  18.5. Post-hoc: Wilcoxon signed rank with Bonferroni correction, sensory evoked vs. extended movement: p = 0.18, z = 1.88; sensory evoked vs volitional whisking: p = 6×10−3, z = 3.06; extended movement vs. volitional whisking: p = 0.08, z = 2.20). The other colored circles indicate the median R values calculated for predictions of CBV during individual periods of behavior, (Two-way ANOVA, p = 9×10−12, F(3,11) = 45.01. Post-hoc: paired t-test with Bonferroni correction. sensory evoked vs. extended movement: p = 0.01, t(11) = 3.81; sensory evoked vs. volitional whisking: p = 6×10−5, t(11) = 7.15, sensory evoked vs rest: p = 6×10−7, t(11) = 11.40, extended movement vs. volitional whisking: p = 1×10−3, t(11) = 5.11, volitional whisking vs. rest: p = 0.13, t(11) = 2.29, extended movement vs rest: p = 1×10−4, t(11) = 6.46). The filled black circles indicate the population medians. b: Same as in a, but corresponding to MUA-derived HRFs (Averaged CBV: Friedman Test, p = 0.04, χ2(2,22) = 6.5, Post-hoc: Wilcoxon signed-rank with Bonferroni correction, sensory-evoked vs. extended movement: p = 0.08, z = 2.19, sensory-evoked vs. volitional whisk: p = 0.12, z = 2.04, volitional whisk vs. rest: p = 0.4, t(11) = −0.86; Individual CBV: Friedman test, p = 4×10−3, χ2(3,33) = 13.2, Post-hoc: Wilcoxon signed rank with Bonferroni correction, sensory evoked vs. extended movement: p = 0.05, z = 2.67, sensory evoked vs. volitional whisk: p = 0.05, z = 2.59, sensory evoked vs. rest: p = 0.09, z = 2.43, extended movement vs. whisk: p = 0.39, z = −0.86, extended movement vs. rest: p = 0.81, z = −0.23, volitional whisk vs. rest: p = 0.53, z = 0.63). Inset waveforms show examples of average sensory-evoked predictions (gray) with high and low correlations to the measured CBV (black). c: Example power spectra of the measured (black) and predicted (blue) CBV over all trials for a single animal. d: The mean power of the resting CBV prediction residuals (using the gamma-band HRF) across all animals (n = 12, black line). For comparison, the mean resting power spectrum following infusion of muscimol was also plotted (n = 9, blue line) was also plotted (see right axis for scale). Shaded regions indicate the population standard deviations. e: Subtraction of the global CBV signal did not substantially improve the gamma-band (paired t-test, p = 0.39, t(11) = 0.90) or MUA derived (Paired t-test, p = 0.03, t(11) = 2.87) prediction of CBV at rest. Colored circles denote single animals (n = 12). Black-filled circles indicate the mean. f: A 2-dimensional histogram of correlation coefficients between the gamma-band power derived predictions and the measured CBV during periods of spontaneous behavior, pooled across all animals, and the normalized CBV variance from the same period. Black line shows the best fit line (Linear mixed-effects regression, p = 1.44×10−8, t(3420) = 5.68) g: same as f, but for MUA derived CBV predictions (Linear mixed-effects regression, p = 0.17, t(3420) = 1.35). h: Slopes from f and g plotted for each animal. Slopes which were significantly different from zero (p<0.05) are black, all others are gray. The average MUA response to whisking was compared between animals with significant and not significant slopes showing differences in the whisking triggered MUA between the groups (inset). i: The changes in sensory-evoked summed MUA power and CBV (circles). Circles and bars outside the axes show the mean and standard deviation of each measure. We observed an 86±3% reduction in MUA standard deviation compared to aCSF controls and an 87±2% reduction following muscimol+CNQX+AP5 infusions, sensory evoked CBV were reduced 23±55% and 44±9% respectively. The reduction in CBV response to sensory stimulation was not significantly different between muscimol and muscimol+CNQX+AP5 (Welch’s t-test, p = 0.15, t(4.5) = 1.13). j: Same as for i, except for measured of summed gamma-band power following stimulation. Sensory-evoked gamma-band power was reduced 69±19% following infusion of muscimol and 82±8% following infusion of muscimol+CNQX+AP5 (unpaired t-test, one-sided, p = 0.12, t(11) = 1.24).

Supplementary Figure 8 Examples of the effects of pharmacological infusions on sensory-evoked and resting responses in individual animals.

a: Top left: image of a thinned-skull window showing the locations of the CBV ROI (yellow shaded region), implanted cannula, and stereotrode scale bar  =  1 mm. Top center: spatial map of the difference between the average aCSF and muscimol CBV responses to a contralateral whisker stimulus 0.5 to 2 seconds after stimulation. Top right: the spatial map of the difference between the standard deviation of CBV at rest following aCSF and muscimol infusions. Bottom left: the average sensory evoked MUA power for the example animal compared between aCSF (black) and muscimol (blue) infusions (bottom left). Bottom right: two examples of the CBV measured during single periods of rest following aCSF (black) and muscimol (blue) infusions (bottom right). b,c: Same as a, but for an animal infused with muscimol+CNQX+AP5 and the muscimol+adrenergic blockers, respectively.

Supplementary Figure 9 Changes in sensory-evoked neural activity following infusion.

a: Example data from two representative trials following aCSF (left panel) and muscimol (right panel) infusions. Movement of the whiskers (orange tick marks) were detected from the whisker angle (top), movement of the body (gray tick marks) and stimulation times (blue triangles) were also tracked. Normalized reflectance changes were averaged over a region of interest within the thinned skull window (second from top). Gamma-band power (third from top) and MUA (bottom) following infusion. b,c: Same as a, but from different animals following muscimol+CNQX+AP5 and muscimol+adrenergic blocker infusions, respectively.

Supplementary Figure 10 CBV fluctuations after muscimol infusions do not propagate from nearby regions.

a: Mean (solid line) and standard deviation (shaded region) of cross-correlograms between the reflectance within the region of interest and the reflectance from the rest of the window (n = 6, three animals omitted which had windows that were spanned by the ROI). b: Comparison of the correlation coefficients between aCSF and muscimol infusions. The correlations were not significantly different (paired t-test, p = 0.12, t(5) = −1.9). c: Comparison of the lags at maximum correlation between aCSF and muscimol infusions. The lags were not significantly different (paired t-test, p = 0.3, t(5) = −1.17).

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Supplementary Figures 1–10

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Supplementary Video 1

Example CBV and neural fluctuations in the context of animal behavior. The normalized reflectance (ΔR/R), measured from a thinned skull window, is shown during rest and behavior. Note that decreased reflectance corresponds to increased CBV. Images of the whiskers are provided for behavioral context. An image of the thinned skull window with the location of the stereotrode (dark blue circle) and whisker barrels (light blue shading) are also given. A time-frequency spectrogram of the measured local field potential, MUA and the detected whisker angle are plotted. The sliding black bar indicates the trial time for each CBV and whisker image.

Supplementary Video 2

Example CBV and neural fluctuations during rest following infusion of aCSF. The normalized reflectance (ΔR/R), measured from a thinned skull window, is shown during rest. Note that decreased reflectance corresponds to increased CBV. Images of the whiskers are provided for behavioral context. An image of the thinned skull window with the location of the stereotrode, cannula and the region of muscimol effect (shaded yellow, compare to Movie 3, see Methods) are also given. A time-frequency spectrogram of the measured local field potential, MUA and the detected whisker angle are plotted. The sliding black bar indicates the time of each CBV and whisker image.

Supplementary Video 3

Example CBV and neural fluctuations during rest following infusion of muscimol. An example of the normalized reflectance (ΔR/R) measured from a thinned skull window during rest. Note that decreased reflectance corresponds to increased CBV. Images of the whiskers are provided for behavioral context. An image of the thinned skull window with the location of the stereotrode, cannula and the region of muscimol effect (shaded yellow, see Methods) are also given. A time-frequency spectrogram of the measured local field potential, MUA and the detected angle are plotted. The sliding black bar indicates the time of each CBV and whisker image.

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Winder, A.T., Echagarruga, C., Zhang, Q. et al. Weak correlations between hemodynamic signals and ongoing neural activity during the resting state. Nat Neurosci 20, 1761–1769 (2017). https://doi.org/10.1038/s41593-017-0007-y

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