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Distal activity patterns shape the spatial specificity of neurovascular coupling

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Abstract

Neurovascular coupling links brain activity to local changes in blood flow, forming the basis for non-invasive brain mapping. Using multiscale imaging, we investigated how vascular activity spatially relates to neuronal activity elicited by single whiskers across different columns and layers of mouse cortex. Here we show that mesoscopic hemodynamic signals quantitatively reflect neuronal activity across space but are composed of a highly heterogeneous pattern of responses across individual vessel segments that is poorly predicted by local neuronal activity. Rather, this heterogeneity is dependent on vessel directionality, specifically in thalamocortical input layer 4, where capillaries respond preferentially to neuronal activity patterns along their downstream perfusion domain. Thus, capillaries fine-tune blood flow based on distant activity and encode laminar-specific activity patterns. These findings imply that vascular anatomy sets a resolution limit on functional imaging signals, where individual blood vessels inaccurately report neuronal activity in their immediate vicinity but, instead, integrate activity patterns along the vascular arbor.

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Fig. 1: Changes in HbT are spatially well correlated to neuronal activity across cortical columns in mouse barrel cortex.
Fig. 2: The selectivity of vessel dilations is highly heterogenous and poorly reflects local excitatory activity in L2/3 and L4.
Fig. 3: Vessel response selectivity does not increase with capillary branch order.
Fig. 4: Vascular perfusion path directionality, but not vessel position per se, shapes vessel dilation selectivity in L4.
Fig. 5: Unlike neurons, vessels show a preference toward activity in the nearest of two diametrically opposite barrels in L4.
Fig. 6: Neuronal activity is more spatially confined than HbT in awake mice.
Fig. 7: Despite a general increase in selectivity, vessel response selectivity in L2/3 remains spatially heterogenous in awake mice.

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Data availability

The dataset necessary to interpret, verify and extend the results presented in this paper is available at https://doi.org/10.6084/m9.figshare.26121076 (ref. 79). The standardized ROI masks delineating the subregions of S1 and the individual barrels are included in this dataset.

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Acknowledgements

This work was supported by a Natural Sciences and Engineering Research Council of Canada discovery grant (RGPIN-2020-05276), a Canadian Institutes of Health Research project grant (no. 455513) and an Azrieli Future Leader in Canadian Brain Research grant from the Brain Canada Foundation through the Canada Brain Research Fund, with the financial support of Health Canada and the Azrieli Foundation to R.L.R. and an ERA-NET NEURON (JTC2022) grant with financial support from Fonds de Recherche du Québec. R.L.R. holds a Canada Research Chair in Neurovascular Interactions. A.M. was supported by a Mitacs Accelerate Studentship (IT28768) and a Bourse de Mérite from the Faculty of Medicine. We thank V. Linder (Maine Medical Center) for PDGFRβ-Cre mice; I. Laplante, P. Kwemo and L. Zana for colony and laboratory management; M. Abran and S. Bélanger for technical assistance; and P. Rompré for statistical advice. Schematics in Figs. 1a, 2a, 5a and 6a were created with BioRender.

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Authors and Affiliations

  1. Centre for Interdisciplinary Research on Brain and Learning (CIRCA), Université de Montréal, Montréal, Quebec, Canada

    Éric Martineau, Antoine Malescot, Nouha Elmkinssi & Ravi L. Rungta

  2. Department of Physiology and Pharmacology, Université de Montréal, Montréal, Quebec, Canada

    Éric Martineau & Antoine Malescot

  3. Department of Neuroscience, Université de Montréal, Montréal, Quebec, Canada

    Nouha Elmkinssi & Ravi L. Rungta

  4. Department of Stomatology, Faculty of Dental Medicine, Université de Montréal, Montréal, Quebec, Canada

    Ravi L. Rungta

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  1. Éric Martineau
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Contributions

É.M. and R.L.R. designed the study. É.M., N.E. and R.L.R. performed experiments. É.M. and A.M. developed analysis procedures, analyzed data and interpreted results, with R.L.R. É.M. and R.L.R. wrote the paper, with feedback from A.M. R.L.R. supervised the research. All authors agreed on the final version of the paper.

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Correspondence to Ravi L. Rungta.

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Nature Neuroscience thanks Tzu-Hao Chao, Anna Devor, Anusha Mishra and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Examples of vessel dilations poorly reflecting the selectivity of neighboring excitatory neurons.

(a) Example image of average jRGECO1a fluorescence in response to the stimulation of the associated whisker (green) superimposed onto an image of the vasculature (magenta), measured in layer 2/3 from a thy1-jRGECO1a-expressing mouse. Vessels are labelled by retro-orbitally injecting Alexa-680 Dextran (2000 kDa). (b) Diameter changes of each vessel segment and (c) average changes in jRGECO1a fluorescence (ΔF/F0) from neurons surrounding these vessels following the stimulation of the associated whisker (blue) or its neighbor (orange). (d) Example images of average GCaMP6f fluorescence (green) in response to the stimulation of the associated whisker (left) or its neighbor (right) superimposed onto an image of the vasculature (red), measured in layer 4 from a Scnn1a-Tg3-Cre::GCaMP6f mouse. Vessels are labelled by retro-orbitally injecting TexasRed Dextran (70 kDa). (e) Diameter changes of each vessel segment and (f) average change in GCaMP6f fluorescence from neurons surrounding these vessels (neuron #1–5) following the stimulation of the associated whisker (blue) or its neighbor (orange). Scale bars = 20 µm (a) and 10 µm (d). Signals were extracted by manually drawing ROIs over the neurons and without neuropil subtraction.

Extended Data Fig. 2 A greater proportion of neurons in the L4 barrel are selective for their associated whisker than in L2/3.

(a-b) False-color images of GCaMP6f fluorescence during the stimulation of the C2 (a) or D2 (b) whisker measured in the C2 barrel in layer 4 of a Scnn1a-Tg3-Cre::GCaMP6f mouse (average of 9 and 11 trials respectively). Dashed lines represent the boundary of the C2 L4 barrel. Fluorescence changes on the right side of the barrel are obscured by surface vessels. (c) Image illustrating the selectivity of neuronal cell bodies within the imaged barrel, segmented with the assistance of suite2P. (d) Average change in GCaMP6f fluorescence (ΔF/F0), after neuropil subtraction, from example neuronal somas following the stimulation of the associated whisker (C2, blue) or its neighbor (D2, orange). A 200 ms moving average was applied to the traces for representation. SI are calculated on unfiltered signals. (e) Global distribution of neuronal selectivity indexes in the barrel ROI in layer 2/3 (left) and layer 4 (right) showing that roughly twice as many neuronal somas were selective for their associated whisker in L4 compared with L2/3 in sedated mice (L2/3: N = 6 mice (4M, 2F), n = 6 recordings, 113 excited neurons out of 251 segmented; L4: N = 10 mice (5M, 5F), n = 12 recordings, 299 excited neurons out of 477 segmented). (f) Percentage of selective neuronal somas (SI > 0.1) in L2/3 and L4, calculated per recording (p = 0.0415; L2/3: n = 6 vs L4: n = 12; Two-tailed Mann-Whitney test). (g) Selectivity of the neuropil signal, extracted by removing the neuronal ROIs from the barrel ROI, in layer 2/3 and layer 4 (p = 0.1675; L2/3: n = 6 vs L4: n = 12; Two-tailed Student’s T-test). Scale bars = 50 µm (a-b). (f-g) *: p < 0.05, ns: non-significant. Error bars (f-g) represent the SEM.

Extended Data Fig. 3 Arteriole dilation onset tends to be faster in L4 than in L2/3.

(a) Normalized arteriole diameter changes in L2/3 and L4 following a 5 Hz 4 second stimulation of the whisker associated with its barrel (L2/3: N = 10 mice (5M, 5F); n = 11 responsive arterioles; L4: N = 9 mice (4M, 5F), n = 14 responsive arterioles). Two responsive arterioles in L2/3 and 1 responsive arteriole in L4 were excluded from this analysis as the fit was too uncertain to accurately calculate an onset. (b-c) Onsets were calculated by fitting a sigmoid to the rise of each response and calculating the time to 10% (b, p = 0.0745, Two-tailed Student’s t-test) and 25% (c, p = 0.106, Two-tailed Student’s t-test) of the peak, as previously described33. Shaded areas (a) and error bars (b-c) represent the SEM. All mice were under dexmedetomidine sedation.

Extended Data Fig. 4 Vessel dilation selectivity across capillary branches in L2/3.

(a) Raster plots of diameter changes of responding vessels following the stimulation of the associated (left) or neighboring (right) whisker in L2/3, split by branch order and sorted by ascending selectivity index. Dashed lines represent stimulation onset and offset. (b) Average vessel diameter changes of different branch orders following the stimulation of the associated (blue) or neighboring (orange) whisker. (c) Vessel dilation selectivity is independent of branch order in L2/3. (d) Correlation between vessel dilation selectivity index and cortical depth. (a-d; N = 13 mice (8M, 5F), arterioles: n = 13/15, 1st order: n = 12/20, 2nd / 3rd order: n = 48/61, 4th / 5th order: n = 29/49, 6th - 8th order: n = 7/11). Shaded areas (b) and error bars (c) represent the SEM. All mice were under dexmedetomidine sedation.

Extended Data Fig. 5 Vessel dilation selectivity across capillary branches in L4.

(a) Raster plots of diameter changes of responding vessels following the stimulation of the associated (left) or neighboring (right) whisker from Scnn1a-Tg3-Cre::GCaMP6f (L4) mice, split by branch order and sorted by ascending selectivity index. Dashed lines represent stimulation onset and offset. (b) Average vessel diameter changes of different branch orders following the stimulation of the associated (blue) or neighboring (orange) whisker. (c) Vessel dilation selectivity is independent of branch order in L4. (d) Correlation between vessel dilation selectivity index and cortical depth. (a-d; N = 10 mice (5M, 5F), arterioles: n = 15/15, 1st order: n = 11/14, 2nd / 3rd order: n = 37/53, 4th / 5th order: n = 39/70, 6th - 8th order: n = 16/36). Shaded areas (b) and error bars (c) represent the SEM. All mice were under dexmedetomidine sedation.

Extended Data Fig. 6 RBC velocity and flow selectivity across branch order and cortical depth.

(a) Example image of average jRGECO1a fluorescence in response to the stimulation of the associated whisker (green) superimposed onto an image of the vasculature (magenta), measured in L2/3. (b) Example image of a linescan (distance/time) within a capillary (left) and average increase in RBC velocity and flow in the capillary (right) following the stimulation of the associated (blue) or neighboring (orange) whisker. (c-d) Average change in RBC velocity (c) and flow (d), across different branch orders (N = 16 mice, (10M, 6F)), following the stimulation of the associated (blue) or neighboring (orange) whisker. (e-f) Correlation between cortical depth and selectivity indexes for increases in RBC velocity (e) or RBC flow (f). Of note, cortical depth had little impact on the selectivity of RBC dynamics, with only increases in RBC velocity being very slightly more selective with depth in higher order capillaries (RBC Velocity: R2adj = 0.08875, Overall regression: p = 0.0204, Depth: p = 0.2007, BranchOrder: p = 0.0154, Depth*BranchOrder: p = 0.0389; RBC Flow: Adjusted R2adj = -0.01495, Overall regression: p = 0.4905, Depth: p = 0.1407, BranchOrder: p = 0.1621, Depth*BranchOrder: p = 0.1463;MLR). Shaded areas (c-d) represent the SEM. Results are pooled from thy1-jRGECO1a-expressing (L2/3) and Scnn1a-Tg3-Cre::GCaMP6f (L4) mice. The numbers of vessels and mice in each group is identical to those in Fig. 3g, h. All mice were under dexmedetomidine sedation.

Extended Data Fig. 7 2D-vessel position relative to the associated and neighboring whisker representation centroid.

(a) Schematic representation of the formula used to calculate the normalized axial position on the C2-D2 axis and the relative proximity of each vessel to the barrel column’s centroid. (b-c) Normalized axial position (b) and relative proximity index (c) values for five hypothetical vessel positions on the cortical surface.

Extended Data Fig. 8 Vessels that were poorly tuned to their associated whisker have a preference towards activity in one of two diametrically opposite barrels in L4.

(a) Example image highlighting two neighboring capillary networks emerging from the same penetrating arteriole, but perfusing spatial domains in different directions. These networks were recorded over two imaging sessions while stimulating a different whisker pair. (b-c) Dilation measurements from each network showing that (b) vessels along network 1 become gradually less selective towards the associated whisker (C2), and more selective to D2, while (c) vessels along network 2 become more selective to C2, and less responsive to either neighbor of C2. (d) Changes in vessel selectivity depending on the stimulated whisker pair (N = 6 mice (3M, 3F), arterioles: n = 7, 1st order: n = 7, 2nd/3rd order: n = 25, 4th/5th order: n = 32, 6th-8th order: n = 12). (e-f) Results in (d) split between vessels that were non-selective (e, N = 6 mice, arterioles: n = 5, 1st order: n = 5, 2nd/3rd order: n = 14, 4th/5th order: n = 22, 6th-8th order: n = 6) or selective (f, N = 6 mice, arterioles: n = 2, 1st order: n = 2, 2nd/3rd order: n = 11, 4th/5th order: n = 10, 6th-8th order: n = 6) toward the associated whisker, showing that vessels with poor selectivity towards the associated whisker were more selective towards one of the two neighbors and vice versa (e-f, StimulatedWhiskerPair: p = 0.0002 and p = 0.0087 respectively, RM-Two-way ANOVA). Non-responsive vessels when stimulating either neighboring whisker were assigned a value of 0. The shaded area in (d) represents the cut-off value (0.2268) for splitting selective and non-selective vessels, corresponding to the mean selectivity index when stimulating the associated-neighbor1 pair (in absolute value). Absolute values were used for this analysis in order to quantify if selectivity improved or deteriorated in each subgroup when stimulating both neighbors. Shaded areas (b-c) represent the stimulation. (e-f) * p < 0.05, Sidak’s multiple comparisons. Refer to Supplementary Tables 38 and 39 for exact p-values. Scale bars = 50 µm.

Extended Data Fig. 9 In awake mice, both neuronal activity and [HbT] elevations are more spatially specific in the late-phase of the response.

(a) jRGECO1a responses during the early and late phase (red shaded areas, AUCt = 4:5 and AUCt = 7:8, respectively) were extracted and compared to (b) changes in [HbT] during the matching phase in awake mice. An offset of +0.479 seconds, was applied to each phase for the [HbT] signal to account for the delay in NVC. This offset was determined by calculating a transfer function between the [HbT] and jRGECO1a signals33,79. (c) Spatial tuning of the neuronal and [HbT] response in awake mice for each phase, showing both neuronal and [HbT] responses are more spatially specific in the late phase of the response (N = 6 mice (3M, 3F); Associated barrel: n = 19 barrels, 1-away: n = 26 barrels, 2-away: n = 14 barrels). (d) The AUC of vessel dilations from two-photon experiments in L2/3 of awake mice during the early and late phase (AUCt = 4.479: 5.479 and AUCt = 7.479: 8.479, respectively) were extracted and compared. (e-g) Dilation selectivity during the early (e) and late phase (f) of the response in awake mice, grouped by branch order (g, N = 5 mice (2M, 3F), arterioles: n = 11/12, 1st order: n = 12/14, 2nd / 3rd order: n = 43/57, 4th / 5th order: n = 25/50, 6th order: n = 1/3 for each phase, RM-Two-way ANOVA with Tukey’s multiple comparisons). (h-i) Changes in dilation selectivity in the early (h) and late (i) phase, as a function of vascular path directionality. Traces in (a, b and d) are identical to those presented in Fig. 6c, d, and Fig. 7c, and serve only to represent the phases used for this analysis. Shaded traces (a,b and d) and error bars (c, e and f) represent the SEM. The single dilating 6th order vessel (e-g) was not included in the analysis. (g) * p < 0.05, ** p < 0.01, **** p < 0.0001 Tukey’s multiple comparisons. Refer to Supplementary Table 41 for exact p-values.

Extended Data Fig. 10 Spatial diversity of L2/3 neuronal responses is maintained in awake mice.

(a, b) Images illustrating the selectivity of neuronal cell bodies in L2/3 within the imaged barrel, segmented with the assistance of suite2P, in sedated (a) and awake (b) thy1-jRGECO1a-expressing mice. (c) Global distribution of neuronal selectivity indexes in the barrel ROI in layer 2/3 in awake mice (L2/3: N = 5 mice (2M, 3F), n = 9 recordings, 293 excited neurons out of 588 segmented). Of note, only recordings from the barrel in the center of the image were included, as the majority of this barrel was visible for segmentation (9 out 12 total recordings). (d) Percentage of selective neuronal somas (SI > 0.1) in awake and sedated mice, calculated per recording, showing that a similar proportion of cells in the L2/3 are selective for their associated whisker (p = 0.8639; Sedated: n = 6 vs Awake: n = 9; Two-tailed Mann-Whitney test). (e) Selectivity of the neuropil signal, extracted by subtracting the neuronal ROIs from the barrel ROI, in awake and sedated mice (p = 0.0169; Sedated: n = 6 vs Awake: n = 9; Two-tailed Student’s T-test). Error bars (d-e) represent the SEM. Scale bars = 50 µm (a-b).

Supplementary information

Supplementary Information

Legends for Supplementary Videos 1–3 and Tables 1–44.

Reporting Summary

Supplementary Video 1

Neuronal activity in L2/3 during C2 or D2 whisker stimulation in sedated mice. Imaging of jRGECO1a expressed in L2/3 excitatory neurons during the stimulation of the C2 (top left, bottom: blue) or D2 (top right, bottom: yellow) whiskers. Whiskers are stimulated from 0 s to 4 s. Images represent the average of seven C2 stimulations and 13 D2 stimulations. Between-frame movement was corrected by applying a rigid alignment algorithm (Methods) on simultaneously acquired images of the vasculature (Alexa 680–dextran) and then applying the same translation vectors to the images from the neuronal channel. Same recording as the example in Fig. 2b. The ROI used to extract signal from the C2 barrel is traced in white.

Supplementary Video 2

Neuronal activity in L2/3 during C2 or D2 whisker stimulation in sedated mice. Imaging of GCaMP6f expressed in L4 neurons during the stimulation of the C2 (top left, bottom: green) or D2 (top right, bottom: magenta) whiskers. Whiskers are stimulated from 0 s to 4 s. Images represent the average of 11 C2 stimulations and nine D2 stimulations. Between-frame movement was corrected by applying a rigid alignment algorithm (Methods) on simultaneously acquired images of the vasculature (Texas Red–dextran) and then applying the same translation vectors to the images from the neuronal channel. Same recording as the example in Fig. 2g. The ROIs used to extract signal from the C2 and D2 barrels are traced in white.

Supplementary Video 3

Neuronal activity in L2/3 during C2 or D2 whisker stimulation in awake mice. Imaging of jRGECO1a expressed in L2/3 excitatory neurons during the stimulation of the C2 (top left, bottom: blue) or D2 (top right, bottom: yellow) whiskers. Whiskers are stimulated from 0 s to 4 s. Images represent the average of 10 C2 stimulations and 10 D2 stimulations. Between-frame movement was corrected by applying a rigid alignment algorithm (Methods) on simultaneously acquired images of the vasculature (Alexa 680–dextran) and then applying the same translation vectors to the images from the neuronal channel. Same recording as the example in Fig. 7a. The ROIs used to extract signal from the C2 and D2 barrels are traced in white.

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Martineau, É., Malescot, A., Elmkinssi, N. et al. Distal activity patterns shape the spatial specificity of neurovascular coupling. Nat Neurosci (2024). https://doi.org/10.1038/s41593-024-01756-7

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