Cortical depth-specific microvascular dilation underlies laminar differences in blood oxygenation level-dependent functional MRI signal Peifang Tiana,1, Ivan C. Tenga, Larry D. Mayb, Ronald Kurzb, Kun Lub, Miriam Scadengb, Elizabeth M. C. Hillmanc, Alex J. De Crespignyc, Helen E. D’Arceuilc, Joseph B. Mandevillec, John J. A. Marotac, Bruce R. Rosenc, Thomas T. Liub, David A. Boasc, Richard B. Buxtonb, Anders M. Dalea,b, and Anna Devora,b,c,2 Departments of aNeurosciences and bRadiology, University of California at San Diego, La Jolla, CA 92093; and cMartinos Center for Biomedical Imaging, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129 Edited by Nikos K. Logothetis, Max Planck Institute for Biological Cybernetics, Tübingen, Germany, and approved July 15, 2010 (received for review May 13, 2010)
Changes in neuronal activity are accompanied by the release of vasoactive mediators that cause microscopic dilation and constriction of the cerebral microvasculature and are manifested in macroscopic blood oxygenation level-dependent (BOLD) functional MRI (fMRI) signals. We used two-photon microscopy to measure the diameters of single arterioles and capillaries at different depths within the rat primary somatosensory cortex. These measurements were compared with cortical depth-resolved fMRI signal changes. Our microscopic results demonstrate a spatial gradient of dilation onset and peak times consistent with “upstream” propagation of vasodilation toward the cortical surface along the diving arterioles and “downstream” propagation into local capillary beds. The observed BOLD response exhibited the fastest onset in deep layers, and the “initial dip” was most pronounced in layer I. The present results indicate that both the onset of the BOLD response and the initial dip depend on cortical depth and can be explained, at least in part, by the spatial gradient of delays in microvascular dilation, the fastest response being in the deep layers and the most delayed response in the capillary bed of layer I. blood flow
| cortical layer | hemodynamic | imaging | somatosensory
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euroglial activation is accompanied by release of vasoactive mediators that dilate and constrict the surrounding arterioles (1, 2) and capillaries (3, 4). These changes in diameter in turn lead to changes in blood flow throughout the vascular matrix and can be detected on the macroscopic level as a positive blood oxygenation level-dependent (BOLD) functional MRI (fMRI) signal when blood flow response exceeds oxygen consumption (5–7). Under the assumption of local neurovascular coupling, the onset of the changes in diameter is determined by the following three factors, any of which may differ as a function of the cortical depth and branching order within the vascular tree: (i) the onset and peak time of the neuronal activity evoking the response; (ii) the time needed to release a vascular messenger [e.g., prostaglandin or NO (8)]; and (iii) the time needed for the target vessel to respond. However, in addition to local neurovascular coupling, vascular responses can propagate within the arteriolar/capillary networks (3, 9, 10). Indeed, propagation of dilation and constriction has been observed on the cortical surface (11–15), in excised cerebral vessels, and in noncerebral preparations (16, 17). Previous studies with single-vessel resolution in vivo have been limited to the cortical surface, but recent improvements in twophoton microscopy technology allow direct imaging of singlevessel diameters and flow velocities within a 3D geometry of vascular trees (1, 2, 18, 19). In the present study, we used this technology to examine microvascular responses to sensory stimulation down to 550 μm below the cortical surface in the rat primary somatosensory cortex (SI). We then compared the results with highresolution BOLD fMRI to investigate the extent to which laminar BOLD profiles reflected the underlying microvascular dynamics. 15246–15251 | PNAS | August 24, 2010 | vol. 107 | no. 34
Specifically, we focus on the following questions. (i) What is the location of the fastest dilation onset within the 3D branching arteriolar/capillary tree? (ii) Can we observe a gradient of microvascular onset and peak times as a function of the cortical depth or branching order? (iii) What features of the cortical depth-resolved BOLD response can be explained by the microscopic vascular measures? Results Spatial Gradient of Dilation Onset and Peak Times Along the Trunks of Diving Arterioles Suggests Propagation Toward the Pial Surface.
We used two-photon microscopy in combination with intravascular injection of a fluorescent contrast agent (fluorescein-dextran) to follow 90 diving arterioles down to 550 μm below the cortical (pial) surface in the rat SI. All measurements were acquired within ∼1.5 mm from the center of the neuronal response, mapped before two-photon imaging using surface potential recordings (1, 2). First, we address the issue of propagation of dilation along main diving arterioles toward the surface. Fig. 1A shows a typical example of the cortical microvasculature. In the expanded view on the right, surface arterioles (red) dive down (red arrows) to supply oxygen and glucose to the capillary bed. Surfacing venules (blue arrows) bring the deoxygenated blood from the capillary bed to draining surface venules (blue). For every arteriole, diameter change in response to a forepaw stimulus (2 s, 3 Hz, ∼1 mA) was measured at multiple depths along the diving trunks and their lateral branches. The majority of diving arterioles and their branches (82%) exhibited 5– 25% dilation, with no significant dependence on the depth (Fig. S1). An example of a set of measurements acquired along an individual arteriolar trunk, including the parent surface arteriole measured close to the diving point, is shown in Fig. 1B. The dilation time-courses measured at different depths were normalized by the peak amplitude (time-courses without normalization are shown in Fig. S1). Trunk measurements for different depth categories, averaged across all measured arterioles (population-averaged), are overlaid in Fig. 1C and Fig. S1A. The number of different vessels in each category is listed in Table S1. We found that the delay in
Author contributions: B.R.R., T.T.L., D.A.B., R.B.B., A.M.D., and A.D. designed research; P.T., I.C.T., E.M.C.H., A.J.D.C., H.E.D., J.B.M., J.J.A.M., and A.D. performed research; L.D.M., R.K., K.L., and M.S. contributed new reagents/analytic tools; P.T., I.C.T., and A.D. analyzed data; and P.T., A.M.D., and A.D. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Freely available online through the PNAS open access option. 1
Present address: Department of Physics, John Carroll University, University Heights, OH 44118.
2
To whom correspondence should be addressed. E-mail:
[email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1006735107/-/DCSupplemental.
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NEUROSCIENCE
Fig. 1. Relative timing of dilation response along diving trunks. (A) (Left) Image calculated as a maximum intensity projection (MIP) of an image stack 0–300 μm in depth using a 4× objective. Individual images were acquired every 10 μm. (Right) Detailed view of the region within the white square on the left, calculated as a MIP of a stack 0–400 μm in depth acquired with 2-μm resolution. (B) An example of a set of temporal diameter change profiles acquired from an individual arteriolar tree. Each curve is an average of eight stimulus trials. The curves are normalized by the peak amplitude. (C) Population-averaged and peak-normalized time-courses of diameter change for the arteriolar trunks from different cortical depths. Insets in B and C show zoomed-in views of the first 3 s following the stimulus and define color coding. There was no statistically significant change in peak amplitude with depth; the same time-courses without normalization are shown in Fig. S1. (D) Onset time of the arteriolar trunk dilation as a function of the cortical depth. Inset shows an example of fitting a line to the rising slope for the estimation of dilation onset. (E) Time-to-peak of the arteriolar trunk dilation as a function of the cortical depth. In both D and E, the straight line depicts the trend obtained from linear regression fitting. The number of measurements for each category is listed in Table S1.
vascular response increased with decreasing cortical depth. For each measurement, we extracted the onset time (Fig. 1D) and timeto-peak (Fig. 1E). The onset was estimated by fitting a line to the rising slope between 20 and 80% to the peak and calculating an intercept with the prestimulus baseline (Inset, Fig. 1D). Although estimation of the onset potentially can be biased by differences in the signal-to-noise ratio of measurements in different layers, the prestimulus SD in our sample was independent of the cortical depth (Fig. S1E). Both the onset and time-to-peak decreased significantly with an increase in the cortical depth (P < 0.05). We estimated a propagation speed of 1,100 and 600 μm/s for the onset and timeto-peak, respectively, based on the linear regression slope. Spatial Gradient of Dilation Onset and Peak Times as a Function of Branching Order Suggests Propagation into Capillary Beds. We fol-
lowed branching arteriolar trees and measured diameter changes evoked by a forepaw stimulus while keeping track of the cortical depth and branching order. An example of a set of measurements is shown in Fig. 2A. Fig. 2 B and C and Fig. S1 B and C show averaged time-courses of arteriolar diving trunks (black), their first-order branches (magenta), and higher-order branches Tian et al.
(yellow) for layer I (0.5 s. Laminar differences in the BOLD response have been demonstrated in an earlier high-resolution fMRI study (28). The authors attributed these differences to neuronal propagation from layer IV, the cortical input layer, to the superficial and deeper layers. However, neuronal activity is known to spread throughout the cortical depth within a few milliseconds, too fast to explain the observed laminar hemodynamic differences of 500 ms or more. For example, in our previous publications we reported tight synchronization of the onset and peak of neuronal activity (both spiking and synaptic currents) throughout the cortical depth evoked by the forepaw or whisker stimulation on a time scale of
