Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex

Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex

^*Department of Physics, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0319; and ^†Bell Laboratories, Lucent Technologies, 700 Mountain Avenue, Murray Hill, NJ 07974

Communicated by Harry Suhl, University of California at San Diego, La Jolla CA (received for review August 13, 1998)

Abstract

Cortical blood flow at the level of individual capillaries and the coupling of neuronal activity to flow in capillaries are fundamental aspects of homeostasis in the normal and the diseased brain. To probe the dynamics of blood flow at this level, we used two-photon laser scanning microscopy to image the motion of red blood cells (RBCs) in individual capillaries that lie as far as 600 μm below the pia mater of primary somatosensory cortex in rat; this depth encompassed the cortical layers with the highest density of neurons and capillaries. We observed that the flow was quite variable and exhibited temporal fluctuations around 0.1 Hz, as well as prolonged stalls and occasional reversals of direction. On average, the speed and flux (cells per unit time) of RBCs covaried linearly at low values of flux, with a linear density of ≈70 cells per mm, followed by a tendency for the speed to plateau at high values of flux. Thus, both the average velocity and density of RBCs are greater at high values of flux than at low values. Time-locked changes in flow, localized to the appropriate anatomical region of somatosensory cortex, were observed in response to stimulation of either multiple vibrissae or the hindlimb. Although we were able to detect stimulus-induced changes in the flux and speed of RBCs in some single trials, the amplitude of the stimulus-evoked changes in flow were largely masked by basal fluctuations. On average, the flux and the speed of RBCs increased transiently on stimulation, although the linear density of RBCs decreased slightly. These findings are consistent with a stimulus-induced decrease in capillary resistance to flow.

Footnotes

↵ § For a temporal window, T, the image data F(x,t) was transformed to a reference frame that moved with velocity c. We constructed the matrix F(x + ct, t) with t ₀ < t < t ₀ + T, where t ₀ is the offset from the start of acquisition. The velocity was defined through a parametric search for value of c at which the RBCs were closest to being stationary in the moving frame, quantified by the fractional variance captured by the largest singular value in a singular value decomposition (19) of F(x +ct, t). If the data were free of noise and the RBCs moved uniformly, this fraction would equal 1 when c corresponded to the velocity of the RBCs. Computationally, this procedure involves interpolation of the data to perform transforms by fractions of pixel units and a singular value decomposition for each transform.
↵ ¶ We checked whether the apparent increase in linear density with increasing flux was an artifact of a systematic change in caliber of the vessels with increasing flux. The density, calculated as linear density/π(caliber/2)², was plotted versus flux for the 21 vessels with known caliber. We still observed a significant trend between density and flux; a linear regression gave Δ(density)/Δ(flux) = 1.5 × 10⁴ ± 0.7 × 10⁴ sec/mm³ (mean ± SEM), as opposed to Δ(linear density)/Δ(flux) = 0.38 ± 0.13 sec/mm (mean ± SEM) for the data in Fig. 3 h.
↵ ‖ The spectral coherence of the flux through an unbranched vessel must equal unity as the frequency approaches zero as a consequence of conserved flux. To the extent that the density of RBCs is relatively constant over a distance of 25 μm, the coherence of the velocity will also approach 1 at low frequencies.
↵ ** In a series of control experiments (17 trials in four capillaries across two animals), we consistently observed that mild electric shock (see Methods) led to changes in speed that were ≈50% of the basal value, ≈3-fold larger than the changes caused by natural stimulation.
ABBREVIATIONS:

ECoG,

electrocorticogram;

RBC,

red blood cell.