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All surgical and experimental procedures were in accordance with NIH guidelines. Details are described elsewhere (7, 8). Cats were anesthetized during the recording phase with continuous infusion of sodium pentothal. A craniotomy overlying area 18 of the visual cortex was performed and the dura removed. Stimulation protocol: (i) blank screen (prestimulus time, 1.5 to 5 s); (ii) moving gratings for 4 s (high contrast, 0.2 cycles per degree, duty cycle = 0.3, 6 cycles per second), and (iii) a blank screen (poststimulus time, at least 30 s). Up to % such trials were averaged.
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The other factors affecting the phosphorescence decay time are temperature, salinity, and pH. By their nature, salinity and temperature changes are much slower than the phenomenon under examination. The upper limit for a pH change should be smaller than the arterovenous pH difference of ΔpH = 0.03. Using pH calibration data (17), the effect of ΔpH (pH = 7.4 was taken as the resting pH) on the decay time is negligible, about 7% of the difference we observed during the deoxygenation phase. The initial dip detected with phosphorescence decay time measurements cannot be attributed to blood volume rearrangements among microvascular compartments, because the latter's onset is delayed by ∼500 ms (Fig. 2B, inset). In addition, in those high-resolution volume measurements (4 μm), the volume increase observed in venules and veins was neither faster nor larger than in the other compartments. Previous (7, 79) and recent (I. Vanzetta, R. Hildesheim, A. Grinvald, unpublished data) fluorescent tracer imaging measurements confirmed these results. The visualization of a stimulus-dependent increase in red blood cell velocity in individual capillaries [D. Weinfeld, P. P. Mitra, F. Helmchen, W. Denk, Proc. Natl. Acad. Sci. U.S.A. 95, 15741 (1998)], also showed that the flow increase starts late (> ∼0.5 s).
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The other factors affecting the phosphorescence decay time are temperature, salinity, and pH. By their nature, salinity and temperature changes are much slower than the phenomenon under examination. The upper limit for a pH change should be smaller than the arterovenous pH difference of ΔpH = 0.03. Using pH calibration data (17), the effect of ΔpH (pH = 7.4 was taken as the resting pH) on the decay time is negligible, about 7% of the difference we observed during the deoxygenation phase. The initial dip detected with phosphorescence decay time measurements cannot be attributed to blood volume rearrangements among microvascular compartments, because the latter's onset is delayed by ∼500 ms (Fig. 2B, inset). In addition, in those high-resolution volume measurements (4 μm), the volume increase observed in venules and veins was neither faster nor larger than in the other compartments. Previous (7, 79) and recent (I. Vanzetta, R. Hildesheim, A. Grinvald, unpublished data) fluorescent tracer imaging measurements confirmed these results. The visualization of a stimulus-dependent increase in red blood cell velocity in individual capillaries [D. Weinfeld, P. P. Mitra, F. Helmchen, W. Denk, Proc. Natl. Acad. Sci. U.S.A. 95, 15741 (1998)], also showed that the flow increase starts late (> ∼0.5 s).
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3 = 7%. These large differences underscore the inherent ambiguities of multiexponential analysis [(3O) and references therein]. Adding constraints to some of the parameters that can be obtained from independent measurements should allow more quantitative analysis.
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The decay constant at zero oxygen tension and the quenching constant were taken for pH = 7.4 and 38°C body temperature, 4% albumin, and 120 mM NaCl from published values (17).
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2 and in oxyhemoglobin and deoxyhemoglobin concentrations were assumed to be small relative to their resting values; (ii) the cooperative binding of oxygen to hemoglobin was approximated by a reaction with constant binding strength; and (iii) mean oxygen saturation in the sampled blood volume was assumed to be ∼85%.
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Supported by a grant from the German Israeli Foundation. We thank R. Buxton, U. Dirnagl, N. Logothetis, I. Steinberg, R. B. H. Tootell, K. Ugurbil, and D. F. Wilson for their useful suggestions and comments, and D. Ettner, I. Lampl, D. Nelson, E. Shtoyerman, D. Sharon, H. Slovin, and C. Wijnbergen for their help during the experiments.
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