Spatial Integration of Vascular Changes with Neural Activity in Mouse Cortex

Abstract

The authors evaluated representations of discretely activated, neighboring brain regions using real-time optical intrinsic signals by transcranial imaging with 540-nm and 610-nm broadband illumination of the mouse barrel cortex. Iron filings were glued to two neighboring whiskers (C2 + D2) that were stimulated magnetically, singly and together. Real-time images were collected, averaged, and analyzed statistically. Postmortem filling of arteries with fluorescent beads was shown in relation to histochemical staining of barrels to accurately relate surface changes to functional cortical columns. Significant optical intrinsic signal changes are related to overlapping distributions of arterioles that feed the two separate areas. Activation of adjacent and interacting cortical columns leads not only to increased magnitude of vascular responses in those columns, but also to wider spatial extent of absorption changes occurring principally in areas of cortex fed by vessels upstream of the active cortex. The localization of changing hemoglobin absorption around upstream blood vessels and their vascular domains suggests that propagated vasodilation of upstream parent vessels is greater when vasodilatory signals from separate areas of active cortex converge on common arterioles that feed them.

Keywords

Optical imaging Intrinsic signals Whisker stimulation Barrels

Neural activity can be inferred from vascular responses resulting from various stimuli (Raichle, 1994). Local perfusion and vessel related signals are the bases for three methods used to assess neural activity: O¹⁵ positron emission tomography (Raichle, 1983), functional magnetic resonance imaging (Belliveau et al., 1991), and intrinsic optical signal (IOS) imaging (Grinvald et al., 1986). Positron emission tomography provides direct, three-dimensional measures of local cerebral blood flow, albeit at relatively low spatial and temporal resolution. Because the method of positron emission tomography is based on injected radioactive isotopes, this limits both the number of studies that can be done in a single patient and the ages of subjects that can be studied. In contrast, functional magnetic resonance imaging is non-invasive, and provides measurements related to changes in the oxygenation of hemoglobin associated with evoked changes in flow but does not measure blood flow directly. Fast, high-resolution blood oxygen-level–dependent imaging (Silva et al., 2000) provides a picture of changes in active regions of the brain in humans and other animals at higher spatial and temporal resolutions than positron emission tomography. Technically, there is virtually no limit to the number of studies that can be performed in persons of any age.

Intrinsic optical signal imaging is invasive because direct observation of the brain is required. At visible wavelengths, the observations are necessarily confined to accessible areas of the brain close to the surface. When measured in the 540-nm (green) wavelength range, IOS detects changes in hemoglobin absorption by erythrocytes in pial vessels and parenchymal capillaries. When measured in the 610-nm (red) wavelength range, IOS detects changes in hemoglobin oxygenation in erythrocytes (Mayhew et al., 2000). Because of the resolution, speed, and sensitivity with which photons can be detected, IOS provides images of active tissues at temporal and spatial resolutions sufficient to follow evoked changes closely (Grinvald et al., 1986). Observations at different time points are limited to experimental animals.

Malonek and Grinvald (1996) characterized the spatial precision and dynamics of blood oxygenation signals with optical imaging spectroscopy. They found that erythrocyte hemoglobin deoxygenation, but not erythrocyte delivery, colocalized to orientation columns in cat V2 (area 18) with stimuli covering the visual field. They concluded that changes in the absorption of green light were blood-flow related, which they likened to “watering the entire garden for the sake of one thirsty flower.” Intraoperative optical imaging with red light (610–620 nm) in humans with independent activation of the tips of three adjacent fingers (Cannestra et al., 1998) showed overlapping representations. Although generally consistent with underlying neurophysiology, IOS is sufficiently different at 540 nm to suggest that the mechanisms of vascular hemoglobin delivery could contribute significantly to changes that do not match known functional representations. We hypothesized that vascular architecture, which has been largely ignored, contributes significantly to apparent mismatches between neural and vascular responses. Accordingly, we investigated vascular and neural activation in functionally discrete but adjacent areas of the cortex separately and together by monitoring the IOS (Dowling et al., 1996; Erinjeri and Woolsey, 2001).

The barrel cortex (Woolsey and Van der Loos, 1970) provides a practical context in which to study the relation between stimulus-evoked activity in focal groups of neurons and resulting vascular responses. A barrel is an anatomical marker in layer IV of the somatosensory cortex for a column of cells activated by a whisker on the contralateral face of the mouse, rat, and many other rodents. Whisker stroking is a simple means by which to alter neural activity in an anatomically identifiable part of the brain. In the mouse, there are five rows of whiskers (A–E), with five to nine whiskers per row (e.g., whisker A1 or E9).

The stereotyped location, size, and shape of barrels (activated by appropriate whiskers) (Simons, 1983, 1985; Woolsey and Wann, 1976) contrasts with the variable vascular trees that feed them (Rovainen et al., 1993). This arrangement permits interpretation of the sources of variation in optical signals. Furthermore, the small size of the mouse brain permits the observation of nearly all of the cortex in a hemisphere (Woolsey, 1967), effective reconstruction of the entire vascular tree feeding the active area, and unique comparisons with responses in larger-scale mammalian systems.

In the present study, we characterize the responses to individual and simultaneous activation of two adjacent mouse whisker barrels in different rows (C2 and D2) with real-time IOS imaging. We describe results for both 540-nm optical signals (light absorption by erythrocytes related to hemoglobin concentration, and resulting from changes in capillary hematocrit levels and pial arterial vasodilation) and 610-nm optical signals (light absorption by erythrocytes related to changes in hemoglobin oxygenation). The findings are consistent with a mechanism of integration of vasodilatory signals by the arteriolar tree.

MATERIALS AND METHODS

Experimental procedures

Animal methods. Animals were housed by the Washington University Division of Comparative Medicine in facilities meeting or exceeding National Institutes of Health, Washington University, and Association for Assessment and Accreditation of Laboratory Animal Care International standards. Animals were studied under protocols approved by Washington University's Institutional Animal Care and Use Committee. Eight female BALB/c mice (age, 60–90 days; weight, 18–20 g) were sedated with 0.5 mg/kg ketamine and 0.2 mg/kg xylazine and anesthetized with 0.5 mg/kg urethane. Animals respired spontaneously and were treated with 0.1 mg/kg atropine to inhibit bronchial secretions. Systolic blood pressure was measured at regular intervals with a tail cuff device (Kent Scientific, Torrington, CT, U.S.A.). Heart rate (503 ± 33 beats/min), blood pressure (110 ± 11 mm Hg), and respiratory rate (202 ± 36 breaths/min) were in the physiologic range. Because measuring blood gases by left ventricular puncture is incompatible with postmortem perfusion in the imaged mice (Dalkara et al., 1995), we measured blood gas concentrations and pH in sham-operated animals. These values were within the normal physiologic range (arterial oxygen tension, 105 ± 5 mm Hg; arterial carbon dioxide tension, 50 ± 6 mm Hg; oxygen saturation, 97.0 ± 0.5%; pH, 7.28 ± 0.04) (Dalkara et al., 1995). Body temperature was maintained at 37°C with a thermostatic heating pad.

Transcranial window. The scalp was reflected after a midline incision to expose the dorsal calvarium. A 3-mm square brass rod was attached to the left temporal bone with cyanoacrylate and held in a manipulator. The unthinned right parietal bone was irrigated with saline and a 12-mm round glass coverslip was placed over it to reduce glare associated with the wet skull. One edge of the coverslip was glued to the brass rod medially with cyanoacrylate, whereas the other edge rested on the exposed scalp rostrally, caudally, and laterally, and remained in position by capillary action of the saline.

Whisker stimulation. All whiskers except the C2 and D2 whiskers on the left side of the face were trimmed at the skin. Iron powder (100 mesh; VWR, Willard, OH, U.S.A.) was glued to each of these two whiskers with cyanoacrylate. A small magnet was affixed just caudal to the animal's whisker pad to conveniently secure a coated whisker that was not being stimulated. Data acquisition sequences were scripted on a WPI Pro4, 4 Channel Timer/Controller (World Precision Instruments, Sarasota, FL, U.S.A.), which triggered a Pulsemaker A300 (World Precision Instruments) to generate square-wave 50-millisecond pulses at 10 Hz (Woolsey et al., 1981) that drove a small, silent magnet (following the general approach of Melzer et al., 1985) placed 2-mm rostral to the whisker to deflect the whisker over a 3-mm anterior-posterior excursion. The Pulsemaker also triggered video capture on a Macintosh G3 computer (Apple Computer, Cupertino, CA, U.S.A.). For most sessions, the surface of the brain was imaged at 30 Hz for 10 seconds (2 seconds before, 3 seconds during, and 5 seconds after the stimulus). A 300-image video clip was captured every 30 seconds in 10 trials for a 5-minute period.

Real-time optical imaging

Details of the imaging system and analysis are provided elsewhere (Dowling et al., 1996; Erinjeri and Woolsey, 2001). Briefly, illumination was provided by filtered light (520–560 or 600–620 nm) supplied by a 150-W, 21-V EKE light bulb (Opti-Quip, Highland Mills, NY, U.S.A.) powered by a regulated power supply. The voltage was further stabilized by placing 10 0.29-F capacitors in parallel with the power supply. A focusing unit (Edmund Scientific, Tonawanda, NY, U.S.A.) increased the intensity of the incident light. The image of the brain surface through the skull was magnified 0.75 to 3 times with a Nikon SMZ-U dissecting microscope (Nikon Corporation, Melleville, NY, U.S.A.) while focusing on surface vessels and captured with a CCD camera (72S; Hamamatsu Corporation, Bridgewater, NJ, U.S.A.) at 30 Hz. These 640 × 480-pixel images were stored digitally using a real-time capture card (AG-5, Scion Corporation, Frederick, MD, U.S.A.) in a Power Macintosh G3 Computer running the public domain National Institutes of Health Image 1.62 program (developed by Wayne Rasband at the US National Institutes of Health and available at http://rsb.info.nih.gov/nih-image), which we optimized by modifying the source code.

Data analysis

Each of the 10 300-image video clips (total size, 900 megabytes; 640 × 480-pixel grayscale images × 300 images per trial × 10 trials) were analyzed with a “first frame analysis.” The prestimulus background absorption was subtracted from each frame of each 300-image movie. After subtracting the background, all 10 video clips were averaged together to yield one 30-Hz, 10-second, 300-frame optical image sequence per animal from which time courses could be followed for particular regions of interest.

To evaluate the significance of changes in 540-nm absorption at locations on the cortex, a repeated-measures analysis of variance was performed at each of the 307,200 locations measured by the CCD array (640 × 480). From this analysis, the F ratio for the optical time course was generated at each location in the cortex and F-map images were produced based on the statistical question posed. The resulting image represents the likelihood that a time course of an IOS at a location on the surface of the brain exhibited an exponential increase after stimulus onset and a subsequent exponential decay (Fig. 2B).

Using this statistical map, we computed the contiguous region of cortex where changes in optical time-course were unlikely to be random (P < 0.05). The areas of these significantly different regions were used for statistical comparisons of IOS resulting from different stimuli or different illumination. Sizes of regions of interest compared included total areas of change, overlapping areas of change with different stimuli, and areas of change upstream to the activated barrel. The upstream area was defined by drawing a line through the center of the activated barrel perpendicular to the principal axis of the vessels feeding that barrel.

Histology

After imaging, the mice were deeply anesthetized and perfused transcardially with heparinized saline, followed by 4% paraformaldehyde in 0.1 mol/L phosphate buffer, followed by 0.1 mol/L phosphate buffer. A mixture of 0.5% 10 μm green fluorescent beads (Duke Scientific, Palo Alto, CA, U.S.A.) to lodge in arterioles and 0.5% TRITC-dextran (Molecular Probes, Eugene, OR, U.S.A.) in 1.5% gelatin to fill arteries, capillaries and veins was then perfused and the animals were placed on ice for 30 minutes (Hedenstierna et al., 2000). The brains were removed and immersed in 4% paraformaldehyde in 0.1 mol/L phosphate buffer for 3 to 7 days at 4°C. Tangential, 150-μm serial sections were cut on a freezing microtome, reacted for cytochrome oxidase activity (Wong-Riley, 1979; Wong-Riley and Welt, 1980) and wet-mounted using Vectashield mountant (Vector Laboratories, Burlingame, CA, U.S.A.). Surface sections with pial arterioles and deeper sections with parenchymal arterioles were captured digitally at two times using a CCD camera attached to a Nikon Labophot microscope (Boyce Scientific, Gray Summit, MO, U.S.A.). The surface sections were aligned to the in vivo image from imaging with Adobe Illustrator 9.0 (Adobe Systems, San Jose, CA, U.S.A.) by translation, rotation, and scaling. The deeper sections in which the cytochrome oxidase-positive barrels were identified and drawn were then added to the overlay using the parenchymal vessels as fiducials. Therefore, the digital overlay represents an alignment of pial vessels, parenchymal arterioles, barrels, and optical-image time courses.

RESULTS

Magnitude of optical changes

To determine the effect of simultaneous activation of neighboring areas of cortex on the strength of evoked vascular response in those regions, the C2 and D2 whiskers were stroked individually and together while 540-nm optical signals from the contralateral hemisphere were recorded (Fig. 1). During single-whisker stimulation, 540-nm absorption increased over the activated whisker barrel in each animal (Figs. 1C and 1D). Peak responses were in surface arterioles feeding the stimulated whisker's barrel column. When both whiskers were stroked together, the magnitude of increase in 540-nm optical changes over both barrels was greater than that evoked by either whisker alone during the last second of stimulus (Figs. 1E and 2A). In addition to local responses, multiwhisker stimulation evoked increases in 540-nm absorption in surface vessels up to 500 μm from the active area. In contrast, 610-nm optical signals had peak magnitudes centered over the two stroked whisker barrels and were not related to the pattern of surface arterioles (Fig. 1F); 610-nm optical signals often changed in veins draining the active region and the cortex around it.

FIG. 1.

C2/D2 whisker stimulation: intrinsic optical signal (IOS) localization. (A) In vivo transcranial image of the right somatosensory cortex showing the locations of two whisker barrels (C2 and D2) identified during postmortem histology from matching images of vascular patterns. Scale bar = 1 mm; medial = up, anterior = right. (B) Arteriolar labeling with fluorescent beads. (C) 540-nm IOS during C2 (red) activation. The response shows a strong pial component and activation beyond the C2 barrel. (D) 540-nm IOS during D2 (red) activation. (E) 540-nm IOS during C2 + D2 stimulation. The spatial extent and intensity of the response is increased relative to the simple sum of panels C and D, mostly related to upstream vessels supplying the active barrel columns. (F) With 610-nm IOS during C2 + D2 stimulation, the response extent is smaller than the 540-nm IOS and is consistent with the combined size of the C2 + D2 barrel columns. Draining veins, but not surface arterioles, show optical changes at 610 nm.

Timing of optical changes

To test how simultaneous activation of neighboring areas of the cortex influenced the timing of evoked vascular changes, the time courses of 540-nm optical changes were measured over the C2 and D2 whisker barrels before, during, and after 3 seconds of C2, D2, or C2 and D2 whisker stimulation (Fig. 2B). The same initial rate (slope) of IOS change throughout the first second of stimulation was found for all three conditions. With stimulation of C2 or D2 alone, the rate slowed between the second and third seconds of stimulation. However, stimulation of C2 and D2 together resulted in a change prolonged throughout the stimulus period close to the initial rate. The IOS change for all three stimuli peaked approximately 500 milliseconds after the stimulus and the rates of decrease for all three curves were similar. As the magnitude of change with C2 and D2 stimulation was higher than the individual cases, the time to baseline was slightly longer.

FIG. 2.

C2/D2 whisker stimulation: magnitude and timing of the 540-nm intrinsic optical signal (IOS). (A) When C2 and D2 are stimulated together, the 540-nm IOS over the C2 and D2 barrels is greater than those obtained during stimulation of either whisker alone. (B) C2, D2, and C2 + D2 stimulation all produce the same initial rate of rise during the first second of the stimulus, but C2 + D2 stimulation evokes a bigger change than stimulation of C2 or D2 alone. Decay time courses are similar for all stimuli.

Spatial extent of optical changes

To test the hypothesis that vascular responses to combinations of stimuli are the spatial combination of vascular responses to individual stimuli, the region of significant (P < 0.05) 540-nm optical change was determined while stimulating the C2 or D2 whiskers or the C2 and D2 whiskers for 3 seconds (Fig. 3). Individual C2 and D2 responses were much larger than the anatomical surface projection of the barrel (Figs. 4A and 4B). Response regions always contained the anatomical region of the stroked whisker barrel, but were not always centered on or symmetrical about the barrel, with a preference for territories upstream of (more proximal to in an arterial sense) the activated barrel. The size and shape were strongly influenced by the branching pattern of arterioles feeding the active barrel. The region of 540-nm optical changes evoked by stimulation of the C2 whisker barrel and the D2 whisker barrel were not mutually exclusive. In fact, there was considerable overlap, particularly regarding arterioles feeding both barrels.

FIG. 3.

C2/D2 whisker stimulation: spatial extent of 540-nm responses. (A) 540-nm intrinsic optical signal (IOS) for C2 stimulation (C2, red; spatial extent, yellow). (B) 540-nm IOS for D2 activation (D2, red; spatial extent, cyan). (C) 540-nm IOS for C2 + D2 stimulation (C2 and D2, red; spatial extent, green). The overlapping area of the IOS is centered over common pial vessels. The response area upstream of the active barrel columns is augmented. (D) Although C2 and D2 are close to a pial arteriole, the 540-nm IOS when both are active is more extensive spatially than when each is active alone.

FIG. 4.

Spatial extent of intrinsic optical signal (IOS). (A) Significant 540-nm IOS obtained during C2, D2, or C2 + D2 stimulation are significantly larger (P < 0.05) than the cytochrome oxidase-stained barrel area, whereas significant 610-nm IOSs are intermediate in size (≈ two times larger) than the projected barrel areas. For C2 + D2 activation, the spatial extent of the 540-nm IOS is less than the sum of the individual responses, confirming overlap of the individual regions. (B) As overlap between 540-nm IOS from individual whisker stimulation increases, the IOS upstream of the relevant whisker barrels increases when both are activated.

Stimulation of C2 and D2 together led to 540-nm responses that were more extensive than the combination of the individual D2 and C2 responses (Figs. 3C and 4A). However, this effect was not due to widespread activation of cortex because significant (P < 0.05) 610-nm optical signals (activity-dependent deoxygenation of hemoglobin) were barrel shaped and nearly barrel sized (Figs. 1F and 4A). The augmented 540-nm area was in regions fed by tributaries of upstream parent vessels common to the C2 and D2 barrels. This effect was greater in cases where the degree of overlap between the individual C2 and D2 response was larger (i.e., when C2 and D2 shared a feeding vessels) (Fig. 3D).

DISCUSSION

These experiments show that activation of adjacent and interacting cortical columns leads not only to increased magnitude of vascular responses in those columns, but also to increased spatial extent of absorption changes occurring principally upstream of the active cortex. The rate (slope) of IOS change with complex stimuli (combination of stroking the C2 and D2 whiskers) was not greater than that of simple stimuli (individual stroking of C2 or D2 whiskers). The localization of changing hemoglobin absorption around upstream blood vessels and their vascular domains suggests that propagated vasodilation of upstream parent vessels is greater when vasodilatory signals from separate areas of active cortex converge on common arterioles that feed them.

Iadecola et al. (1997) demonstrated that activation of discrete areas of cerebellar cortex led to both local increases in cerebellar blood flow and delayed vasodilation of blood vessels feeding, but remotely located from, the active area. Both effects were proportional to the intensity of local activity. Here, we demonstrate that propagated vasodilation is augmented through combined activation of the same blood vessel by downstream branches to separate but nearby areas of active cortex. This effect is proportional to the extent of local neural activity. We conclude that blood vessels summate upstream vasodilatory responses to provide increased flow in capillary beds within the active area of the brain.

In the mouse barrel cortex, a barrel column is the group of neurons that fire in response to stroking a particular whisker. Although neurons in barrels (in layer IV) respond principally to one whisker, neurons in supragranular and infragranular layers above and below a barrel column respond to more than one whisker (Bernardo et al., 1990; McCasland and Woolsey, 1988; Simons and Woolsey, 1979). A barrel column can be thought of as a hyperboloid rather than a cylinder: the narrow “waist” of the hyperboloid corresponds to a barrel in layer IV, whereas the superficial and deep portions of a whisker barrel column in the supragranular and infragranular layers are broader. Therefore, paired whisker deflections such as those used in the present experiments lead to firing rates in each of the stroked whisker barrel columns that are higher than that of independently fired rates (Goldreich et al., 1998; McCasland and Woolsey, 1988; Simons and Woolsey, 1979). We found that the rate of onset and offset of 540-nm signals was identical for C2, D2, and C2 plus D2 stimulation. However, when the strength of a stimulus is increased by spatial summation of neural activity, vascular responses are matched to the intensity of the stimulus not by increasing the rate of vascular response, but by increasing the magnitude. In contrast, Blood et al. (1995) found that the frequency of whisker stroking leads to a faster time to peak (i.e., rate) of vascular responses. Brain vessel responses apparently differ depending on whether neural activity is increased as a result of temporal summation (increasing stroking frequency) or as a result of spatial summation (simultaneous activation of neighboring areas). To meet energy needs, the rate of adenosine triphosphate hydrolysis should be greater in the former, whereas its spatial extent should be wider in the latter (e.g., Yarowsky et al., 1983).

Our results suggest that during simple stimuli (single whisker stimulation), maps of neural activity are related to maps of vascular responses through a transformation that depends on the pattern of the vascular tree. For stimulation of multiple whiskers, the transformation of maps of neural activity into maps of vascular responses is dominated by temporospatial integration of vasodilatory signals in the vascular tree. Local activity in cortex produces resistance decreasing vasodilatory signals (Iadecola, 1993), which hyperpolarize the membranes of vascular endothelium and smooth muscle (Knot et al., 1996; McGahren et al., 1998; Tyml et al., 1997). Vasodilatory signals are propagated electrotonically through the vasculature by gap junctions along arterioles via the cellular path of least resistance (Little et al., 1995a,b), namely, upstream feeding vessels. By this mechanism, spatially distinct neural activations can have overlapping areas of vascular response when the same upstream arterioles are dilated. In the case where shared arterioles are simultaneously signaled by activation of separate but nearby neural ensembles, these common arterioles presumably summate vasodilatory signals through increasing hyperpolarization. This summed electrical signal is conducted passively further upstream through the vessels to provide an appropriate level and spatial extent of blood flow to both neural ensembles; the greater the summation of local responses, the farther the vascular responses extend upstream.

The vascular responses in arterioles less than 100 μm in diameter on the surface of mouse brains are analogous to those responses occurring in comparably sized parenchymal blood vessels in human cortex. Diffusion of oxygen and nutrients are similar regardless of scale. Indeed, the density of precapillary arterioles we observed in the mouse is only twice the density in humans (Duvernoy et al., 1981). Therefore, the present findings indicate that the limit of the precision of the 540-nm signal is the extent to which a single feeding arteriole feeds a single neuronal module. A whisker barrel column in a mouse has approximately 10% of the surface projection of a somatosensory cortical column in the monkey (Powell and Mountcastle, 1959). Several 100-μm arterioles ought to feed a single functional column in humans in contrast to a single arteriole supplying several functional columns in mice. With stimuli activating a single cortical column (e.g., equivalent to a barrel column) as opposed to an entire functional area (i.e., area 18), the more robust 540-nm IOS can provide adequate resolution to map function in larger animals because the number of arterioles per cortical column is five to 10 times greater in humans than in the mouse.

At its core, IOS imaging is digital-subtraction light microscopy, and should in theory only be able to give spatially focused information from superficial layers of cortex, despite that (1) most optical imaging systems are wide-field, low-magnifications systems that have a low numerical aperture and large depth of field of 100 μm or more, and (2) longer-wavelength red light can penetrate deeply through the cortex. Although the idea of imaging deep layers of the cortex by focusing several hundred microns into the cortex is attractive (Ratzlaff and Grinvald, 1991), the brain strongly scatters light within it and will lead to a mixture of some light from deep in cortex combined with blurred surface images. To avoid these limitations, confocal (Dirnagl et al., 1992) or multiphoton (Kleinfeld et al., 1998) strategies may be used. Because this study concentrates on the integration of vascular signals in proximal upstream arterioles evoked by coactivation of neighboring areas of cortex, we focused on surface vessels.

The IOS does not measure cerebral blood flow and, at the capillary level, cerebral blood flow may not be the only determinant of erythrocyte distribution in the brain. In addition to blood flow, hematocrit levels, vessel-tissue oxygen gradients, transit time and path length, and diffusion coefficients determine oxygen release from erythrocytes within a capillary bed (Hudetz, 1997, Hudetz et al., 1996; Mayhew et al., 2000). Path lengths (Boero et al., 1999), oxygen gradients (Moskalenko et al., 1997), transit times (Woolsey et al., 1996), and blood flow (Adachi et al., 1994; Woolsey et al., 1996) have been measured in discrete locations in the barrel cortex related to neural activity evoked by stimulating a single whisker. As the principal oxygen carrier, hemoglobin in erythrocytes reflected in the 540-nm IOS indicate changes in oxygen delivery to meet need. Alternatively, changes in erythrocyte distribution may not indicate a specific need for oxygen, but instead may be a byproduct of mechanisms for increasing flow and erythrocyte delivery to active brain (Ido et al., 2001; Mintun et al., 2001). Spatial integration of signals conducted via vessels could explain the relation between hemoglobin changes and vascular architecture. Therefore, IOS at 540 or 610 nm are functional imaging modalities that are blood-delivery based, but not necessarily blood-flow based (Dunn et al., 2001). We have shown specific flow responses confined to particular whisker and barrel columns (Woolsey et al., 1996). Because barrels provide a template for direct comparison of results with different methods from separate studies, we conclude that blood flow coincides better with the changes seen at 610 nm rather than at 540 nm. Therefore, the 540-nm IOS does not represent flow per se, and hence is not “water for the garden” (Malonek and Grinvald, 1996).

Footnotes

Acknowledgments:

The authors thank Brad Miller and Nicquet Blake for helpful discussions, and Kathy Diekmann for assistance with manuscript preparation.

References

Adachi

Takahashi

Melzer

Campos

K.L.

Nelson

Kennedy

Sokoloff

(1994) Increases in local cerebral blood flow associated with somatosensory activation are not mediated by NO. Am J Physiol 267:H2155–H2162.

Belliveau

J.W.

Cohen

M.S.

Weisskoff

R.M.

Buchbinder

B.R.

Rosen

B.R.

(1991) Functional studies of the human brain using high-speed magnetic resonance imaging. J Neuroimaging 1:36–41.

Bernardo

K.L.

McCasland

J.S.

Woolsey

T.A.

Strominger

R.N.

(1990) Local intra and interlaminar connections in mouse barrel cortex. J Comp Neurol 291:231–255.

Blood

A.J.

Narayan

S.M.

Toga

A.W.

(1995) Stimulus parameters influence characteristics of optical intrinsic signal responses in somatosensory cortex. J Cereb Blood Flow Metab 15:1109–1121.

Boero

J.A.

Ascher

Arregui

Rovainen

Woolsey

T.A.

(1999) Increased brain capillaries in chronic hypoxia. J Appl Physiol 86:1211–1219.

Cannestra

A.F.

Black

K.L.

Martin

N.A.

Cloughesy

Burton

J.S.

Rubinstein

Woods

R.P.

Toga

A.W.

(1998) Topographical and temporal specificity of human interoperative optical intrinsic signals. Neuroreport 9:2557–2563.

Dalkara

Irikura

Huang

Panahian

Moskowitz

M.A.

(1995) Cerebrovascular responses under controlled and monitored physiological conditions in the anesthetized mouse. J Cereb Blood Flow Metab 15:631–638.

Dirnagl

Villringer

Einhaupl

K.M.

(1992) In vivo confocal scanning laser microscopy of the cerebral microcirculation. J Microsc 165:147–157.

Dowling

J.L.

Henegar

M.M.

Liu

Rovainen

C.M.

Woolsey

T.A.

(1996) Rapid optical imaging of whisker responses in the rat barrel cortex. J Neurosci Methods 66:113–122.

10.

Dunn

A.K.

Bolay

Moskowitz

M.A.

Boas

D.A.

(2001) Dynamic imaging of cerebral blood flow using laser speckle. J Cereb Blood Flow Metab 21:195–201.

11.

Duvernoy

H.M.

Delon

Vannson

J.L.

(1981) Cortical blood vessels of the human brain. Brain Res Bull 7:519–579.

12.

Erinjeri

J.P.

Woolsey

T.A.

(2001) Real-time optical imaging of whisker evoked cerebrovascular regulation in mouse barrel cortex. Soc Neurosci Abstr 27:1386.

13.

Goldreich

Peterson

B.E.

Merzenich

M.M.

(1998) Optical imaging and electrophysiology of rat barrel cortex. Part 2: Responses to paired-vibrissa deflections. Cereb Cortex 8:184–192.

14.

Grinvald

Lieke

Frostig

R.D.

Gilbert

C.D.

Wiesel

T.N.

(1986) Functional architecture of cortex revealed by optical imaging of intrinsic signals. Nature 324:361–364.

15.

Hedenstierna

Hammond

Mathieu-Costello

Wagner

P.D.

(2000) Functional lung unit in the pig. Respir Physiol 120:139–149.

16.

Hudetz

A.G.

(1997) Regulation of oxygen supply in the cerebral circulation. Adv Exp Med Biol 428:513–520.

17.

Hudetz

A.G.

Feher

Kampine

J.P.

(1996) Heterogeneous autoregulation of cerebrocortical capillary flow: Evidence for functional thoroughfare channels? Microvasc Res 51:131–136.

18.

Iadecola

(1993) Regulation of the cerebral microcirculation during neural activity: Is nitric oxide the missing link? Trends Neurosci 16:206–214.

19.

Iadecola

Yang

Ebner

T.J.

Chen

(1997) Local and propagated vascular responses evoked by focal synaptic activity in cerebellar cortex. J Neurophysiol 78:651–659.

20.

Ido

Chang

Woolsey

T.A.

Williamson

J.R.

(2001) NADH: Sensor of blood flow need in brain, muscle, and other tissues. FAESB J 15:1419–1421.

21.

Kleinfeld

Mitra

P.P.

Helmchen

Denk

(1998) Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex. Proc Natl Acad Sci U S A 95:15741–15746.

22.

Knot

H.J.

Zimmermann

P.A.

Nelson

M.T.

(1996) Extracellular K(+)-induced hyperpolarizations and dilatations of rat coronary and cerebral arteries involve inward rectifier K(+) channels. J Physiol 492:419–430.

23.

Little

T.L.

Beyer

E.C.

Duling

B.R.

(1995a) Connexin 43 and connexin 40 gap junctional proteins are present in arteriolar smooth muscle and endothelium in vivo. Am J Physiol 268:H729–H739.

24.

Little

T.L.

Xia

Duling

B.R.

(1995b) Dye tracers define differential endothelial and smooth muscle coupling patterns within the arteriolar wall. Circ Res 76:498–504.

25.

Malonek

Grinvald

(1996) Interactions between electrical activity and cortical microcirculation revealed by imaging spectroscopy: Implications for functional brain mapping. Science 272:551–554.

26.

Mayhew

Johnston

Berwick

Jones

Coffey

Zheng

(2000) Spectroscopic analysis of neural activity in brain: Increased oxygen consumption following activation of barrel cortex. Neuroimage 12:664–675.

27.

McCasland

J.S.

Woolsey

T.A.

(1988) High-resolution 2-deoxyglucose mapping of functional cortical columns in mouse barrel cortex. J Comp Neurol 278:555–569.

28.

McGahren

E.D.

Beach

J.M.

Duling

B.R.

(1998) Capillaries demonstrate changes in membrane potential in response to pharmacological stimuli. Am J Physiol 274:H60–H65.

29.

Melzer

Van der Loos

Dorfl

Welker

Robert

Emery

Berrini

J.C.

(1985) A magnetic device to stimulate selected whiskers of freely moving or restrained small rodents: Its application in a deoxyglucose study. Brain Res 348:229–240.

30.

Mintun

M.A.

Lundstrom

B.N.

Snyder

A.Z.

Vlassenko

A.G.

Shulman

G.L.

Raichle

M.E.

(2001) Blood flow and oxygen delivery to human brain during functional activity: Theoretical modeling and experimental data. Proc Natl Acad Sci U S A 98:6859–6864.

31.

Moskalenko

Y.E.

Woolsey

T.A.

Rovainen

C.M.

Erinjeri

J.P.

Liu

Weinstein

G.B.

Semernia

V.N.

(1997) Dynamics of LCBF, PO2, electrical impedance (EIM) and intrinsic optical signal (IOS) in rat barrel cortex during whisker stimulation. Soc Neurosci Abstr 23:1578.

32.

Powell

T.P.S.

Mountcastle

V.B.

(1959) Some aspects of the functional organization of the cortex of the postcentral gyrus of the monkey: A correlation of findings obtained in a single unit analysis with cytoarchitecture. Bull Johns Hopkins Hosp 105:133–162.

33.

Raichle

M.E.

(1983) Positron emission tomography. Ann Rev Neurosci 6:249–67.

34.

Raichle

M.E.

(1994) Visualizing the mind. Sci Am 270:58–64.

35.

Ratzlaff

E.H.

Grinvald

(1991) A tandem-lens epifluorescence macroscope: Hundred-fold brightness advantage for wide-field imaging. J Neurosci Methods 36:127–137.

36.

Rovainen

C.M.

Woolsey

T.A.

Blocher

N.C.

Wang

D.B.

Robinson

O.F.

(1993) Blood flow in single surface arterioles and venules on the mouse somatosensory cortex measured with videomicroscopy, fluorescent dextrans, nonoccluding fluorescent beads, and computer-assisted image analysis. J Cereb Blood Flow Metab 13:359–371.

37.

Silva

A.C.

Lee

S.P.

Iadecola

Kim

S.G.

(2000) Early temporal characteristics of cerebral blood flow and deoxyhemoglobin changes during somatosensory stimulation. J Cereb Blood Flow Metab 20:201–206.

38.

Simons

D.J.

(1983) Multi-whisker stimulation and its effects on vibrissa units in rat SmI barrel cortex. Brain Res 276:178–182.

39.

Simons

D.J.

(1985) Temporal and spatial integration in the rat SI vibrissa cortex. J Neurophysiol 54:615–635.

40.

Simons

D.J.

Woolsey

T.A.

(1979) Functional organization in mouse barrel cortex. Brain Res 165:327–332.

41.

Tyml

Song

Munoz

Ouellette

(1997) Evidence for K+ channels involvement in capillary sensing and for bidirectionality in capillary communication. Microvasc Res 53:245–253.

42.

Wong-Riley

(1979) Changes in the visual system of monocularly sutured or enucleated cats demonstrable with cytochrome oxidase histochemistry. Brain Res 171:11–28.

43.

Wong-Riley

M.T.

Welt

(1980) Histochemical changes in cytochrome oxidase of cortical barrels after vibrissal removal in neonatal and adult mice. Proc Natl Acad Sci U S A 77:2333–2337.

44.

Woolsey

T.A.

(1967) Somatosensory, auditory and visual cortical areas of the mouse. Johns Hopkins Med J 121:91–112.

45.

Woolsey

T.A.

Van der Loos

(1970) The structural organization of layer IV in the somatosensory region (SI) of mouse cerebral cortex. The description of a cortical field composed of discrete cytoarchitectonic units. Brain Res 17:205–242.

46.

Woolsey

T.A.

Wann

J.R.

(1976) Areal changes in mouse cortical barrels following vibrissal damage at different postnatal ages. J Comp Neurol 170:53–66.

47.

Woolsey

T.A.

Durham

Harris

R.M.

Simons

D.J.

Valentino

K.L.

(1981) Sensory development. In: Development of perception, vol 1 ( Aslin

R.N.

Alberts

J.R.

Petersen

M.R.

, eds), New York, Academic Press, pp 259–292.

48.

Woolsey

T.A.

Rovainen

C.M.

Cox

S.B.

Henegar

M.H.

Liang

G.E.

Liu

Moskalenko

Y.E.

Sui

Wei

(1996) Neuronal units linked to microvascular modules in cerebral cortex: Response elements for imaging the brain. Cereb Cortex 6:647–660.

49.

Yarowsky

Kodeko

Solkoff

(1983) Frequency-dependent activation of glucose utilization in the superior cervical ganglion by electrical stimulation of cervical sympathetic trunk. Proc Nat Acad Sci U S A 80:4179–4183.