1 Laboratory for Integrative Neural Systems, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan, 2 Present address: Department of Neurobiology, Brain Imaging Research Center, University of Pittsburgh, 3025 East Carson Street, PA 15203, USA, 3 Present address: Laboratory for Cognitive Brain Mapping, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan, 4 Present address: Advanced Technology Research Laboratories, Matsushita Electric Industrial Co., Ltd., 3-10-1 Higashimita, Tama-ku, Kawasaki-shi, 214-8501, Japan
Address correspondence to Manabu Tanifuji, Laboratory for Integrative Neural Systems, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan. Email: tanifuji{at}postman.riken.go.jp.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: cerebral blood flow functional MRI hemodynamic response intrinsic signal imaging orientation column spectroscopic analysis
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Circumstantial evidence suggests that hemodynamic responses, such as changes in deoxyhemoglobin (Hbr) concentration and blood volume changes, are the major sources of intrinsic signals at visible wavelengths (Frostig et al., 1990; Bonhoeffer and Grinvald, 1996
; Malonek and Grinvald 1996
). Decreases in light reflection (i.e. increases in light absorption) at 600630 nm, where the absorption coefficient of Hbr is 510 times higher than that of oxyhemoglobin (HbO2) (Fig. 1 inset), suggest that increases in Hbr concentration are accompanied by the oxygen consumption of activated neurons (Silver, 1978
; Sibson et al., 1998
; Thompson et al., 2003
). The decrease in light reflection at the wavelength where the absorption coefficient of HbO2 equals that of Hbr (i.e. hemoglobin's isosbestic point) suggests that an increase in total hemoglobin (Hbt) concentration (blood volume change) is another component of the signal. In addition to these hemodynamic components, activity-dependent light scattering (Ls) changes (MacVicar and Hochman, 1991
; Holthoff and Witte, 1996
) may also be involved in intrinsic signals at wavelengths >700 nm, at which the absorption coefficients of both HbO2 and Hbr are relatively small (Maheswari et al., 2003
).
|
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animal Preparations
Cats were initially anesthetized with an inhalation of isoflurane (22.5%) in a mixture of 50% N2O and 50% O2. After tracheal cannulation, anesthesia was maintained with a mixture of 70% N2O and 30% O2 supplemented with 12% isoflurane. The cephalic vein was catheterized and neuromuscular blockade was carried out by continuous infusion of pancuronium bromide (0.2 mg/kg/h) mixed with dexamethazone (0.05 mg/kg/h) and 7.5% glucose in lactose-containing Ringer's solution. The cats were then artificially ventilated. To prevent the cornea from drying, contact lenses were fitted to the eyes. We continuously monitored rectal temperature, electroencephalogram (EEG), electrocardiogram (ECG), and expired CO2 to assess the depth of anesthesia. Rectal temperature was maintained at 37.538.5 °C with a feedback-regulated heating pad system. The expired CO2 was maintained between 3.0 and 4.0%.
On the first day of recording, a cat was placed in a stereotaxic apparatus (SN-3N, Narishige). Under aseptic surgery, we first attached with dental acrylic cement a metal post to fix its head and a stainless steel chamber (18 mm inner diameter, Nakazawa-Seisaku, Japan) for optical imaging to the skull. The metal post was placed approximately above the bregma. The chamber was placed such that it included area 17 or the border between areas 17 and 18 (in HorsleyClarke coordinates, approximately A5P10 for area 17 and A10P5 for the border between areas 17 and 18). We then performed craniotomy inside the chamber, and resected the dura mater. The inside of the chamber was then filled with 1.52.0% agarose (Agarose-HGS, gel strength 1.5%; Nacalai Tesque, Japan) containing dexamethazone (0.1 mg) and an antibiotic (gentamicin, 0.25 mg). Finally, the chamber was covered with a round glass coverslip and sealed with a screw-top lid including a silicone gasket. We could observe the cortical surface clearly through the glass coverslip and could visualize the same functional structures repeatedly for 23 weeks without cleaning the inside of the chamber. After the surgery and the recordings, an appropriate antibiotic (cefodizime sodium, 60 mg/kg i.m.) was administered to the cat before returning the animal to its home cage.
During recordings on the first and subsequent days, the cat's head was immobilized with a head post instead of ear bars. Pupils were dilated by applying 0.5% tropicamide and 0.5% phenylephrine hydrochloride. The isoflurane concentration was maintained at 0.51.0% during the recordings. The other conditions were the same as those in the initial surgery described above.
Visual Stimuli
Square wave gratings (white: 8 cd/m2; black: 0 cd/m2) were generated with a VSG2/3 graphics video board (Cambridge Research Systems, Rochester, UK), controlled by homemade software, and were presented on a monitor screen (640 x 480 pixels and 100 Hz refresh rate, GDM-20SE3T, Sony). The spatial frequency and the drifting velocity of the gratings were 0.5 cycles/deg and 4 deg/s for area 17, and 0.15 cycles/deg and 15 deg/s for area 18 (Bonhoeffer et al., 1995). The drifting direction was reversed every 0.5 s during a 2 s stimulus presentation. Two or four stimuli [orientations, 0° (horizontal), 45°, 90° and 135°] together with a blank screen (homogenous gray, 4 cd/m2) as a control were presented in a pseudorandom order. In the experiment using awake cats, we used stationary grating patterns flickering at 5 Hz (8 Hz in one cat) to minimize the effect of eye movements following the grating motion. The same flickering gratings were also used in the examination of the same cats under anesthesia for comparison.
The center of a cat's visual field was estimated by projecting images of optic disks and patterns of surrounding vessels onto the monitor screen in front of the cat. The screen was placed at a distance ranging from 20 to 40 cm, where the best focus of the optic disks and the patterns of surrounding vessels were obtained for each cat. At these distances the size of the screen corresponded to 43°86° (width) x 36°71° (height) of the cat's visual field. In the experiment with awake cats, the screen was placed 20 cm in front of the animals.
Optical Imaging of Intrinsic Signals
We developed a multiple wavelength imaging system equipped with three identical cameras (Sanso-Seisaku, Japan) that enabled us to simultaneously record intrinsic signals at three different wavelengths (Fig. 1). We used two sets with the same configuration: one was equipped with CID-2221D video cameras (CIDTEC, Liverpool, NY), and the other was equipped with CS8310 video cameras (Tokyo Electric Industry, Japan). The exposed cortical surface was illuminated with white light using eight fiber optic bundles placed around the chamber, which were connected to two tungstenhalogen bulbs (82 V, 300 W; Philips) driven by stabilized DC power supplies (PD110-5D; Kenwood, Japan). The duration of exposure of the cortical surface to the light was restricted to 10 s using a mechanical shutter, which opened 2 s before starting image acquisition. Reflected light from the cortical surface was collected by an objective lens, and divided into three separate paths by prisms. Each light beam passed through an interference filter tuned to one of three different wavelengths (538, 569 and 620 ± 10 nm; Asahi Spectra, Japan) and focused onto one of three video cameras by each projection lens. A combination of objective and projection lenses constituted the tandem-lens optics (Ratzlaff and Grinvald, 1991). Using different combinations of a projection lens (50 mm, f1.2; Nikon, Japan) and an objective lens (35 mm, f1.4 or 50 mm, f1.2; Nikon, Japan), imaging areas were 4.9 x 4.9 mm2 or 7.0 x 7.0 mm2 for the CID-2221D camera (256 x 256 pixels) and 8.8 x 6.6 mm2 for the CS8310 camera (640 x 480 pixels). The imaging areas of the three cameras were adjusted to overlap in order to record signals from the same region of the cortical surface. Video signals from the three cameras were separately digitized with 10-bit video A/D converter boards (Pulsar, Matrox Graphics Inc., Canada) on three computers. These computers synchronously acquired 240 frames at a frame rate of 1/30 s for the CS8310 camera or 1/60 s for the CID-2221D camera. Fifteen consecutive frames for the CS8310 camera or 15 alternate frames for the CID-2221D camera were averaged on-line. Consequently, 16 images were acquired with a temporal resolution of 500 ms (i.e. 8 s). Acquired images were stored on hard disks without binning for the CID-2221D camera (256 x 256 pixels) and with binning (2 x 2 pixels were combined into a single pixel) for the CS8310 camera (i.e. 320 x 240 pixels).
Images of the cortical surface were taken using these cameras and the focal plane was changed from the cortical surface to 700800 µm below. The maximum intensities of video signals from the three cameras were adjusted to near-saturation level by changing the intensity of incident light and camera gains. The black levels of each video signal (a video signal obtained under complete darkness, 10% of saturation level) were then recorded for off-line data processing. In the experiment with anesthetized cats, data acquisition was started at a certain phase of respiration in synchrony with heartbeat. A visual stimulus appeared 1 s after the onset of image acquisition. To allow the relaxation of vascular responses to the previous stimulation, the interstimulus interval (ISI) was set at 30 s. Data of four trials using the same stimulus were averaged online and saved as one block. We recorded 20 blocks in one experiment; altogether, responses for 80 trials (20 blocks, 4 trials per block) were acquired for each stimulus.
In the recordings with awake cats, the body of the cat was placed into a loosely fitting pouch and the head was immobilized by the implanted head post. The cats quickly became accustomed to the restriction of movements and presented no signs of discomfort during the experiments. To minimize visual input from surroundings, the experiments were performed in a dark room and mechanical shutters were placed in front of the cat's eyes. The shutters were only opened during the stimulus presentation. Unlike the experiment under anesthesia, data acquisition was not synchronized with respiration and heartbeat. The total recording period was restricted to 1.5 h/day to maintain the cats' alertness level. Data from 40 trials were averaged (20 blocks, 2 trials per a block) for each stimulus in one day. The experiments were repeated for two successive days and the data from the two days were finally averaged. The temporal and spatial patterns of intrinsic signals on the first day were almost identical to those on the second day (data not shown). The data from the same cat under anesthesia were also collected. To ensure that recordings were taken from the same region, we did not change the camera position relative to the head post until a series of experiments for the cat had been completed. We did not observe a misalignment of cortical vascular patterns in the series of experiments.
Imaging of Changes in Blood Volume with Intravascular Absorption Dye
For the measurement of changes in blood volume, we injected a light-absorbing dye (Nigrosin, water-soluble Acid Black 2; Sigma) through the cat's cephalic vein and recorded the stimulus-evoked absorption changes at 620 nm. We chose absorption dye instead of fluorescent dyes, which have previously been used for the measurement of blood volume (Frostig et al., 1990; Narayan et al., 1995
; Cannestra et al., 1998
), because (i) fluorescent dye signals are in general too weak to resolve small changes such as stimulus-specific components, (ii) there is minimal effect of dye bleaching, and (iii) the same optics can be employed to assess the dye-specific responses immediately after the intrinsic signal imaging. The dye was dissolved in saline, filtered using a Millipore filter (0.22 µm pore size, Millipore Co. Bedford, MA), and injected prior to recording (final dosage, 2034 mg/kg nigrosin for five cats). The physiological conditions (e.g. heart rate) of the cats did not change following the injection of the dye. Since the dye absorbed the incident light, the reflected light intensity from the cortical surface decreased after the dye injection. The intensity of the reflected light was readjusted to the saturation level of video signals prior to recording by increasing the incident light intensity. Data from 2080 trials were averaged (5 to 20 blocks, 4 trials per block) for each stimulus.
Data Analysis
We analyzed all images pixel by pixel using IDL 5.4 (Research Systems, Inc.). The statistical significance of the data was evaluated by t-test (two-tailed, paired). The first step in the data analysis was to extract reflected light intensity from video signals by subtracting the image obtained in complete darkness (the black level) from the 16 consecutive images (8 s at 0.5 s/image). A change in reflected light intensity (intrinsic signals) was then expressed as the change in the optical density (OD) as follows:
![]() | (1) |
To demonstrate the spatial patterns of iso-orientation domains, differential images were generated by subtracting responses to one orientation from those to the orthogonal orientation. The differential images were then temporally averaged from 1 to 7 s after stimulus onset and processed using a Gaussian spatial filter (cutoff frequencies, = 10/mm for a high cutoff frequency and 1/mm for a low cutoff frequency for images obtained by the CS8310 camera, and
= 5/mm for high cutoff and 1/mm for low cutoff for images obtained by the CID-2221D camera). The similarity between two differential images obtained at different wavelengths was quantified by calculating a correlation coefficient on pixel-by-pixel basis. In particular, when two pairs of orthogonal stimuli (the combination of 0° and 90° or that of 45° and 135°) were used at these wavelengths, a correlation coefficient of differential images was calculated for each pair separately, and then an average of the correlation coefficients was used to evaluate the similarity of differential images obtained at these wavelengths.
To quantitatively examine the intensities of intrinsic signals we averaged pixels in the region of interest (ROI). Pixels covering surface vessels thicker than 50 µm and those located outside of the cortex were excluded from the ROI. We divided ROI into active and less-active domains and averaged pixels of the active domains separately from pixels of the less-active domains. The active and less-active domains were determined on the basis of a differential image at 620 nm processed with the spatial filter. Pixels having positive values in the differential image were assigned to the active domains and the remaining pixels were assigned to the less-active domains. The difference in the signal intensity between two domains was calculated by subtracting the average pixel value for the less-active domains from that for the active domains.
To quantify the spatial resolution of intrinsic signals, we measured the distance between neighboring iso-orientation domains in differential images. The distance between neighboring iso-orientation domains in the differential image was evaluated using an auto-correlation map of the differential image using NIH Image software (Scion Corporation) since the pattern of iso-orientation domains seems to have a periodic structure. The differential image used for this analysis was not processed by any spatial filter. The autocorrelation map was calculated for an ROI of 128 x 128 pixels (2.5 x 2.5 mm2 for the CID-2221D camera with a 35 mm objective lens and 3.5 x 3.5 mm2 for the CID-2221D and CS8310 cameras with a 50 mm objective lens) in the differential images. We then extracted the profile of the autocorrelation map along the central and secondary largest peaks. We assigned the distance between these two peaks as the distance between neighboring iso-orientation domains assuming that the periodic structure of the iso-orientation domains is the most dominant one in the map. The size of iso-orientation domains was estimated by measuring full width at half-maximum (FWHM) of the center peak of the profile.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Figure 2A,B shows the spatiotemporal patterns of the intrinsic signal at 620 nm in cat visual cortex induced by full-field grating stimuli. The grating stimuli evoked initial increases in light absorption (darkening of the cortex), which were followed by absorption decreases across the baseline (lightening of the cortex). These absorption changes were not spatially confined to domains specific for particular stimulus orientations. As reported previously (Grinvald et al., 1986), two orthogonal orientations elicited a common absorption increase over the entire range (stimulus-nonspecific component), which was locally modulated in a stimulus-specific manner (stimulus-specific component) (Fig. 2D). Since this stimulus-specific modulation is complementary in two orthogonal orientations (Fig. 2D, red and blue lines), the region showing this stimulus specificity was extracted by subtracting the response for one orientation from that for the orthogonal orientation (Fig. 2C, and Fig. 2D, green line). We define regions where a stimulus elicited a larger absorption than the orthogonal stimulus, such as the shaded regions shown in Figure 2D, as active domains for the stimulus. On the other hand, we define regions where the stimulus elicited smaller increases in absorption than the orthogonal stimulus as less-active domains for the stimulus. For the analysis of the stimulus-nonspecific component, we averaged the intrinsic signals regardless of whether the domain was active or less active. For the analysis of the stimulus-specific component, we subtracted the intrinsic signal for the less-active domains from the signal for the active domains (see also Materials and Methods).
|
As an approximation, we assume that Hbr and HbO2 concentration changes are the major sources of intrinsic signals at visible wavelengths. Intrinsic signals at 620 and 569 nm then correspond approximately to changes in Hbr concentration and that of total hemoglobin (Hbt) concentration (the sum of Hbr and HbO2 concentrations), respectively. This is because at 620 nm, the absorption coefficient of Hbr is about 10 times larger than that of HbO2 and 569 nm corresponds to the isosbestic point of Hbr and HbO2 absorption (Fig. 1 inset). In addition, we recorded intrinsic signals at 538 nm, where HbO2 has a higher absorption coefficient than Hbr. Figure 3A shows the time courses of stimulus-nonspecific components of the intrinsic signals obtained at these wavelengths. At 620 nm, we consistently observed biphasic time courses, in which the light absorption increased after stimulus onset, reached a maximum after 2 s and decreased, going below the baseline (time to reach the minimum, 5 s). On the other hand, the time courses of the signals at 538 and 569 nm were monophasic: the light absorption increased and returned to the baseline without crossing it (time to reach the maximum, 3.5 s). In accordance with the absorption coefficients of hemoglobin at 538 and 569 nm, light absorption changes at 538 nm were slightly larger than those at 569 nm. The biphasic time course of the signal at 620 nm suggests that Hbr concentration initially increased due to oxygen consumption of activated neurons, which was followed by a decrease in Hbr concentration below the baseline due to blood inflow outstripping oxygen consumption. On the other hand, the absorption increase at 569 nm can be explained by the increase in Hbt resulting from the increase in blood inflow.
|
Unlike the stimulus-nonspecific components of the intrinsic signals, the polarity of the stimulus-specific components did not change at these wavelengths (Fig. 3B). Thus, to visualize the spatial patterns of the stimulus-specific components, we first temporally averaged the intrinsic signals from 1 to 7 s after stimulus onset. Then, the differential images of the signals were calculated by subtracting the temporally averaged images for one orientation from those for the orthogonal orientation (Fig. 4A). Even if the physiological sources of the signals seem to be different among these wavelengths, the spatial patterns of the stimulus-specific components were almost identical. The correlation coefficients between the differential images obtained at 620 nm and those at other wavelengths calculated on a pixel-by-pixel basis were significantly high (0.86 and 0.90 for the images obtained at 538 and 569 nm respectively; P < 0.01). We obtained consistent results for the other 13 cats: the average correlation coefficients for the 14 cats were 0.78 ± 0.08 for the images at 538 nm and 0.71 ± 0.09 for the images at 569 nm. These values indicate that there is a statistically significant correlation between differential images obtained at 620 nm and those obtained at other wavelengths (P < 0.01). These results indicate that the intrinsic signals recorded at these three wavelengths have a sufficient spatial resolution to resolve orientation-specific columnar organizations.
|
Measurement of Blood Volume Changes Based on Dye-specific Absorption Changes
Since intrinsic signals at 569 nm are considered to be proportional to Hbt concentration, the above analysis indicates that the blood volume component, as well as the Hbr component, has sufficient spatial precision to visualize individual iso-orientation domains in areas 17 and 18 of the cat visual cortex.
To further confirm our interpretation, we injected an absorption dye into the bloodstream and measured changes in blood volume on the basis of dye-specific absorption changes. Three observations provided evidence that the dye-specific absorption changes reflected changes in blood volume. First, the injections of the dye into the bloodstream caused a 17 ± 3% (mean ± SD, n = 5 cats) increase in light absorption. Second, the dye injections also increased the amplitude of the stimulus-nonspecific component of changes in absorption (Fig. 5A). The ratio of peak amplitudes before and after injections was 2.1 ± 1.1 (mean ± SD, n = 5). Thirdly, we found that the time courses of the stimulus-nonspecific component for the intrinsic signal at 569 nm were the same as those for the dye-specific absorption changes (Fig. 5B).
|
|
To examine the blood volume component of intrinsic signals also in awake cats, we compared the intrinsic signals at 569 nm in anesthetized and awake states from the same cat. To avoid the effect of eye movements following the grating stimulus motion on the intrinsic signal in the awake state, we recorded the signal evoked by a flickering grating stimulus instead of a moving grating stimulus both in anesthetized and awake states in this experiment. We confirmed beforehand that these two stimuli elicited nearly the same responses in temporal patterns and identical response in the spatial patterns of iso-orientation domains in anesthetized cats (data not shown).
The time courses of intrinsic signals at 569 nm in the awake state were similar to those in the anesthetized state. The time to reach the peak (mean ± SD, n = 5 cats) of the intrinsic signal at 569 nm was 4.2 ± 0.6 s in the anesthetized state and 4.1 ± 0.46 s in the awake state. The spatial patterns of iso-orientation domains revealed by differential images at 569 nm in both states were almost identical (Fig. 7). Quantitatively, the correlation coefficient between the two images was 0.77 in this example. This value and the correlation coefficients obtained from the other four cats indicate that there is a statistically significant correlation between these images [average correlation coefficient: 0.74 ± 0.11 (mean ± SD); P < 0.01]. The average distance between neighboring iso-orientation domains and the average FWHM for the five awake cats was 1.26 ± 0.31 and 0.60 ± 0.10 mm respectively (mean ± SD). These values are similar to those obtained from the above-mentioned anesthetized cats. These results suggest that stimulus-specific changes in blood volume are not specific to the anesthetized state.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Several studies revealed the submillimeter-scale spatial localization of Hbr concentration changes in visual cortex (Malonek and Grinvald, 1996; Kim et al., 2000
; see also Thompson et al., 2003
). In this study, we examined the spatial precision of changes in blood volume using two different methods of analyzing reflection changes elicited by neural activation: (i) intrinsic signal imaging at the wavelength of the isosbestic point of hemoglobin, and (ii) analysis of stimulus-induced absorption changes of an intravenously infused dye. These analyses provide concrete evidence supporting a previous proposal that changes in blood volume are spatially localized (Frostig et al., 1990
). We showed that blood volume, as well as Hbr concentration, is precisely controlled at a submillimeter-scale resolution in areas 17 and 18 of the cat visual cortex in anesthetized and awake states.
It should be pointed out that a blood volume increase includes an intravascular space increase accompanied by the dilation of vessels (increase in plasma volume) and an increase in the number of red blood cells. The measurement of the intrinsic signal at 569 nm is sensitive to the increase in the number of red blood cells. On the other hand, it is considered that the increase in dye-specific absorption change reflects the increase in plasma volume. However, we cannot exclude the possibility that the dye-specific responses also reflect the increased number of red blood cells, since we did not quantitatively examine the possibility that the dye molecules were accumulated in or bound to red blood cells. Taking into account a previous in vivo microscopy study (Villringer et al., 1994) demonstrating that increase of red blood cells in a capillary is associated with capillary dilation (i.e. increase of plasma volume) during hypercapnia, it is more plausible that these two effects the increase in intravascular space and the increase in the number of red blood cells are coupled and show similar behavior even when these changes are elicited by neural activation.
From the measurement of spatial separation and the size of the iso-orientation domains, we estimated the spatial resolution of blood volume changes to be as high as 0.6 mm. Recently, Duong et al. (2001) have reported, using a CBF-based fMRI technique, that the size of iso-orientation domains was 0.47 mm. These results suggest that localization of blood flow changes is stronger than that of blood volume changes. In fact, unlike blood volume changes, Duong et al.'s experiment did not show any apparent stimulus-nonspecific component of CBF changes. They reported that the average CBF percentage-change ratio of the active to inactive domains was 3.3. This means that the stimulus-specific component of CBF changes was
70% of the stimulus-nonspecific component. In contrast, this percentage was 2.3% in our measurement of blood volume changes. Further examinations are required to confirm these findings.
Limitation of Analyses of Intrinsic Signals at Multiple Wavelengths
Our analysis of intrinsic signals at multiple wavelengths is based on the assumption that intrinsic signals at visible wavelengths mainly reflect absorption changes of hemoglobin. According to this assumption, we used intrinsic signals at 569 nm as a measure of blood volume changes. We found that the time course of the intrinsic signals at 569 nm was similar to that of the dye-specific absorption changes, which supported the findings of this approach.
Although there was some supporting evidence for intrinsic signals at the isosbestic point of hemoglobin as a measure of blood volume, the isolation of components in intrinsic signals by the recording wavelengths may not be exclusive. For example, components other than hemoglobin absorption changes, such as Ls changes (MacVicar and Hochman, 1991; Kreisman et al., 1995
; Holthoff and Witte, 1996
; Momose-Sato, 1998
; Andrew et al., 1999
; Maheswari et al., 2003
; Sato et al., 1997
; see also Tomita et al., 1983
), may be involved in intrinsic signals. One way to better isolate individual components is to use a model that describes light reflection from the cortical surface (Malonek and Grinvald, 1996
; Mayhew et al., 1998
; Nemoto et al., 1999
; Lindauer et al., 2001
). We have also analyzed our result using such a model and obtained results consistent with results described above (see Appendix). However, such analyses are not accurate and only provide semi-quantitative results because there is no exact model for changes in reflection from cortical surfaces.
Underlying Mechanisms of Stimulus-specific and Stimulus-nonspecific Components of the Blood Volume Component
In the present study, we have demonstrated that in the cat visual cortex the blood volume changes were resolved in individual iso-orientation domains 0.6 mm in size. Taking into account the fact that the spatial separation of arteries is larger than that of functional domains, this finding suggests that fine mechanisms of blood volume control exist in fine vessel components, such as precapillary arterioles and capillaries whose spatial separations are definitely <0.6 mm (Pawlik et al., 1981
). The presence of such blood volume components is supported by anatomical studies showing contractile structures that may control blood flow and/or volume at the branching points of capillaries (Nakai et al., 1981
; Kuschinsky and Paulson, 1992
; Shepro and Morel, 1993
; Harrison et al., 2002
). This stimulus-specific component of blood volume changes, however, is only a small fraction of their stimulus-nonspecific component. To extract stimulus-specific changes in blood volume, for example, subtracting responses evoked by one orientation from those evoked by the orthogonal orientation is necessary in the visual cortex (see also for rodent barrel cortex, Hess et al., 2000
). There are three possibilities that explain the origins of stimulus-nonspecific components of the blood volume changes.
First, light scattering can limit the spatial resolution of optical measurement. Orbach and Cohen (1983) demonstrated that the light from a small 40 µm diameter spot spread to
200 µm in diameter 500 µm away from the spot in cortical tissue. Because of this light scattering, stimulus-specific absorption changes can also spread into cortical domains related to the orthogonal stimulus. Thus, the stimulus-nonspecific component of the signals may be explained by the spread of the stimulus-specific component. If this is the case, however, it is difficult to explain the different ratios of signal magnitudes in the awake state to those in the anesthetized state between stimulus-specific and stimulus-nonspecific components (2.7 versus 3.6 in Fig. 8C).
Second, the distinction between stimulus-specific and stimulus-nonspecific components may be related to the specificity of neural activities coupled to the intrinsic signals. Assuming that synaptic activities are coupled to blood volume changes, blood volume changes can be elicited not only at regions where action potentials are generated but also at regions where subthreshold synaptic potentials are generated. The stimulus-nonspecific component may reflect subthreshold synaptic potentials elicited regardless of the stimulus orientation. The mechanisms of coupling between neural activities and intrinsic signals remain issues for future investigations.
Finally, as the most plausible possibility, we consider the contribution of distinct mechanisms of blood flow control in small (precapillary, capillary) and large vessels (artery). Neural activations induce blood flow increases in arteries (Ngai et al., 1988, 1995
; Akgoren and Lauritzen, 1999
). The arteries do not necessarily govern a particular iso-orientation domain. Blood flow increases in arteries should then induce nonspecific blood volume increases in downstream small vessels (precapillary, capillary and also probably arteriole), though these vessels have a submillimeter spatial precision (but see Iadecola et al., 1997
). Assuming supplemental blood flow control mechanisms in a capillary bed, we can expect stimulus-specific and -nonspecific components of blood volume changes. Because this proposal assumes two distinct control mechanisms, we can explain the different ratios of signal amplitudes in awake state to those in anesthetic state between stimulus-specific and stimulus-nonspecific components (Fig. 8C). Furthermore, the involvement of different vascular systems in the stimulus-specific and -nonspecific components of blood volume changes also explains the absence of correlation (r = 0.43, P = 0.12, n = 14) between the peak amplitudes of stimulus-specific and -nonspecific components.
![]() |
Conclusions |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Appendix |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() | (2) |
To calculate the concentration changes of HbO2, Hbr and the Ls component from intrinsic signals at 538, 569 and 620 nm, we analytically solved equation (2). The total hemoglobin (Hbt) concentration change was calculated as the sum of HbO2 and Hbr concentration changes. The calculations were performed for individual stimuli and for control (blank screen) separately. Then, we subtracted the result calculated for the control (blank screen) from those for the stimuli. The product of the light path length, d, and the concentration of each component was used as a measure of each component, because d cannot be determined in the reflection measurement.
Figure 9A shows stimulus-induced changes in these three components. The time courses of the Ls component were biphasic, and did not vary greatly among the cats. The time courses of the Hbt concentration changes were monophasic and largely varied among the cats. To confirm whether the Hbt components faithfully represent blood volume changes, we compared the Hbt concentration changes with the stimulus-nonspecific components of the intrinsic signal at 569 nm (green line) and the dye-specific change (black line), and found these three signals showed very similar time courses (Fig. 9B). The time courses of the Hbr also varied among the cats. In 4 of 14 cats, the Hbr concentration initially increased after stimulus onset, reached a maximum and decreased below the baseline. However, in most cats, the changes in Hbr concentration did not cross the baseline. Consequently, the average time course of the Hbr concentrations for 14 cats was monophasic. The decrease in the Hbr concentration from the baseline in the late phase was not prominent in our study, but was in the previous studies (Malonek and Grinvald, 1996; Malonek et al., 1997
; Nemoto et al., 1997
, 1999
; Mayhew et al., 1999
, 2000
, 2001
; Shtoyerman et al., 2000
, Jones et al., 2001
, Lindauer et al., 2001
). The discrepancy may be due to the following reasons. First, the solution based on the BeerLambert equation is largely affected by light pathlengths that may not be necessarily the same across the recording wavelengths used for the analysis (Mayhew et al., 1999
, Lindauer et al., 2001
). Second, time courses of intrinsic signals varied (e.g. at 607 nm, data not shown) since the relative contributions of Hbr, Hbt and Ls to the intrinsic signals may not be the same under different experimental conditions, such as individual specificity, anesthetic agents (Lindauer et al., 1993
), surgical procedure, recording area, stimulus type and species difference. For example, the time to reach the peak of the intrinsic signals depends on the stimulus frequency and duration (data not shown). Finally, there was no way to evaluate errors associated with the calculation of equation (2), since measurements from three wavelengths are used to obtain three parameters, the concentration changes of HbO2, Hbr and the Ls component. Thus, in some of our measurements, we might fail to obtain reliable values of Hbr concentration changes, particularly late in the time course.
|
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Andrew RD, Jarvis CR, Obeidat AS (1999) Potential sources of intrinsic optical signals imaged in live brain slices. Methods 18:185196, 179.[CrossRef][ISI][Medline]
Bonhoeffer T, Grinvald A (1991) Iso-orientation domains in cat visual cortex are arranged in pinwheel-like patterns. Nature 353:429431.[CrossRef][ISI][Medline]
Bonhoeffer T, Grinvald A (1993) The layout of iso-orientation domains in area 18 of cat visual cortex: optical imaging reveals a pinwheel-like organization. J Neurosci 13:41574180.[Abstract]
Bonhoeffer T, Grinvald A (1996) Optical imaging based on intrinsic signals: the methodology. In: Brain mapping: the methods (Toga AW, Mazziotta JC, eds), pp. 5597. San Diego, CA: Academic Press.
Bonhoeffer T, Kim DS, Malonek D, Shoham D, Grinvald A (1995) Optical imaging of the layout of functional domains in area 17 and across the area 17/18 border in cat visual cortex. Eur J Neurosci 7:19731988.[ISI][Medline]
Cannestra AF, Pouratian N, Shomer MH, Toga AW (1998) Refractory periods observed by intrinsic signal and fluorescent dye imaging. J Neurophysiol 80:15221532.
Cohen LB, Keynes RD (1971) Changes in light scattering associated with the action potential in crab nerves. J Physiol 212:259275.[ISI][Medline]
Duong TQ, Kim DS, Ugurbil K, Kim SG (2001) Localized cerebral blood flow response at submillimeter columnar resolution. Proc Natl Acad Sci USA 98:1090410909.
Frostig RD, Lieke EE, Ts'o DY, Grinvald A (1990) Cortical functional architecture and local coupling between neuronal activity and the microcirculation revealed by in vivo high-resolution optical imaging of intrinsic signals. Proc Natl Acad Sci USA 87:60826086.
Ghose GM, Ts'o DY (1997) Form processing modules in primate area V4. J Neurophysiol 77:21912196.
Grinvald A, Lieke E, Frostig RD, Gilbert CD, Wiesel TN (1986) Functional architecture of cortex revealed by optical imaging of intrinsic signals. Nature 324:361364.[CrossRef][ISI][Medline]
Harrison RV, Harel N, Panesar J, Mount RJ (2002) Blood capillary distribution correlates with hemodynamic-based functional imaging in cerebral cortex. Cereb Cortex 12:225233.
Hess A, Stiller D, Kaulisch T, Heil P, Scheich H (2000) New insights into the hemodynamic blood oxygenation level-dependent response through combination of functional magnetic resonance imaging and optical recording in gerbil barrel cortex. J Neurosci 20:33283338.
Holthoff K, Witte OW (1996) Intrinsic optical signals in rat neocortical slices measured with near-infrared dark-field microscopy reveal changes in extracellular space. J Neurosci 16:27402749.[Abstract]
Iadecola C, Yang G, Ebner TJ, Chen G (1997) Local and propagated vascular responses evoked by focal synaptic activity in cerebellar cortex. J Neurophysiol 78:651659.
Jones M, Berwick J, Johnston D, Mayhew J (2001) Concurrent optical imaging spectroscopy and laser-Doppler flowmetry: the relationship between blood flow, oxygenation, and volume in rodent barrel cortex. Neuroimage 13:10021015.[CrossRef][ISI][Medline]
Kim DS, Duong TQ, Kim SG (2000) High-resolution mapping of iso-orientation columns by fMRI. Nat Neurosci 3:164169.[CrossRef][ISI][Medline]
Kreisman NR, LaManna JC, Liao SC, Yeh ER, Alcala JR (1995) Light transmittance as an index of cell volume in hippocampal slices: optical differences of interfaced and submerged positions. Brain Res 693:179186.[CrossRef][ISI][Medline]
Kuschinsky W, Paulson OB (1992) Capillary circulation in the brain. Cerebrovasc Brain Metab Rev 4:261286.[ISI][Medline]
Lindauer U, Villringer A, Dirnagl U (1993) Characterization of CBF response to somatosensory stimulation: model and influence of anesthetics. Am J Physiol 264:H12231228.[ISI][Medline]
Lindauer U, Royl G, Leithner C, Kuhl M, Gold L, Gethmann J, Kohl-Bareis M, Villringer A, Dirnagl U (2001) No evidence for early decrease in blood oxygenation in rat whisker cortex in response to functional activation. Neuroimage 13:9881001.[CrossRef][ISI][Medline]
Maheswari RU, Takaoka H, Kadono H, Homma R, Tanifuji M (2003) Novel functional imaging technique from brain surface with optical coherence tomography enabling visualization of depth resolved functional structure in vivo. J Neurosci Methods 124:8392.[CrossRef][ISI][Medline]
MacVicar BA, Hochman D (1991) Imaging of synaptically evoked intrinsic optical signals in hippocampal slices. J Neurosci 11:14581469.[Abstract]
Malonek D, Grinvald A (1996) Interactions between electrical activity and cortical microcirculation revealed by imaging spectroscopy: implications for functional brain mapping. Science 272:551554.[Abstract]
Malonek D, Tootell RB, Grinvald A (1994) Optical imaging reveals the functional architecture of neurons processing shape and motion in owl monkey area MT. Proc R Soc Lond B Biol Sci 258:109119.[ISI][Medline]
Malonek D, Dirnagl U, Lindauer U, Yamada K, Kanno I, Grinvald A (1997) Vascular imprints of neuronal activity: relationships between the dynamics of cortical blood flow, oxygenation, and volume changes following sensory stimulation. Proc Natl Acad Sci USA 94:1482614831.
Mayhew J, Hu D, Zheng Y, Askew S, Hou Y, Berwick J, Coffey PJ, Brown N (1998) An evaluation of linear model analysis techniques for processing images of microcirculation activity. Neuroimage 7:4971.[CrossRef][ISI][Medline]
Mayhew J, Zheng Y, Hou Y, Vuksanovic B, Berwick J, Askew S, Coffey P (1999) Spectroscopic analysis of changes in remitted illumination: the response to increased neural activity in brain. Neuroimage 10:304326.[CrossRef][ISI][Medline]
Mayhew J, Johnston D, Berwick J, Jones M, Coffey P, Zheng Y (2000) Spectroscopic analysis of neural activity in brain: increased oxygen consumption following activation of barrel cortex. Neuroimage 12:664675.[CrossRef][ISI][Medline]
Mayhew J, Johnston D, Martindale J, Jones M, Berwick J, Zheng Y (2001) Increased oxygen consumption following activation of brain: theoretical footnotes using spectroscopic data from barrel cortex. Neuroimage 13:975987.[ISI][Medline]
Momose-Sato Y, Sato K, Hirota A, Kamino K (1998) GABA-induced intrinsic light-scattering changes associated with voltage-sensitive dye signals in embryonic brain stem slices: coupling of depolarization and cell shrinkage. J Neurophysiol 79:22082217.
Nakai K, Imai H, Kamei I, Itakura T, Komari N, Kimura H, Nagai T, Maeda T (1981) Microangioarchitecture of rat parietal cortex with special reference to vascular sphincters. Scanning electron microscopic and dark field microscopic study. Stroke 12:653659.[Abstract]
Narayan SM, Esfahani P, Blood AJ, Sikkens L, Toga AW (1995) Functional increases in cerebral blood volume over somatosensory cortex. J Cereb Blood Flow Metab 15:754765.[ISI][Medline]
Nemoto M, Nomura Y, Tamura M, Sato C, Houkin K, Abe H (1997) Optical imaging and measuring of local hemoglobin concentration and oxygenation changes during somatosensory stimulation in rat cerebral cortex. Adv Exp Med Biol 428:521531.[ISI][Medline]
Nemoto M, Nomura Y, Sato C, Tamura M, Houkin K, Koyanagi I, Abe H (1999) Analysis of optical signals evoked by peripheral nerve stimulation in rat somatosensory cortex: dynamic changes in hemoglobin concentration and oxygenation. J Cereb Blood Flow Metab 19:246259.[CrossRef][ISI][Medline]
Ngai AC, Ko KR, Morii S, Winn HR (1988) Effect of sciatic nerve stimulation on pial arterioles in rats. Am J Physiol 254:H133139.[ISI][Medline]
Ngai AC, Meno JR, Winn HR (1995) Simultaneous measurements of pial arteriolar diameter and laser-Doppler flow during somatosensory stimulation. J Cereb Blood Flow Metab 15:124127.[ISI][Medline]
Orbach HS, Cohen LB (1983) Optical monitoring of activity from many areas of the in vitro and in vivo salamander olfactory bulb: a new method for studying functional organization in the vertebrate central nervous system. J Neurosci 3:22512262.[Abstract]
Pawlik G, Rackl A, Bing RJ (1981) Quantitative capillary topography and blood flow in the cerebral cortex of cats: an in vivo microscopic study. Brain Res 208:3558.[CrossRef][ISI][Medline]
Ratzlaff EH, Grinvald A (1991) A tandem-lens epifluorescence macroscope: hundred-fold brightness advantage for wide-field imaging. J Neurosci Methods 36:127137.[CrossRef][ISI][Medline]
Roe AW, Ts'o DY (1995) Visual topography in primate V2: multiple representation across functional stripes. J Neurosci 15:36893715.[Abstract]
Salzberg BM, Obaid AL, Gainer H (1985) Large and rapid changes in light scattering accompany secretion by nerve terminals in the mammalian neurohypophysis. J Gen Physiol 86:395411.[Abstract]
Sato K, Momose-Sato Y, Arai Y, Hirota A, Kamino K (1997) Optical illustration of glutamate-induced cell swelling coupled with membrane depolarization in embryonic brain stem slices. Neuroreport 8:35593563.[ISI][Medline]
Shepro D, Morel NM (1993) Pericyte physiology. FASEB J 7:10311038.
Shtoyerman E, Arieli A, Slovin H, Vanzetta I, Grinvald A (2000) Long-term optical imaging and spectroscopy reveal mechanisms underlying the intrinsic signal and stability of cortical maps in V1 of behaving monkeys. J Neurosci 20:81118121.
Sibson NR, Dhankhar A, Mason GF, Rothman DL, Behar KL, Shulman RG (1998) Stoichiometric coupling of brain glucose metabolism and glutamatergic neuronal activity. Proc Natl Acad Sci USA 95:316321.
Silver IA (1978) Cellular microenvironment in relation to local blood flow. Ciba Found Symp:4967.
Thompson JK, Peterson MR, Freeman RD (2003) Single-neuron activity and tissue oxygenation in the cerebral cortex. Science 299:10701072.
Tomita M, Gotoh F, Yamamoto M, Tanahashi N, Kobari M (1983) Effects of hemolysis, hematocrit, RBC swelling, and flow rate on light scattering by blood in a 0.26 cm ID transparent tube. Biorheology 20:485494.[ISI][Medline]
Ts'o DY, Frostig RD, Lieke EE, Grinvald A (1990) Functional organization of primate visual cortex revealed by high resolution optical imaging. Science 249:417420.[ISI][Medline]
Tsunoda K, Yamane Y, Nishizaki M, Tanifuji M (2001) Complex objects are represented in macaque inferotemporal cortex by the combination of feature columns. Nat Neurosci 4:832838.[CrossRef][ISI][Medline]
Vanzetta I, Grinvald A (1999) Increased cortical oxidative metabolism due to sensory stimulation: implications for functional brain imaging. Science 286:15551558.
Villringer A, Them A, Lindauer U, Einhaupl K, Dirnagl U (1994) Capillary perfusion of the rat brain cortex. An in vivo confocal microscopy study. Circ Res 75:5562.[Abstract]
Wang G, Tanaka K, Tanifuji M (1996) Optical imaging of functional organization in the monkey inferotemporal cortex. Science 272:16651668.[Abstract]
Wang G, Tanifuji M, Tanaka K (1998) Functional architecture in monkey inferotemporal cortex revealed by in vivo optical imaging. Neurosci Res 32:3346.[CrossRef][ISI][Medline]
|