INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Rigorous design and interpretation of functional imaging studies using techniques such as positron emission tomography (PET) and functional magnetic resonance imaging (fMRI) presupposes a thorough understanding of neurovascular coupling. Use of these techniques is based on the premise that there is a spatial correlation between hemodynamic responses and underlying neuronal activity (Fox et al. 1988; Roy and Sherrington 1890; Sokoloff 1981). There is evidence, however, that under certain conditions there may be substantial spatial uncoupling between these two processes, indicating that neurovascular coupling requires further investigation (Blood and Toga 1998).

Optical intrinsic signal imaging (OIS) is an ideal technique for studying neurovascular relationships because it allows detection of vascular-related activity over broad cortical regions with high spatial and temporal resolution and can be paired with electrophysiological measurements in the same subject. Although OIS signals have been obtained using bloodless preparations (Cohen et al. 1971, 1972; MacVicar and Hochman 1991), there is evidence that, depending on imaging wavelength, a significant proportion of OIS signals in the intact brain can be attributed to changes in vascular activity, including blood flow, blood volume, and hemoglobin oxygenation levels (Frostig et al. 1990; Grinvald 1992; Grinvald et al. 1986; Malonek and Grinvald 1996; Narayan et al. 1994, 1995).

OIS and other imaging techniques, such as laser-Doppler flowmetry, vascular beads, vascular dyes, PET, and fMRI, have been used to characterize properties of regional cerebral perfusion during functional neuronal activity (Cox et al. 1993; Grinvald et al. 1986; Lindauer et al. 1993; Narayan et al. 1994; Ngai et al. 1988; Rovainen et al. 1993). The precision of neurovascular coupling varies among temporal, magnitude, and spatial dimensions and is also affected by stimulus parameters and baseline physiological conditions. For example, Lindauer et al. (1993) used laser-Doppler flowmetry to characterize the delayed and prolonged time course of activity-related perfusion relative to electrophysiological activity in rodent barrel cortex. Peak cerebral blood flow (CBF) responses occurred 2-3 s following stimulation onset as compared with the millisecond resolution of electrophysiological responses. Imaging spectroscopy and laser-Doppler flowmetry were used to demonstrate that during functional activation, changes in deoxygenated hemoglobin levels precede changes in CBF by more than 1 s (Malonek et al. 1997). In addition, variables such as stimulus frequency or regional CBF response amplitude have been shown to correlate with time course of the vascular response (Ances et al. 1998; Blood et al. 1995).

Magnitude of vascular perfusion is related both to characteristics of the perfusion time course (Frahm et al. 1997; Kruger et al. 1996) and to stimulus parameters. Using PET, Fox and Raichle (1985) observed a relationship between photic stimulus rate and percent regional CBF increases in striate cortex. Specific relationships have also been observed between stimulus frequency or strength and pial arteriolar diameter changes in rodent sensory hindlimb cortex (Ngai et al. 1988). Cannestra et al. (1998) described decreased OIS response magnitude to a somatosensory stimulus when stimulation occurred during vascular "refractory periods." Vascular response magnitude is also influenced by baseline physiological conditions; for example, hypercapnia causes increased CBF in response to somatosensory stimulation in rats (Schmitz et al. 1996).

Spatial relationships between microvascular anatomy and cortical functional units have been illustrated by co-localization of dense capillary beds and individual whisker barrels in layer IV of the rat barrel cortex (Cox et al. 1993; Woolsey et al. 1996). However, arteriolar supply and venular drainage of blood volume to a given region results in local uncoupling of the vascular response outside the boundaries of receptive fields or neuronal activity (Cox et al. 1993; Narayan et al. 1995; Woolsey et al. 1996). For example, OIS and vascular dye responses in rodent primary somatosensory cortex (SI) have been shown to spread over wider areas than center receptive fields mapped by single-unit recordings (Narayan et al. 1995). Spatial extent of perfusion-related responses may also increase due to the fact that neurovascular coupling appears to be mediated, at least in part, by freely diffusible substances such as nitric oxide (NO) (Dirnagl et al. 1993; Wahl and Schilling 1993).

While spatial uncoupling of neuronal and vascular activity most often occurs locally, there is increasing evidence for "passive" overspill across functional boundaries of cerebral cortex. It appears that CBF recruited by neural activity in a given cerebral region can diffuse into vessel branches that are peripheral to the branches overlying, feeding, or draining the functionally active region. This is mechanically feasible when CBF is recruited through an upstream parent artery that bifurcates to perfuse multiple cerebral regions. A passive overspill response is thus functional in origin but is not recruited by functional neural activity in the region to which it flows (Blood and Toga 1998). Note that the term passive is used here to distinguish this type of overspill from the local spatial incongruities produced by vessels feeding or draining a functionally active region.

Previous studies have demonstrated evidence for passive overspill. For example, Ngai et al. (1988) showed that under some conditions, arteriolar diameter changes were observed outside hindlimb sensory cortex following sciatic nerve stimulation. Similarly, we recently observed OIS responses over and around branches of the MCA feeding barrel cortex, following forelimb stimulation only (Blood and Toga 1998). These barrel cortex OIS responses were seen in the absence of whisker stimulation or detectable barrel cortex evoked potentials (EPs). This dramatic spatial uncoupling also appeared to influence the net magnitude of the barrel cortex OIS signal during simultaneous whisker and forelimb stimulation because there were complex changes in OIS response magnitude (including both increases and decreases) during simultaneous whisker and forelimb stimulation. Because barrel cortex EPs also decreased during simultaneous whisker and forelimb stimulation, it was unclear whether OIS magnitude decreased only as a result of neuronal modulation or if other nonfunctional vascular modulation also contributed to magnitude decreases. For example, magnitude decreases may have been caused partially by "shunting" of blood flow from barrel cortex to forelimb SI on activation of this region (Shmuel et al. 2001). However, the hypothesis tested in the present study was that vascular overspill is the dominant form of nonfunctional vascular modulation that occurs when vascular response curves overlap, leading to increases rather than decreases in OIS response magnitude.

Regions of rodent SI corresponding to whisker and forelimb are likely candidates for vascular overspill because they are each perfused by branches of the middle cerebral artery (MCA) arising from a common parent vessel, which is located in the most lateral portion of SI (Blood and Toga 1998; Cox et al. 1993; Ngai et al. 1988). SI representation of the rat's 25 primary mystacial whiskers is centered 2.4 mm posterior and 5.5 mm lateral to bregma (Chapin and Lin 1984) and is perfused by a major posterolateral MCA arteriole (Cox et al. 1993; Woolsey and Rovainen 1990) (Fig. 1A). This region is referred to as "barrel cortex" because of the barrel-shaped cytoarchitecture corresponding to somatotopic representation of individual mystacial whiskers of the rodent (Chapin and Lin 1984). SI representation of the rat forelimb is centered 1.0 mm anterior and 4.0 mm lateral to bregma and is perfused by a major anteromedial arteriole arising from the same MCA parent vessel (Cox et al. 1993) (Fig. 1A). Barrel and forelimb SI regions are also particularly appropriate for investigation of neurovascular relationships because their high level of anatomical and functional organization has already been well studied using electrophysiological and autoradiographic techniques (Durham and Woolsey 1977; Santori et al. 1986; Simons 1978; Welker 1971; Woolsey and Van der Loos 1970).

View larger version (98K): [in this window] [in a new window]   Fig. 1. A: schematic illustration of middle cerebral artery (MCA) branches overlying/feeding barrel cortex, forelimb primary and secondary somatosensory cortex (SI, and SII) (modified from Cox et al. 1993). These branches arise from a single parent vessel that enters somatosensory cortex anterolaterally. The box drawn over the vessels indicates approximate field of view for optical intrinsic signal (OIS) images in the present study. Direction is indicated by M (medial), L (lateral), A (anterior), and P (posterior) in the top right-hand corner. OIS images were divided between major anteromedial and posterolateral MCA arterioles, indicated here by the gray dotted line, to create masks for data analyses (see B for individual subject divisions). There was some anatomical variability in the exact location of MCA branches across subjects, but all subjects adhered to the general branching pattern indicated in the schematic diagram here. A, the most lateral branch of the posterolateral MCA arteriole feeding barrel cortex. B: the more medial branch of the posterolateral MCA arteriole feeding barrel cortex. C: the anteriomedial MCA arteriole feeding forelimb SI. D, the MCA parent vessel bifurcation into these posterolateral and anteromedial arterioles. B: raw, unfiltered OIS image, indicating field of view of the OIS camera used for data collection and showing configuration of vascular anatomy, similar to A. A-D: as in A. C: raw OIS images from each of the nine individual subjects. Each raw image, viewed through an 850-nm filter, indicates the field of view of the OIS camera used for data collection. All images illustrate 2 prominent MCA branches over barrel cortex. The black diagonal line on each image indicates the division between barrel and forelimb regions (barrel to the left, and forelimb to the right of each line, respectively) used to generate masks for all principal component analysis (PCA) and magnitude/spatial extent analyses. Note that because images were filtered at 850 nm, vessels were not easily visible on raw images. Therefore image contrast was increased in this figure to illustrate location of MCA vessels; lighting levels were more uniform in original images. D: illustration of regionally distinct OIS responses to lone-whisker (top) and lone forelimb (bottom) stimulation, corresponding with stereotaxic coordinates of barrel cortex and forelimb SI, respectively (see A). Note that in the lone forelimb stimulation condition OIS responses are visible in both the forelimb SI and the barrel cortex regions, while in the lone-whisker stimulation condition there are visible OIS responses only in the barrel cortex region. While OIS response time course was characterized both in barrel cortex and in forelimb SI regions, all magnitude and spatial extent analyses were performed only for the barrel cortex region.

Secondary somatosensory cortex (SII), which may respond to activity in either barrel cortex or forelimb SI, is located just lateral to barrel cortex (Chapin and Lin 1984). Although SII is most likely fed by vessels diverging from the most lateral branch of the posterolateral MCA arteriole feeding barrel cortex, the coordinates of SII itself usually lie lateral to this MCA branch (Welker and Sinha 1972). Thus the vascular supply to SII may, at least in some cases, overlap with the supply to barrel cortex. This offers an alternative and/or additional explanation for the origin of barrel cortex OIS responses to lone forelimb stimulation.

To further investigate the hypothesized influence of neurovascular uncoupling on the magnitude and time course of OIS responses in rodent somatosensory cortex, the present study was conducted using OIS and a modified version of the stimulus paradigm used by Blood and Toga (1998). In this modified paradigm, whisker and forelimb stimulation were temporally staggered rather than simultaneous (Fig. 2). Because the temporal resolution of neuronal and vascular responses differs substantially (milliseconds vs. seconds, respectively), we designed this stimulus paradigm based on the premise that staggered stimulation should result in significant temporal overlap of vascular responses, but not primary neuronal responses, in whisker and forelimb SI. EP measurements were used to support this premise. Thus we were able to observe evidence for the effects of vascular overspill on spatial, magnitude, and temporal properties of the OIS signal, while minimizing temporal overlap of neural activity in the two regions. We discuss these properties and their practical implications.

View larger version (8K): [in this window] [in a new window]   Fig. 2. Whisker and forelimb stimulation conditions used in the present study. Lines represent deflection of whisker C1 at a constant rate of 10 Hz () or constant vibration of forelimb digits 3 through 5 (dashed line; vibratory device driven at 80 Hz, 150 V) over corresponding time periods (seconds) indicated on x axis. Each condition shown here was repeated 3 times (3 subtrials) per trial and averaged across time epochs. During each subtrial, images were collected every 150 ms for 8.25 s, for a total of 55 image frames. There were 30-s intervals between all subtrials to allow cortical activity to return to baseline. The 6 stimulus conditions shown here were presented in pseudorandom order within each trial set (order of conditions listed in this figure does not indicate actual order of presentation). A: 1.5-s lone-whisker stimulation: whisker was stimulated for 1.5 s. B: staggered whisker-forelimb stimulation: whisker was stimulated for 1.5 s directly followed by stimulation of forelimb for 1.5 s. C: staggered forelimb-whisker stimulation: forelimb was stimulated for 1.5 s directly followed by stimulation of whisker for 1.5 s. D: lone forelimb stimulation: forelimb was stimulated for 1.5 s. E: 3.0-s lone-whisker stimulation: whisker was stimulated for 3.0 s. F: simultaneous whisker/forelimb stimulation (replication of paradigm from Blood and Toga 1998): whisker and forelimb were stimulated at the same time, each for 1.5 s.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animal preparation

Nine adult male Sprague-Dawley rats (Charles River Laboratories) weighing between 200 and 350 g were prepared with thinned skulls (Blood et al. 1995). During surgery, rats were initially anesthetized with gaseous halothane and the right femoral vein was cannulated with PE-50 tubing. Rats were then given 0.4-0.5 ml of 0.5 M urethan during 0.5 h, and urethan administration was continued throughout data collection to maintain anesthesia, while still allowing withdrawal from toe pinch. Halothane administration was discontinued once urethan anesthesia stabilized and at least 1 h before imaging began.

Anesthetized rats were placed in a stereotaxic frame, heads shaved, and a midline incision made in the scalp. The skull was cleaned and the right temporalis muscle retracted. A small metal scraping instrument (Biomedical Research Instruments, Rockville, MD) was then used to thin the bone over the primary somatosensory region of the right hemisphere of the brain until branches of the superior cerebral vein and the MCA were visible (Fig. 1, B and C). Thinning was done uniformly so that the entire imaging field was viewed through a homogenous region of bone. Silicone oil was applied to the skull after thinning and as needed during imaging sessions to make the bone more translucent. After each application of silicone oil, excess oil was wiped off with a dry cotton swab, and oil was allowed to soak in for several minutes to minimize the likelihood of significant changes in the quality and properties of the optical interface across time. Furthermore, overall reflectance was recorded at the beginning and end of each trial to ensure that there were no large global changes in reflectance across time. All animal and experimental protocols were approved by the Animal Research Committee of the University of California, Los Angeles.

Experimental protocol

Following surgery, anesthetized rats with thinned skulls were placed, still in the stereotaxic frame, under the CCD camera (Princeton Instruments) and secured in place so that cortical vessels were visible on a computer screen display of the camera's field of view. The field of view was placed such that both barrel cortex and forelimb SI regions were visible. To more precisely characterize OIS responses in barrel cortex, however, it was not possible to include the entire forelimb SI region in this field of view. Because a substantial portion of the forelimb response could be detected, however, we opted to calculate and compare OIS response time course for this portion of the forelimb SI responses. Unlike measurements of magnitude and spatial extent, time course is measured within individual trials and is made as a relative rather than an absolute measurement. It should be noted, however, that the measurements of time course in forelimb SI are only estimations and are not fully conclusive. OIS response magnitude and spatial extent in forelimb SI were not calculated due to the limited field of view.

During imaging, the cortex was illuminated with voltage-stabilized white light and images were filtered at 850 nm. Filtering images at different wavelengths emphasizes detection of signals associated with different physiological processes (Frostig et al. 1990; Grinvald 1992; Haglund et al. 1994; Holthoff and Witte 1996; Malonek and Grinvald 1995; Narayan et al. 1994, 1995). An 850-nm filter was used in the present study, emphasizing intrinsic signals due largely to light scattering from changes in blood volume, blood flow, vascular morphology, and/or cell swelling. Although the absorbance spectra for oxy- and deoxyhemoglobin extend into the 850-nm range, the light scattering component of the signal at this wavelength is significantly larger than the absorbance due to either oxy- or deoxyhemoglobin. Therefore it is generally assumed that changes in hemoglobin oxygenation contribute negligibly to the signal at 850 nm (Narayan et al. 1995, Nemoto et al. 1999).

The left whisker C1 was mechanically deflected using a motorized whisker nudger, while left forelimb digits 3-5 were vibrated with a piezoelectric device (Narayan et al. 1994). The 8-V electromechanical motorized whisker nudger had an angle of deflection of approximately 30° and was driven at 10 Hz, 80V. Velocity of whisker deflection was 1.2 m/s in an anterior to posterior direction. Forelimb stimulus intensity driving the vibratory device was set at 80 Hz, 150 V.¹ Stimulus duration was either 1.5 or 3.0 s (see following text). Frequency, duration, voltage, and other stimulus parameters were controlled with a Grass stimulator (model 888, Quincy, MA). Images were collected and digitized on an IBM personal computer (PC) and analyzed on PC and Macintosh computers and/or sent via network to a UNIX workstation for data analysis.

Images were collected before, during, and after lone whisker, lone forelimb, or staggered whisker and forelimb stimulation. During the staggered-stimulation condition, OIS images were obtained in barrel cortex before, during, and after staggered stimulation of whisker and forelimb (Fig. 2). Forelimb stimulation occurred either immediately before (forelimb-whisker condition) or immediately after (whisker-forelimb condition) whisker stimulation. Stimulus duration was adjusted so that although the whisker and forelimb stimuli themselves did not overlap, the vascular response curves were still temporally overlapping (Cannestra et al. 1996). During staggered stimulation, each of the two stimuli lasted 1.5 s, for a total of 3.0 s of stimulation. An average OIS response to 1.5-s whisker stimulation lasts approximately 4-5 s, peaking at 2.0-2.5 s following stimulus onset (Fig. 3A) (see Cannestra et al. 1996 for more detailed description of OIS response time course). Thus onset of the second stimulus preceded the OIS response peak of the first stimulus, while the peak of the second stimulus occurred just before the first stimulus OIS response returned to baseline. For comparison, the lone whisker was stimulated for both 1.5 and 3.0 s, and lone forelimb was stimulated for 1.5 s; 1.5 whisker stimulation was used as the main comparison with staggered stimulation so that whisker stimulation time was identical between conditions. The only difference was addition of the forelimb stimulation. As an additional control stimulus, and for replication of previous findings (Blood and Toga 1998), whisker and forelimb were stimulated simultaneously (whisker/forelimb) for 1.5 s.

View larger version (81K): [in this window] [in a new window]   Fig. 3. Time course comparison of OIS responses to 1.5-s lone-whisker, staggered, and lone forelimb conditions from individual trials in a single subject. Barrel cortex OIS responses are shown during and following lone-whisker stimulation (A, 1.5 s), whisker-forelimb stimulation (B), forelimb-whisker stimulation (C), and lone forelimb stimulation (D). Note that whisker stimulus onset is synchronized for all 4 time courses; thus forelimb stimulation in C occurred before displayed images. A raw image from this subject is displayed at the bottom of the figure to illustrate that the OIS signal overlies vascular anatomy. The area inside the yellow box on this raw image indicates the field of view for the time course images in this figure; field of view was restricted here to barrel cortex to more clearly exhibit signal contrast in this region over time. Examples of whole OIS images, including forelimb SI, are illustrated in Fig. 6. To the right of each time course, correponding peak barrel cortex magnitude (white) and area (yellow) ROIs are displayed on top of whole raw images. Diagonal black lines indicate the barrel/forelimb region division as described in Fig. 1. Ratio images were colorized on a scale of reflectance decrease ×103 (see color bar at bottom left of figure). Note that this scale was chosen for illustrative purposes and is not related to statistical significance. Images were collected every 150 ms, and each image is the average of 3 subtrials with prestimulus control images subtracted from stimulated and poststimulated images.

Data were collected for each stimulus condition in groups of three subtrials which, when averaged across time epochs (see Data analysis), comprised one trial. During each subtrial, images were collected every 150 ms for 8.25 s for a total of 55 image frames. Camera exposure time for each frame was 50 ms. There were 30-s intervals between all subtrials and trials to allow cortical activity to return to baseline. Trials were acquired in groups of six, comprising one trial set (see Blood et al. 1998): 1.5-s lone-whisker stimulation; staggered whisker-forelimb stimulation; staggered forelimb-whisker stimulation; lone forelimb stimulation; 3.0-s whisker stimulation; and simultaneous whisker/forelimb stimulation. Data for each trial set were collected within a time period less than 1 h to minimize variations in anesthetic level. Stimulus conditions within each trial set were presented pseudorandomly to ensure that there were no order or "learning" effects. Trial sets were used to counterbalance stimulus conditions across time and to make the desired comparisons within a given period of time; subjects had no way of knowing when one trial set ended and the next began. Thus each stimulus was equally likely to occur at any time, and there was no possible way that subjects could anticipate an upcoming condition. To add a further element of randomization, as well as to keep trial sets within the designated 1-h period, 5 of the 16 trial sets included in the study did not include all six stimulus conditions; however, all trial sets included the 1.5-lone -whisker, whisker-forelimb, and forelimb-whisker conditions.

Following OIS data collection, EPs were recorded from barrel cortex in three OIS subjects to verify that there were no changes in barrel cortex field potentials with addition of the forelimb stimulus. EPs were chosen as the most appropriate comparative measurement of electrical activity in this study because OIS measures activity over broad regions rather than from individual neurons.

To measure EPs, a craniotomy was performed over barrel cortex to allow electrode insertion. Recordings were made from just below the surface of the cortex using SNEX-200 bipolar electrodes with 100-µm tip diameter (Rhodes Medical, CA). Electrodes were placed in stereotaxically defined coordinates corresponding to barrel C1 (Chapin and Lin 1984). Additional measurements also were made in a 3 × 3 grid at 1-mm intervals around the C1 barrel. EPs were averaged using a Nicolet model 527 signal averager (Nicolet Instrument, Madison, WI), and recorded with a Model 7D polygraph (Grass Instruments).

EPs were measured from barrel cortex during each of the following stimulation periods: during 1.5-s lone-whisker stimulation, during whisker stimulation in the forelimb-whisker condition, during forelimb stimulation in the whisker-forelimb condition, during lone forelimb, and during simultaneous whisker/forelimb stimulation. Eight traces were averaged for each condition. Note that in staggered-stimulation conditions, EPs were measured during the second stimulus only (during forelimb stimulation in the whisker-forelimb condition and during whisker stimulation in the forelimb-whisker condition). Because there was no temporal overlap of the staggered whisker and forelimb stimuli, the first stimulus in the staggered-stimulation conditions was identical to the lone-whisker or lone forelimb stimulation conditions; thus these measurements did not provide any further information and were not included. The objective of the EP measurements was to determine first whether barrel cortex EPs observed during lone-whisker stimulation were altered when a forelimb stimulus preceded whisker stimulation and, second, whether there were any detectable barrel cortex EPs during forelimb stimulation that had been preceded by whisker stimulation. These questions were answered entirely by measurement of EPs during the second stimulus in staggered-stimulation conditions.

Data analysis

OIS IMAGES. Ratio subtraction images (Blood and Toga 1998; Blood et al. 1995) were calculated for each subtrial and the three subtrials in each trial were averaged to increase signal-to-noise ratio. Similar to our previous study (Blood and Toga 1998), there were spatially distinct OIS responses in barrel and forelimb SI regions (Fig. 1D). We analyzed time course, magnitude, and spatial extent in these two regions separately by dividing raw images between the major anteromedial and posterolateral arterioles of the MCA which overlie forelimb SI and barrel cortex, respectively (Fig. 1A) (Cox et al. 1993). This division clearly separated barrel and forelimb SI regions as defined by stereotaxic coordinates (Chapin and Lin 1984). This division was made for each subject, and then used to create masks to isolate either the barrel cortex region or the forelimb SI region for all subsequent time course, magnitude, and spatial extent analyses in that subject. As noted earlier, it was not possible to include the entire forelimb SI region in the camera field of view and thus only time course was analyzed in this region. While there was some variation in the exact configuration of vascular anatomy between subjects, the major anteromedial and posterolateral MCA arterioles could be observed in all subjects. The posterolateral arteriole, feeding barrel cortex, bifurcated into at least two additional branches (1 medial and 1 lateral) in all subjects as well (Fig. 1B).

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EVOKED POTENTIALS. EP response amplitude and timing across the 3 × 3 grid were compared between staggered-stimulation and 1.5-s lone-whisker or forelimb conditions and between EP and OIS techniques.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Staggered forelimb stimulation altered temporal, magnitude, and spatial extent properties of barrel cortex OIS responses to whisker stimulation in the absence of detectable changes in underlying EPs. OIS response time course in barrel cortex was dramatically broadened during staggered stimulation, and OIS response peaks were temporally shifted toward the relative time of forelimb stimulation. Barrel cortex peak OIS response magnitude and spatial extent were significantly greater in the staggered-stimulation conditions compared with lone-whisker controls. Barrel cortex OIS response time courses in staggered-stimulation conditions were highly correlated with the sum of barrel cortex OIS response time courses in lone-whisker and forelimb conditions. No significant changes in time course were observed in forelimb SI OIS responses during staggered stimulation. Similar to our previous study (Blood and Toga 1998), distinct OIS responses were observed in barrel cortex and forelimb SI regions, corresponding to whisker or forelimb stimulation, respectively (Fig. 1D). As also seen in our previous study, OIS responses were often observed in the barrel cortex region, as well as in forelimb SI, during forelimb stimulation. As in our previous study (Blood and Toga 1998), OIS response intensity was greatest over branches of the MCA.

OIS response time course in barrel cortex

Overall OIS response time course in barrel cortex was dramatically broadened in the temporal direction of the forelimb stimulus in both staggered-stimulation conditions (Figs. 3 and 4). Furthermore, time-to-peak OIS response as defined by PCA analysis was either shortened or delayed relative to whisker stimulation onset, depending on whether forelimb stimulation occurred before or after whisker stimulation, respectively (Table 1). When whisker C1 was stimulated independently for 1.5 s, average OIS response time to peak in barrel cortex was 2.10 s (relative to stimulus onset). During the forelimb-whisker stimulation condition, this time to peak was 1.50 s following whisker stimulus onset (600 ms earlier), whereas in the whisker-forelimb condition it was 3.3 s (1,200 ms later). A one-way ANOVA showed a significant effect of stimulus condition (1.5-s lone whisker, whisker-forelimb, forelimb-whisker) on OIS response time to peak (F = 44.590; P < 0.0001). When OIS response time to peak for individual trials was paired within trial sets and compared between 1.5-s lone-whisker and staggered-stimulation conditions, these differences were statistically significant both for 1.5 s lone whisker versus whisker-forelimb and for 1.5 s lone whisker versus whisker-forelimb (t = 4.58 for lone whisker vs. whisker-forelimb, P < 0.001, 2-tailed; t = 9.79 for lone whisker vs. forelimb-whisker, P < 0.0001, 2-tailed; paired t-tests). Note that these effects on time course were very consistent: SEs were small and quite similar between 1.5-s lone-whisker and staggered-stimulation conditions (Fig. 4). SD of individual time-to-peak values (Table 1) was also consistent between whisker and staggered-stimulation conditions.

View larger version (26K): [in this window] [in a new window]   Fig. 4. Comparison of average OIS signal time courses in barrel cortex following lone-whisker vs. staggered whisker-forelimb or forelimb-whisker stimulation. OIS response time courses were determined by plotting the 1st component of PCA analysis (Cannestra et al. 1996) and are expressed as percent of maximum signal for each trial. Because all responses were normalized to the maximum signal rather than reported as absolute signal change from baseline, response values here are unitless. Whisker stimulus onset is synchronized at time 0.00 for all 3 time courses; thus forelimb stimulation for the whisker-forelimb curve occurred from 1.5 to 3.0 s and forelimb stimulation for the forelimb-whisker curve occurred during the 1.5 s preceding time 0.00. Error bars indicate standard error of the mean. Each curve represents the average of data from 9 subjects and 18 trials. Exposure time for each image was 50 ms. Images were collected every 150 ms with 55 frames per subtrial. Graphs represent relative values within stimulus conditions only; magnitude differences between conditions are not indicated (see Fig. 5 for magnitude).

We also compared OIS response time to peak for the 3.0-s whisker stimulation condition with staggered-stimulation conditions. A one-way ANOVA showed a significant effect of stimulus condition (3.0-s lone whisker, whisker-forelimb, forelimb-whisker) on OIS response time to peak (F = 18.010; P < 0.001). When OIS time to peak for individual trials was paired within trial sets and compared between 3.0-s lone-whisker and staggered-stimulation conditions, the difference between 3.0-s lone whisker versus forelimb-whisker time to peak was statistically significant (P < 0.01, 2-tailed; paired t-test).

When OIS response time to peak was compared between 1.5- and 3.0-s lone-whisker stimulation conditions, there was no significant difference between conditions. There was also no significant difference in OIS response time to peak between simultaneous whisker/forelimb and 1.5-s lone-whisker stimulation conditions. Finally, there was no significant difference in OIS response time to peak between 1.5-s lone-whisker and 1.5-s forelimb stimulation conditions.

As a complementary analysis, ROI magnitude analysis was used to identify the peak barrel cortex OIS response frame for 1.5-s lone-whisker and staggered-stimulation conditions. Using this analysis, average time to peak for the 1.5-s lone-whisker condition was 2.40 ± 0.099 (SD) s. Average time to peak for the staggered whisker-forelimb condition was 3.39 ± 0.200 s. Average time to peak for the staggered forelimb-whisker condition was 1.50 ± 0.279 s. There was a significant effect of stimulus condition on time to peak (ANOVA: F = 74.4; P < 0.0001). Time to peak for the 1.5-s lone-whisker condition was also significantly different from time to peak in both the whisker-forelimb (t = 7.26; P < 0.0001) and forelimb-whisker (t = 5.86; P < 0.0001) conditions.

OIS response magnitude in barrel cortex

We calculated magnitude differences in barrel cortex OIS responses between 1.5-s lone-whisker and staggered-stimulation conditions as a comparison with overall magnitude decreases observed previously during simultaneous whisker/forelimb stimulation (Blood and Toga 1998). During both staggered-stimulation conditions, OIS magnitude increased relative to the 1.5-s lone-whisker condition. Average peak magnitude following 1.5-s lone-whisker stimulation, staggered whisker-forelimb, and staggered forelimb-whisker stimulation was, respectively, 2.986 × 10³, 3.533 × 10³, and 3.759 × 10³ reflectance decrease from unstimulated baseline (Fig. 5A). A one-way ANOVA showed a significant effect of stimulus condition (1.5-s lone whisker, whisker-forelimb, forelimb-whisker) on OIS response magnitude (F = 3.345; P < 0.05). When OIS response magnitude for individual trials was paired within trial sets and compared between 1.5-s lone-whisker and staggered-stimulation conditions, these differences were statistically significant both for 1.5-s lone whisker versus whisker-forelimb and for 1.5-s lone-whisker versus whisker-forelimb (P < 0.05, 2-tailed; paired t-tests). Replicating previous findings, barrel cortex OIS responses were frequently observed during lone forelimb stimulation (Figs. 3D and 6), and were not accompanied by detectable EPs in barrel cortex (see following text).

View larger version (20K): [in this window] [in a new window]   Fig. 5. Average barrel cortex magnitude differences among 1.5-s lone whisker (wh), whisker-forelimb (wh  fl), and forelimb-whisker (fl  wh) stimulus conditions. Average magnitude is displayed for each stimulus condition. Statistical comparisons of OIS response peak magnitude were made between 1.5-s lone-whisker and staggered-stimulation conditions using values from individual trials (paired t-tests; *P < 0.05, 2-tailed). Magnitude was determined using statistically defined region of interest (ROI) analysis (Blood et al. 1995) for peak OIS responses as identified by PCA analysis (Fig. 4) and is expressed as reflectance decrease ×103. Error bars indicate SE for each average value.

View larger version (44K): [in this window] [in a new window]   Fig. 6. An example of peak OIS responses during 4 stimulation conditions (from a different subject than images illustrated in Fig. 3) and comparison with barrel C1 evoked potential (EP) measurements made during these conditions. Due to differences in vascular and electrophysiological temporal characteristics, EP measurements in this figure were made during the relevant stimulation and thus do not necessarily correspond in time to the peak OIS responses depicted here. A: OIS responses. B: corresponding barrel cortex and forelimb SI magnitude (white) and area (yellow) ROIs, superimposed on a raw image from this subject (note that ROIs were calculated here in the forelimb SI region for illustrative purposes only; no statistics were calculated using these ROIs); C: EP responses in barrel C1 during 1.5-s lone whisker, lone forelimb, or the second stimulus of staggered stimulation. D: superimposed barrel C1 EP responses for all 4 stimulus conditions in C. EPs were measured during the 2nd stimulus only in staggered-stimulation conditions (forelimb stimulation in the whisker-forelimb condition, and whisker stimulation in the forelimb-whisker condition). Measurements were made in the region of barrel cortex stereotaxically corresponding to whisker C1 (Chapin and Lin 1984), and in a 3 × 3 grid around this region separated by 1 mm. Multiple EP measurements were acquired during 1.5-s stimulation periods and averaged. EPs (C) were measured from barrel C1 during whisker stimulation alone (a), during forelimb stimulation in the whisker-forelimb condition (b), during whisker stimulation in the forelimb-whisker condition (c), and during lone forelimb stimulation (d). Color bar indicates reflectance decrease ×103. Note that to best resolve the morphology of the large magnitude barrel cortex OIS responses in b, the color scale for all 4 images was adjusted to a level such that in this particular example the response to forelimb stimulation is not easily visible (i.e., threshold values were chosen for illustrative purposes and are not related to statistical significance). Thus the small response in the forelimb SI region does not indicate an absence of OIS response in this region. The horizontal line in the upper right corner indicates time in milliseconds; the vertical line indicates EP amplitude in microvolts.

The effect of staggered stimulation on OIS response magnitude was robust: on average, magnitude increased approximately 20% between 1.5-s lone-whisker and staggered conditions. Even low-magnitude late-onset OIS responses to whisker stimulation were enhanced by staggered forelimb stimulation. In Fig. 3B, for example, the OIS response to whisker stimulation is quite faint and begins later than the OIS response to lone-whisker stimulation in Fig. 3A (note that this initial difference in time course between 3, A and B, occurred before forelimb stimulus onset). After forelimb stimulus onset, however, the barrel cortex OIS response in Fig. 3B increased to a magnitude much greater than that following lone-whisker stimulation in Fig. 3A, and extended temporally beyond Fig. 3A as well.

When PCA curves were multiplied by peak OIS response magnitude for each trial, summated for lone stimulation conditions, and compared with similar curves from each of the staggered-stimulation conditions, there was a high degree of similarity between the curves (Fig. 7). The correlation coefficient for the whisker-forelimb condition was r = 0.898 (Fig. 7A) and for the forelimb-whisker condition was r = 0.895 (Fig. 7B). In the whisker-forelimb condition, however, there was a slight delay in the early part of the time course in comparison to the summated lone condition response curves (Fig. 7A) that was not observed in either the lone-whisker condition or the summated lone-whisker and lone forelimb curves.

View larger version (30K): [in this window] [in a new window]   Fig. 7. Comparison of OIS responses in staggered-stimulation conditions with summated responses from the lone-whisker and forelimb conditions. Response magnitude was calculated as mean pixel intensity over the entire barrel cortex region of each image and multiplied by the percent maximum signal values from PCA analysis (see Fig. 4). Summated lone whisker and forelimb curves in appropriate temporal register are compared here with the whisker-forelimb condition (A) and the forelimb-whisker condition (B). Summated lone-whisker and lone-forelimb responses are indicated with squares, and staggered-stimulation responses are indicated with circles. Error bars indicate SE. Lines under the y axis indicate the temporal register of each stimulus in this graph for lone (squares) and staggered (circles) conditions; solid lines represent whisker stimulation and dotted lines represent forelimb stimulation.

Magnitude in the staggered-stimulation conditions was not significantly different from the summation of lone-whisker and forelimb magnitude at whisker-forelimb or forelimb-whisker peak response time points. However, magnitude at the time point of the lone-whisker peak in the whisker-forelimb condition was significantly different from (less than) magnitude of summated lone-whisker and lone forelimb responses (P < 0.05; t = 2.70), consistent with the time course lag seen in this condition in the previous paragraph (Fig. 7A). This difference was not significant at whisker peak time in the forelimb-whisker condition. Average magnitude of responses in the whisker-forelimb condition at the time of the whisker-forelimb peak was 2.20 × 10³ ± 0.748 × 10³ reflectance decrease from unstimulated baseline, and the sum of whisker and forelimb responses at corresponding time points was 2.43 × 10³ ± 0.983 × 10³ reflectance decrease from unstimulated baseline. Average magnitude of responses in the forelimb-whisker condition at the time of the forelimb-whisker peak was 2.71 × 10³ ± 0.590 × 10³ reflectance decrease from unstimulated baseline, and the sum of whisker and forelimb responses at corresponding time points was 2.53 × 10³ ± 1.04 × 10³ reflectance decrease from unstimulated baseline. Average magnitude of responses in the whisker-forelimb condition at the time of the lone-whisker peak was 1.52 × 10³ ± 0.730 × 10³ reflectance decrease from unstimulated baseline, and the sum of whisker and forelimb responses at corresponding time points was 2.45 × 10³ ± 0.747 × 10³ reflectance decrease from unstimulated baseline. Average magnitude of responses in the forelimb-whisker condition at the time of the lone-whisker peak was 2.24 × 10³ ± 0.760 × 10³ reflectance decrease from unstimulated baseline, and the sum of whisker and forelimb responses at corresponding time points was 2.89 × 10³ ± 0.986 × 10³ reflectance decrease from unstimulated baseline.

OIS response morphology and spatial extent in barrel cortex

OIS RESPONSE MORPHOLOGY. In all stimulation conditions, OIS signal intensity was greatest over MCA branches (compare raw images and OIS responses in Figs. 3 and 6). OIS responses to whisker stimulation were usually observed over at least two branches of the posterolateral MCA arteriole (Figs. 3 and 6) as observed in our previous studies (Blood and Toga 1998; Blood et al. 1995). In most cases, staggered stimulation led to broadened time courses and magnitude increases over both a more medial and the most lateral of these branches (compare Figs. 6, A with B and C; and 3, A with C). OIS responses were sometimes uniform over both branches (Fig. 6, A vs. B), but often they were greater over the more lateral branch (Fig. 3, A vs. B and C). Barrel cortex OIS responses during forelimb stimulation also occurred more often, although not exclusively, over the more lateral of these two branches (Figs. 3D and 6D).

OIS SPATIAL EXTENT. In the 1.5-s lone-whisker, staggered whisker-forelimb, and staggered forelimb-whisker stimulation conditions, average peak spatial extent as defined using magnitude ROIs (number of pixels in the ROI) was, respectively, 3,700, 4,174, and 3,760. A one-way ANOVA indicated that the effects of stimulus condition on spatial extent were just under significance (F = 3.09; P < 0.0554). In contrast, there was a significant effect of stimulus condition on spatial extent as defined using area ROIs (examples of magnitude and area ROIs from 2 different subjects can be seen in Figs. 3 and 6). In the 1.5-s lone-whisker, staggered whisker-forelimb, staggered forelimb-whisker, and simultaneous whisker/forelimb stimulation conditions, average peak spatial extent of area ROIs (number of pixels in the ROI) was, respectively, 3,780, 9,381, 10,369, and 8,009 (Fig. 8). A one-way ANOVA showed a significant effect of stimulus condition (1.5-s lone whisker, whisker-forelimb, forelimb-whisker) on OIS response spatial extent (F = 6.030; P < 0.01). When OIS response spatial extent for individual trials was paired within trial sets and compared between 1.5-s lone-whisker and staggered-stimulation conditions, these differences were statistically significant both for 1.5-s lone whisker versus whisker-forelimb, and for 1.5-s lone whisker versus whisker-forelimb (P < 0.01, 2-tailed; paired t-tests). Spatial extent as defined using area ROIs was also significantly different between 1.5-s lone-whisker and simultaneous whisker/forelimb conditions (P < 0.05, 2-tailed; paired t-test).

View larger version (15K): [in this window] [in a new window]   Fig. 8. Average barrel cortex peak spatial extent differences among 1.5-s lone whisker (wh), whisker-forelimb (wh  fl), whisker-forelimb (fl  wh), and simultaneous whisker/forelimb (sim) stimulus conditions. Average peak spatial extent as determined by area ROIs (expressed as number of pixels in the ROI) is displayed for each stimulus condition. Statistical comparisons of peak spatial extent were made between 1.5-s lone-whisker and staggered or simultaneous stimulation conditions using values from individual trials (paired t-tests; *P < 0.05, **P < 0.01, 2-tailed). Error bars indicate SE for each average value.

EPs in barrel cortex

Because OIS measures activity over broad regions, rather than individual neurons, EPs were chosen as the most appropriate comparative measurement of electrical activity in this study. In contrast to OIS responses, no quantitative or qualitative EP amplitude or timing changes were observed in barrel C1 or the surrounding 3 × 3 grid when a temporally staggered forelimb stimulus was added before or after the whisker stimulus. Figures 6 and 8 show examples of EP measurements from two different subjects. Note that EPs were measured during the second stimulus only in staggered-stimulation conditions (forelimb stimulation in the whisker-forelimb condition and whisker stimulation in the forelimb-whisker condition).

There were no changes in amplitude or timing of barrel cortex EPs during whisker stimulation in the forelimb-whisker condition, relative to the 1.5-s lone-whisker condition. There were also no detectable EPs in barrel cortex during forelimb stimulation, either alone or during the forelimb stimulus in the whisker-forelimb condition. Replicating our previous findings (Blood and Toga 1998), EP responses in barrel cortex decreased relative to 1.5-s lone-whisker stimulation during simultaneous whisker/forelimb stimulation.

EP response amplitude was in the range of 50-150 µV, and the time course of EP responses was approximately 100-ms duration with an average latency of approximately 10 ms. Similar to our previous studies (Blood and Toga 1998; Narayan et al. 1994), the maximal EP response amplitude was always seen at the middle (C1) recording electrode, and there was very little EP activity in the surrounding 3 × 3 grids (Fig. 9). Although there was a small amplitude EP response at electrode 8 in Fig. 9, this response was similar during lone-whisker and staggered forelimb-whisker conditions.

View larger version (28K): [in this window] [in a new window]   Fig. 9. Barrel cortex EPs for the subject illustrated in Fig. 3. Black circles and numbers on the raw image in the bottom left-hand corner correspond to the 9 locations where EP measurements were made. The center measurement (5) was made at the stereotaxic location of whisker C1 (Chapin and Lin 1984). EPs are shown for each of the 5 stimulation conditions indicated in the key at the bottom right of the figure. EP measurements were made only during the stimulus or stimuli underlined in this key (during the 2nd stimulus only in staggered-stimulation conditions). Note that increased amplitude was seen only when whisker was stimulated and that EP responses did not differ between lone whisker and forelimb-whisker conditions. The horizontal line at the bottom center of the figure indicates time in milliseconds; the vertical line indicates EP amplitude in microvolts.

OIS responses in forelimb SI

While forelimb responses could not be fully observed or quantified due to the camera position, OIS response time course in the portions of forelimb SI included in the images was not significantly different between lone-forelimb and staggered-stimulation conditions. Time-course changes observed in forelimb SI following staggered-stimulation conditions were very small (Table 1, Fig. 10). When whisker stimulation followed forelimb stimulation, OIS responses in forelimb SI peaked, on average, at the same time as OIS responses to lone forelimb stimulation. When whisker stimulation preceded forelimb stimulation, average OIS responses in forelimb SI peaked only 150 ms earlier than following lone-forelimb stimulation. A very slight broadening of OIS response curves in forelimb SI was detectable during staggered stimulation, but this broadening was considerably less than that observed in barrel cortex. In addition, no significant forelimb OIS responses were observed following lone-whisker stimulation. In some particular cases, however, forelimb SI response magnitude appeared to increase with staggered stimulation in a pattern similar to that seen in barrel cortex (compare Fig. 6, B-D).

View larger version (28K): [in this window] [in a new window]   Fig. 10. Comparison of average OIS signal time courses in forelimb SI following lone forelimb vs. staggered forelimb-whisker or whisker-forelimb stimulation. OIS response time courses were determined by plotting the first component of PCA analysis (Cannestra et al. 1996) and are expressed as percent of maximum signal for each trial. Because all responses were normalized to the maximum signal, rather than reported as absolute signal change from baseline, response values here are unitless. Forelimb stimulus onset is synchronized at time 0.00 for all 3 time courses; thus whisker stimulation for the forelimb-whisker curve occurred from 1.5 to 3.0 s, and whisker stimulation for the whisker-forelimb curve occurred during the 1.5 s preceding time 0.00. Error bars indicate SE. Each curve represents the average of data from 9 subjects and 18 trials. Exposure time for each image was 50 ms. Image frames were collected every 150 ms, with 55 frames per subtrial. OIS response time courses were determined by plotting the first component of PCA analysis (Cannestra et al. 1996). Graphs represent relative values within stimulus conditions only; magnitude differences between conditions are not indicated.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Temporal, magnitude, and spatial properties of barrel cortex OIS responses were modulated by nonoverlapping forelimb stimulation, in the absence of detectable changes in barrel cortex EPs. Barrel cortex OIS response time courses broadened, OIS response peaks shifted temporally in the direction of the forelimb stimulus, and OIS response magnitude and spatial extent increased significantly during staggered stimulation relative to lone-whisker stimulation. The high degree of correlation between magnitude response curves in staggered-stimulation conditions and summated lone-whisker and forelimb curves suggests that when stimuli do not overlap temporally, modulation of response properties by vascular overspill occurs in an approximately linear fashion. Together, these data provide further evidence for uncoupling of vascular and electrophysiological responses in barrel cortex.

Time-course broadening and magnitude increases in the absence of electrophysiological changes suggest that passive vascular overspill may have been the physiological mechanism underlying modulation of OIS response properties in barrel cortex. We hypothesize that vascular responses functionally recruited to forelimb SI overspilled into MCA branches overlying barrel cortex. Because neural activity in barrel cortex and/or forelimb SI may have triggered neural activity in SII, it is also possible that overspill originated from vascular responses recruited to SII. In either case, vascular overspill crossed functional boundaries: in the first case within SI and in the second case between SI and SII.

While it cannot be ruled out that neural activity in SII enhanced the OIS response in barrel cortex to some degree, we believe that this was not the only source of enhancement because in most cases, activity increased over the medial as well as the more lateral branch of the posterolateral MCA arteriole feeding barrel cortex (see Figs. 3, A vs. C, and 6, A vs. B and C). It is unlikely that this medial branch feeds SII. Another possibility that must be considered is that activity in SII altered barrel cortex activity itself, resulting in increased OIS response magnitude over the more medial branch. However, this scenario is not consistent with the absence of changes in EP responses during these conditions.

Mechanisms of overspill

What previous evidence is there for mechanisms of passive vascular overspill and how might these mechanisms have influenced OIS responses in this particular study?

Vascular anatomical and morphological characteristics are fundamental to the concept of vascular overspill. While brain capillaries are known to be organized in functional modules with high spatial resolution, arterioles that feed and venules that drain these regions may be somewhat spatially unrelated to specific neuronal units. For example, Cox et al. (1993) and Woolsey et al. (1996) have described that, while there are capillary tufts which are highly localized to individual whisker