High-resolution fMRI of macaque V1

Magnetic Resonance Imaging 25 (2007) 740 – 747

Research articles

High-resolution fMRI of macaque V1 Jozien B.M. Goense4, Anne-Catherin Zappe, Nikos K. Logothetis Department of Physiology of Cognitive Processes, Max-Planck Institute for Biological Cybernetics, 72076 Tu¨bingen, Germany Accepted 11 January 2007

Abstract To understand the physiological mechanisms underlying the blood-oxygenation-level-dependent (BOLD) signal, the acquisition of data must be optimized to achieve the maximum possible spatial resolution and specificity. The term bspecificityQ implies the selective enhancement of signals originating in the parenchyma, and thus best reflecting actual neural activity. Such spatial specificity is a prerequisite for imaging aimed at the elucidation of interactions between cortical micromodules, such as columns and laminae. In addition to the optimal selection of functional magnetic resonance imaging pulse sequences, accurate superposition of activation patterns onto corresponding anatomical scans, preferably acquired during the same experimental session, is necessary. At high resolution, exact functional-to-structural registration is of critical importance, because even small differences in geometry, that arise when different sequences are used for functional and anatomical scans, can lead to misallocation of activation and erroneous interpretation of data. In the present study, we used spin-echo (SE) echo planar imaging (EPI) for functional scans, since the SE-BOLD signal is sensitive to the capillary response, together with SE-EPI anatomical reference scans. The combination of these acquisition methods revealed a clear spatial colocalization of the largest fractional changes with the Gennari line, suggesting peak activity in Layer IV. Notably, this very same layer coincided with the largest relaxivity changes as observed in steady-state cerebral blood volume measurements, using the intravascular agent monocrystalline iron oxide nanoparticles (MION). D 2007 Elsevier Inc. All rights reserved. Keywords: Spin echo; CBV; Primate; Monkey; Cortex; Visual system; Cortical architectonics

1. Introduction High-resolution functional magnetic resonance imaging (fMRI) can potentially improve our understanding of the physiological origin of the blood-oxygenation-level-dependent (BOLD) signal by resolving differences in functional activation within the cortical sheet. Moreover, localization of activity patterns limited in any of the well-known tangential and laminar cortical modules may help generate useful hypotheses regarding the functional principles of cortical microcircuits. To resolve differences in function within the cortical sheet, fMRI activation needs to be highly specific (i.e., to have a point-spread function that is less than a millimeter). It has been shown that conventional gradientecho (GE) BOLD signal arises to a large extent from areas near veins, even at high magnetic fields [1], and thus is not suitable for resolving functional differences within the

4 Corresponding author. Tel.: +49 7071 601 1704; fax: +49 7071 601 652. E-mail address: [email protected] (J.B.M. Goense). 0730-725X/$ – see front matter D 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.mri.2007.02.013

cortical microarchitecture. The spin-echo (SE) BOLD signal is more sensitive to functional signals arising from the capillary bed, and is thus more specific to the actual site of neural activation. The BOLD signal is an indirect measure of neural activity [2]. Its usefulness as a surrogate signal strongly depends on the understanding of the neurovascular coupling, and on the optimization of methodologies that increase signal sensitivity to those vascular changes that are most relevant to the activation of neural populations. Enhancement of neural activity causes an increase in local blood flow, which results in a decreased concentration of deoxyhemoglobin. Because the latter is paramagnetic, its fractional reduction decreases local susceptibility gradients and leads to a signal increase [3]. The spatial scale at which differences in activation can be observed depends on physiological characteristics, such as the scale of blood flow regulation, and on technical considerations, such as the specificity of the fMRI method. It has been shown that cerebral blood flow (CBF) is regulated at submillimeter scales or smaller, at the level of microvessels [4–6] or

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single capillaries [7]. SE-BOLD, cerebral blood volume (CBV) and CBF methods have been successfully applied at high field and high resolution to resolve columnar structure [8–12]. Based on modeling, it has been suggested that SE methods be highly specific to the microvasculature [13– 16]. Experimentally, SE-BOLD fMRI has indeed been shown to be more specific than GE-BOLD fMRI [17–20]. Yet in a direct comparison, the specificity of the SE-BOLD method was found to be lower than that of CBV-based methods, which depicted a clearer difference in cortical lamination [21,22]. Using superparamagnetic intravascular contrast agents such as monocrystalline iron oxide nanoparticles (MION), it is possible to determine CBV [23–25]. Relaxivity changes before and after the injection of MION depend on steadystate CBV. When GE-based methods are used, relaxivity changes (DR 2*) are more sensitive to static susceptibility effects around large veins, but DR 2 (using SE) is more weighted towards small vessels [13,16]. Thus, like BOLD fMRI, SE-CBV and GE-CBV methods are sensitive to different fractions of the vasculature. The effect of a contrast agent, such as deoxyhemoglobin or MION, on the relaxivity depends on the concentration of the contrast agent [26] and on the properties of the vasculature [27,28]. Contributions to the signal changes arise from both intravascular and extravascular water and are affected by vessel geometry. The contrast mechanisms of GE-CBV imaging rely mainly on changes in relaxivity of the blood, and dephasing of protons in static susceptibility gradients, while those of SE-CBV depend more on intravascular changes and on dynamic susceptibility effects, arising from diffusion of the protons in local susceptibility gradients, with static susceptibility gradients refocused by the 1808 pulse [14–16]. The aim of this project is to elucidate the origin of fMRI signals by comparing changes in functional activation in the different laminae (ca. 100–500 Am) with electrical signals obtained in extracellular recordings. Differences in laminaspecific functional activation are likely to reflect differences in neural activation (e.g., activity potentially elicited by feedforward or feedback connections), but also differences in vascularization. Insights might be gained by comparing the SE-BOLD response to the steady-state CBV; this can give an indication as to whether the BOLD response increases in proportion to the vascularization, or whether the BOLD response is more heterogeneous, and independent of baseline blood flow, for instance, if activation-induced blood flow increases are larger in specific layers than in others. Towards this aim, here we have extended our earlier results [29] and compare SE echo planar imaging (EPI) fMRI with the steady-state CBV determined with MION. Firstly, highresolution SE-fMRI of the primary visual cortex (V1) in macaque was performed. The specificity of the SE-BOLD signal at 4.7 T in the macaque was found to be comparable to that reported for cats at 9.4 T using CBV methods. Furthermore, we compared the pattern of functional activa-

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tion to the relaxivity changes (DR 2) after injection of MION, which is a measure of the steady-state CBV [16]. The primary visual cortex was evidently selected as region of interest because of its clear lamination, exquisitely pronounced by the two Baillarger lines, the outer of which is the well-known Gennari line. Lamination of V1 is characteristic in both monkeys [30] and humans [31]. 2. Methods Imaging was performed on an upright 4.7 T Bruker BioSpec 47/40v scanner with a 40-cm-diameter bore (Bruker, Ettlingen, Germany). The scanner is equipped with an actively shielded gradient coil of 26 cm diameter, which can attain gradients up to 50 mT m1 within a rise time of 180 As. Experiments were performed on six healthy monkeys (Macaca mulatta) weighing 3.5–8 kg. All experiments were approved by local authorities (Regierungspr7sidium) and were in full compliance with the guidelines of the European Community (EUVD 86/609/EEC) for the care and use of laboratory animals. The experimental setup and procedures are described in detail in Logothetis et al. [32,33]. Monkeys were positioned in the magnet using a specially designed primate chair. Experiments were performed under general anesthesia. Animals were intubated after induction with fentanyl (31 Ag kg1), thiopental (5 mg kg1) and succinyl chloride (3 mg kg1), and ventilated with a Servo Ventilator 900C (Siemens, Germany) maintaining an end-tidal CO2 of 33 mm Hg and oxygen saturation of over 95%. Anesthesia was maintained with remifentanyl (0.5–2 Ag kg1 min1). Mivacurium chloride (3–6 mg kg1 h1) was used to ensure paralysis of the eye muscles. Body temperature was maintained at 38–39.58C. Lactated Ringer’s solution with 2.5% glucose was infused at a rate of 10 ml kg1 h1. Intravascular volume and blood pressure were maintained by administering colloids (30–50 ml of hydroxyethylene starch over 1–2 min, as needed). Two drops of 1% cyclopentolate hydrochloride were administered to each eye to achieve mydriasis, and the animal was fitted with hard contact lenses (hard PMMA lenses; Firma Wfhlk, Kiel, Germany) to bring the eyes to focus on the stimulus plane. Visual stimuli were presented binocularly using an SVGA fiber-optic system (AVOTEC; Silent Vision SV7021, Stuart, FL) with a resolution of 640480 pixels and a frame rate of 60 Hz. The stimulus was a full-field rotating polar checkerboard, presented to both eyes. For MION [34] injection, 8 mg kg1 MION (Center for Molecular Imaging Research, MGH, Boston, MA) was infused as a slow bolus. A volume coil was used for transmission, with an actively and geometrically decoupled surface receiver coil of 25 mm diameter placed over the occipital cortex of one hemisphere [33]. Typically 9–13 slices were acquired of one hemisphere of V1. To minimize partial volume effects, the slices were oriented perpendicular to the cortical surface. Homogeneity along the thickness of the slice was confirmed on a sagittal

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scan. For GE-EPI and SE-EPI functional scans, the field of view (FOV) was 64 or 48 mm in the read direction (L–R) and 64 or 48 mm in the phase-encoding direction (A–P). The SEEPI was optimized to be less sensitive to T 2* effects and residual susceptibility artifacts [29]. A 16-segment EPI was acquired with a 192192 matrix and a receiver bandwidth (BW) of 100 kHz. This resulted in an EPI acquisition window of 23 ms. The spatial resolution for functional scans ranged from 333333 to 250187 Am. T E was 46 ms and T R was 2000 ms for SE-EPI, and T E/T R was 20/750 ms for GE-EPI. The flip angle for GE-EPI was 408. For anatomical reference, a 16-segment SE-EPI was used, with an FOV of 4848 mm and a slice thickness of 1– 2 mm. The matrix was 192256, resulting in a spatial resolution of 250187 Am. The T E was 70 ms and the T R was 3000 ms. To ensure that functional and anatomical scans can be overlaid perfectly, the sequence parameters were adjusted such that anatomical and functional images had exactly the same distortion. This was done by matching the effective BW in the read and phase-encoding directions. In the read direction, the BW was 1560 Hz mm1 for the functional (520 Hz voxel1) and anatomical EPI (390 Hz voxel1); in the phase-encoding direction, the BW was 130 Hz mm1 for the functional (39 Hz voxel1) and anatomical scan (24 Hz voxel1). The same parameters were used for the scans with MION, except that T E was 64 ms. A regular gradient echo fast imaging (GEFI) sequence (Bruker; a customized fast low-angle shot FLASH sequence [35]) was also used for anatomical reference. This highresolution GE sequence was used with an FOV of 51.2 38.4 mm and an image matrix of 512384, resulting in an in-plane resolution of 100 Am. T E was 20 ms, T R was 2000 ms, the flip angle was 508 and the receiver BW was 25 kHz. 2.1. Data analysis Data analysis was performed using custom-written MATLAB routines (The MathWorks) and SPM2 [36] (http://www.fil.ion.ucl.ac.uk/spm). Image registration (SPM) was used to correct for scanner drift during the functional scan. The drift was zero to one voxel for a 34-min SE-EPI functional scan. Because scanner drift leads to a shift in the phase-encoding direction only and because the voxels are highly anisotropic, image realignment was restricted to y-translations only. For functional maps, significantly activated voxels were calculated using a t-test, and the percent signal change was calculated for significant voxels. Voxels were thresholded at P b.05, and maps were clustered. The change in relaxivity DR 2 = R 2,postMIONR 2,preMION was calculated according to: DR2 ¼

1 Spost ln TE Spre

with R 2 =1/T 2. Images were coregistered (SPM) prior to calculation of DR 2.

The profiles over the activated areas were calculated normal to the cortical surface, based on the anatomical or raw EPI images. A circle was fitted to part of the cortex, defined between a begin point and an end point, between which the average radial profiles were calculated. The exact location of the cortical surface was determined from the actual image (instead of being based on the fitted circle). For the high-resolution anatomical images, profiles were calculated every 0.258, and data points were interpolated to an inplane spatial resolution of 75–100 Am. For the profiles of the functional activation, the coordinates specifying the trajectory of the cortical profiles were generated from the raw EPI images, and the functional activation was calculated along each profile. Profiles were calculated every 0.258 over an area defined based on the anatomical reference, where partial volume effects were minimal, and interpolated to 94–125 Am spatial resolution perpendicular to the cortical surface. The average percent change functional activation was calculated over the activation map between the begin point and the end point without thresholding or clustering. The number of significantly activated voxels in the same area was determined after thresholding at P b.05. For the profiles of the activation, the cortical depth was referenced to the original raw EPI image, which matches the SE-EPI anatomy exactly, given that the pixel BW was the same for both scans. When comparisons were made between different scans, images were coregistered (SPM). 3. Results Fig. 1 shows high-resolution functional images of V1 using SE-EPI (Fig. 1A) and GE-EPI (Fig. 1B). Anatomical images (Fig. 1C and D) clearly show the laminar structure of the striate cortex. The GEFI (Fig. 1D) is sensitive to T 2*, and thus distinctly shows blood vessels and intracortical vessels, while the SE-EPI (Fig. 1C) better delineates the Gennari line; contrast is also high in areas with CSF. A comparison of the functional and anatomical SE-EPI shows that the distortion is the same and that the SE-EPI anatomical image can be used as an exact reference. As known from the literature, the GE signal shows the largest percent changes at the surface, with activation due to veins clearly visible in the GE-EPI functional map (blue arrows). In contrast, the largest changes in the SE-BOLD signal were located in Layer IV, with only little activation at the surface. Comparison of the functional and structural scans shows that activation occurs along the Gennari line, i.e., in Layer IV. The effect of partial volume can be appreciated here, by comparison of these scans. In locations where the Gennari line has a fuzzier appearance in the anatomical scan, the functional activation is also broader (purple arrows). This feature permits the selection of regions with little partial volume effects for further analysis. By using the acquisition conditions discussed above, we found an SE-BOLD specificity that was even higher than that reported in Area 17 of the cat at 9.4 T [20–22]. In fact, our SE-BOLD activation showed

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Fig. 1. High-resolution percent change functional maps of V1 using SE-EPI (Column A) and GE-EPI (Column B). The spatial resolution was 333250 Am for GE and 333326 Am for SE, with a slice thickness of 2 mm. The GE-EPI shows maximal activation at the surface, indicating that at 4.7 T, GE-BOLD activation is sensitive to veins. For SE, the maximal activation is located in Layer IV. The SE-EPI anatomy (Column C) has improved contrast, i.e., shows the Gennari line more clearly, and has the same distortion as the functional map. Resolution = 250187 Am. The GEFI anatomy at 100100 Am (Column D) shows the Gennari line and intracortical veins.

specificity similar to that of SE-CBV activation in cat striate cortex [22] and monkey V1 [37]. Yet SE-EPI was still found to be somewhat sensitive to venous signals, as indicated by some activation near surface veins. However, the remaining activation near veins was minimal and could be due to intravascular T 2 changes, as well as residual T 2* effects resulting from the EPI acquisition window. The SE-EPI anatomical image has exactly the same distortion as the functional EPI; thus, using it as an anatomical reference results in more accurate localization of the activation. Although distortion and registration errors due to susceptibility gradients are small for the high degree of segmentation used here, distortions are still present (green arrows). Because of the high resolution, even minor errors can cause mislocalization. Because the SE-EPI anatomical scan maps exactly to the functional scan, it entirely avoids registration errors. Using the SE-EPI anatomical scan as a reference will also improve mapping to a GE-EPI functional scan, as long as the EPI module is the same. However, the GE-EPI can in addition suffer from signal dropout due to short T 2*s. For the calculation of intensity profiles, a region was selected as shown in Fig. 2G (arrows). The selection criterion

was maximum contrast at the Gennari line and the gray–white matter boundary, indicating minimal partial volume effects. Lack of partial volume effects was confirmed in the sagittal reconstruction of the three-dimensional volume in SPM; the slices were oriented exactly perpendicular to the cortex in such areas. At this resolution, irregularities in cortical thickness can also decrease the accuracy of the profiles, necessitating the selection of a limited area. The profiles indicate that for GE-BOLD fMRI (Fig. 2B), the largest signal changes occur at the cortical surface, while for SE-BOLD fMRI (Fig. 2E), the largest signal changes occur in the center of the gray matter, coincident with the Gennari line. The profile of the percent change shows a behavior different from the profile of the number of activated voxels. While the percent change for the GE is high at the surface, the number of voxels is constant up to a depth of about 1 mm (Fig. 2C). No peak in the activation profile was seen for the GE at the level of Layer IV because of the large signal from the surface. The SE shows a comparable number of activated voxels between depths of 0.5 and 1.5 mm (Fig. 2F), but the percent change is largest in Layer IV. The anatomical SE-EPI image (Fig. 2G) shows a clear delineation of the Gennari line and gray–white matter

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Fig. 2. Percent change activation maps (A and D), anatomical reference images (G and I) and a comparison of functional (B, C, E and F) and anatomical profiles (H and J) through the cortex. (A) GE-BOLD activation map; resolution = 333250 Am. For GE-BOLD fMRI, the activation map and the profile of the percent change (B) show maximal activation at the surface, while the number of activated voxels (C) is comparable at depths up to ~1 mm. The SE-BOLD map (D; resolution = 333326 Am) and the profile (E and F) show maximal activation in Layer IV. (G) SE-EPI anatomical image (resolution = 250187 Am) and profile (H). Profiles were calculated over the area between the arrows. The anatomical scan indicates that there is little partial volume effect and that the cortical thickness is uniform over this area. (I) GE image (resolution = 100100 Am) and profile (J).

boundary. The Gennari line is approximately 200 Am thick and lies between cortical Layers IVA and IVC. It is formed by axons of pyramidal and spiny stellate cells contained in Layer IVB. It is one of the two well-known Baillarger lines characterizing most of neocortex, albeit very pronounced only in Area V1. The second stripe of Baillarger lies deep in Layer V, consisting also of intrinsic myelinated cortical fibers. The contrast-to-noise ratio of the images in Fig. 2G and I is just sufficient to depict this fiber network, and it is visible in the profile of the SE-EPI (Fig. 2H). In addition to the aforementioned anatomical landmarks, a bright rim along the surface, presumably CSF, was visible.

The GEFI was coregistered to the SE-EPI, and although the location of the Gennari line coincided for the GEFI and SE-EPI anatomy (Fig. 2H and J), the location of the surface did not. This is because vessels and CSF are dark in the GEFI and larger vessels can cause susceptibility-related dropout. Therefore, if a GE anatomy profile is used as a reference for the SE functional profile, this can introduce an error of up to a few hundred micrometers. Comparison of the profile of the SE-BOLD signal (Fig. 2E) and the anatomical profiles (Fig. 2H and J) shows that the activation is highly specific and is located at the level of the Gennari line. The activity in more superficial layers and in the infragranular layers is lower. The small

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Fig. 3. SE-EPI anatomical image and profile before and after MION injection. (A) SE-EPI anatomical image before MION injection; spatial resolution = 250187 Am. After MION injection (B), the Gennari line becomes more pronounced, and more detailed lamination is visible. This is reflected in the profiles (D and E). The map of the relaxivity changes DR 2 (C) and profile (F) show that that largest relaxivity changes occur at the surface and in Layer IV.

peak at the white matter border (at 2.5 mm in Fig. 2B and E) is possibly due to a small vein running along the gray–white matter boundary. Whether smaller peaks (at 0.6 and 1.8 mm; Fig. 2E) in the activation profile represent physiologically meaningful phenomena is as yet unclear and is a topic for further study. Fig. 3 shows SE-EPI images before (Fig. 3A) and after (Fig. 3B) MION injection. The changes in contrast in the anatomical images result from R 2 changes. Since R 2 of the blood increases after MION injection, DR 2 is a metric of vascularization and CBV; R 2 is more sensitive to the microvasculature [16] than is R 2*. This can be seen by comparison of the SE image after MION injection with a GEFI image (see Smirnakis et al. [25]). The GE- and SEEPI show a similar laminar structure, but the GEFI image shows in addition signal dropout near superficial and intracortical veins. R 2* based functional CBV also peaked in the deeper layers [25]. The profile (Fig. 3F) shows that DR 2 is largest at the surface and at the level of the Gennari line. The exact dependence of DR 2 on vascularization is unknown; it is expected to scale with vascular volume fraction, but the geometry of the vessels in a voxel likely plays a role also.

A comparison of Figs. 2 and 3 shows that the steady-state DR 2 profile reflects the SE-EPI functional activity profile (i.e., the largest changes occur in Layer IV and at the surface, and smaller changes are seen in Layers II and III and towards the white matter). Although white matter is less vascularized than gray matter, the peak at ~2.5 mm depth may be due to veins running along the gray–white matter boundary, which produces locally large R 2 changes. The correspondence between the SE-BOLD profile and the DR 2 profile suggests that the SE-BOLD signal reflects the intracortical vasculature, with more vascularized regions displaying a larger SE-BOLD signal. 4. Discussion In this study, we showed that the SE-BOLD functional activation at 4.7 T is highly localized and is sufficiently specific to show the different cortical laminae, confirming and extending our earlier results [29]. To determine the exact location of the maximum SE-BOLD activation, activation was measured at high spatial resolution (187–333 Am), and the accuracy of the registration to anatomical reference scans was improved. We found that the maximum of the SE-BOLD

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signal was colocalized with the Gennari line, indicating that the functional signal is largest in Layer IV, which is the site of thalamic input. Although significant activation was observed from ~0.5 to 2 mm depth, the percent change was largest at the level of the Gennari line. Little activation at the surface was observed for SE-BOLD fMRI, indicating little contribution from pial vessels. Functional activation in the more superficial layers was lower, with fewer significantly activated voxels. The GE-EPI activation at high resolution confirmed earlier results in cats [20–22], rats [1,17,23,38] and monkeys [25,29], where the largest activation was observed at the cortical surface. Occasionally a small peak was observed at the level of Layer IV in the GE-BOLD signal as well, but usually this signal was drowned out by the strong signal from the surface. At 9.4 T in cat V1, the SE-BOLD method was reported to be less specific than the results reported here, and the specificity of MION-based methods exceeded the specificity of SE-BOLD activation [21,22]. However, we found that in monkey V1 at 4.7 T, the SEBOLD signal was more specific than MION-based GE methods [25,37], and it is comparable to the MION-based SE-CBV activation reported at 9.4 T [22]. This indicates that SE-based methods are preferable for high-resolution fMRI or when a high specificity of the functional signal is required. To accurately determine the site of activation at these resolutions, the method used to reference functional data to the anatomical scan becomes important. Differences in contrast and distortion between anatomical and functional scans can lead to misalignment of activation. We used an SEEPI to accurately reference the fMRI results to the anatomy because anatomical and functional SE-EPI have exactly the same distortion. Using an MR image as a reference improves accurate localization of the activity, since comparison with histological slices suffers from problems resulting from tissue shrinkage during fixation. At ultrahigh-resolution fMRI, the match between anatomical and functional scans needs to be nearly perfect. This is not guaranteed because EPI distortions can be considerable at high field, depending on sequence parameters. Although 16-segment EPI images did not suffer much from distortion, at high resolution, even minor distortion can result in decreased accuracy of the registration. Another factor affecting registration is contrast; the exact location of the cortical surface can be difficult to determine, particularly in a GE scan, because of the veins at the cortical surface, which have a short T 2* and low signal. For these reasons, our reference image was an SE-EPI, that has the same BW in both read direction and phase-encoding direction as the functional scans, and all images were coregistered. This improved the accuracy of the assignment of the functional activation. To determine whether the laminar profile of the SEBOLD signal was related to the vasculature and steady-state CBV, we measured R 2 changes after MION injection. The long half-life of MION allows high-resolution steady-state

CBV measurement. The SE-EPI used for the CBV measurement is more sensitive to MION-induced changes in the microvasculature, and DR 2 reflects differences in CBV [16]. R 2 changes after MION injection were largest at the surface, where large vessels are located, and in Layer IV, coinciding with the Gennari line. Lower steady-state CBV was observed in the more superficial layers and near the white matter. Similar patterns were observed in rats [26,39] and cats [22], although laminar DR 2 differences were less pronounced than in monkeys. The profile of R 2 changes after MION injection shows a qualitative similarity to the SE-BOLD activation profile. The large change in Layer IV is likely correlated with the higher vascularization of Layer IV, although differences in capillary density within V1 [40] are not as large as the DR 2 profile suggests. The DR 2 profile is qualitatively similar to quantitative measures of vascularization [25,40], with the highest vascular density in Layer IV, a small peak in Layer VI and a decrease of vascularization in white matter. However, differences in the R 2 profile are larger than expected based on differences in vascular density. There are, several factors that can possibly explain these differences, for example, DR 2 is dependent on capillary density and blood volume, but also on vessel geometry [13]. Furthermore, the acquisition window of the SE-EPI confers some T 2* sensitivity, and the contribution of T 2* changes to the signal and possible T 1 changes after injection of the contrast agent were not taken into account. The qualitative similarity between the SE-BOLD functional activation profile, the DR 2 profile and cortical vascularization [40] suggests that the SE-BOLD signal reflects the intracortical vasculature and that baseline CBV and SE-BOLD activation are correlated. Although there are also differences, for instance, the BOLD change in superficial layers is smaller than expected based on CBV and vascularization. A detailed quantitative analysis of changes in MION signal during baseline and activation and a comparison with the SE-BOLD activation are subjects of further study.

Acknowledgments We would like to thank Mark Augath for technical support and Kathrin Guhr, Mirko Lindig and Christian Samhaber for preparing and maintaining animal anesthesia. This work was supported by the Max-Planck Society.

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