Cerebral Cortex October 2008;18:2318--2330
Advance Access publication January 24, 2008
The Microvascular System of the Striate
and Extrastriate Visual Cortex of the
Bruno Weber1,2, Anna Lena Keller1, Johannes Reichold3and
Nikos K. Logothetis1
1Max-Planck Institut fu ¨ r biologische Kybernetik, Spemannstr.
38, 72076 Tu ¨ bingen, Germany,2University of Zurich, Institute
for Pharmacology and Toxicology, Ra ¨ mistrasse 100, 8091
Zurich, Switzerland and3Swiss Federal Institute of Technology,
Institute for Fluid Dynamics, Sonneggstrasse 3, 8092 Zurich,
In functional neuroimaging, neurovascular coupling is used to
generate maps of hemodynamic changes that are assumed to be
surrogates of regional neural activation. The aim of this study was
to characterize the microvascular system of the primate cortex as
a basis for understanding the constraints imposed on a region’s
as area- and layer-specific variations. In the macaque visual cortex,
an array of anatomical techniques has been applied, including
corrosion casts, immunohistochemistry, and cytochrome oxidase
(COX) staining. Detailed measurements of regional vascular length
density, volume fraction, and surface density revealed a similar
vascularization in different visual areas. Whereas the lower cortical
layers showed a positive correlation between the vascular and cell
density,this relationshipwasveryweak in theupperlayers.Synapse
density values taken from the literature also displayed a very
moderate correlation with the vascular density. However, the
vascular density was strongly correlated with the steady-state
metabolic demand as measured by COX activity. This observation
suggests that although the number of neurons and synapses
determines an upper bound on an area’s integrative capacity, its
vascularization reflects the neural activity of those subpopulations
that represent a ‘‘default’’ mode of brain steady state.
Keywords: capillaries, cerebral blood flow, collagen, monkey,
Despite its relatively small size the brain consumes roughly
a quarter of the body’s total glucose and a fifth of the oxygen.
The high energy demand in combination with the fact that
brain tissues lack any substantial capacity to store energy
requires a tight spatiotemporal control of the energy supply.
Changes in neural activity are indeed followed by precisely
controlled changes in hemodynamics, as hypothesized more
than a century ago (Mosso 1881; Roy and Sherrington 1890).
This remarkable site- and time-specific neurovascular coupling
has been systematically exploited to generate detailed maps of
hemodynamic changes that are assumed to be surrogates of the
actual regional neural activation. A recent celebrated example
is the so-called blood oxygenation level--dependent (BOLD)
contrast (Ogawa et al. 1990; Kwong et al. 1992) of magnetic
resonance imaging (MRI), which reflects a complicated in-
terplay of changes in blood volume, blood flow, and oxygen
consumption (Logothetis and Wandell 2004). Because BOLD
functional MRI (fMRI) is now the mainstay of biomedical
neuroimaging, a lot of research has been focused recently on
the functional aspects of neurovascular coupling, including the
underlying signaling mechanisms, and the biochemistry of the
neurometabolic link. Yet understanding neurometabolic and
neurovascular coupling also requires a comprehension of the
specific variation in the vascular system.
In response to this, a great deal of research is currently being
devoted to understanding and modeling the link between
neural activation and BOLD or perfusion fMRI (Logothetis et al.
2001; Devor et al. 2003; Jones et al. 2004; Sheth, Nemoto,
Guiou, Walker, Pouratian, Toga, 2004; Thomsen et al. 2004;
Martindale et al. 2005) or to describing the spatiotemporal
dynamics of the hemodynamic response (Sheth, Nemoto,
Guiou, Walker, Pouratian, Hageman, et al. 2004; Vanzetta
et al. 2004; Weber et al. 2004; Sheth et al. 2005).
Ultimately, the spatial resolution and specificity of the
hemodynamic response maps rely not only on the cascade of
neurovascular signaling, but also on the vascular architecture
and spatial level of blood flow regulation. Little is known,
however, about the principles of flow regulation at different
vascular scales, about the spatial distribution of vascular
densities, and about the dependence of vasculature on cortical
areas or brain sites in general. In their seminal study, Duvernoy
et al. (1981) demonstrated area-specific vascular densities in
the human brain, but unfortunately failed to provide precise
More recently, it was shown that areas with higher steady-
state metabolic demand show a higher degree of vascular
density (Zheng et al. 1991; Cox et al. 1993; Riddle et al. 1993;
Woolsey et al. 1996; Tieman et al. 2004). In a study employing
the corrosion cast technique together with optical imaging,
Harrison et al. (2002) postulated a close relationship between
the hemodynamic response map and both vascular density and
the localization of perivascular flow control elements. All of
these studies quantified the vasculature within a primary
sensory area, or at best compared it with a secondary region.
Investigations of a more complete hierarchical system—such as
large parts of the visual system—have yet to be carried out.
Because fMRI has the great advantage of measuring brain
activity with full coverage, countless studies have compared
the activity in different cortical areas. However, the completely
different vascular densities and architectures of these regions
would impede direct comparisons of the local readouts based
on the hemodynamic response.
In this work, we describe and quantify the microvasculature
in the macaque visual cortex, applying 2 different and
complementary techniques. Scanning electron microscopy
was used to image vascular corrosion casts for a qualitative
assessment of the microvascular organization, for an estimation
of the ratio between arteries and veins, as well as for an
investigation of perivascular structures. At the same time,
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double histochemical stains were performed on a large data set
from 3 monkeys to evaluate quantitative layer- and area-specific
vascular densities. To compare the vascular density with the
basal metabolic demand of a given region, cytochrome oxidase
(COX) stains were prepared in 3 additional subjects.
Materials and Methods
The brains of 7 adult monkeys (Macaca mulatta) were used in this
study. The animals were involved in chronic combined physiological
and behavioral experiments. At the end of the experiments and as part
of our standard protocol the animals were killed with an overdose of
pentobarbital (120 mg/kg). The euthanasia methods correspond to the
guidelines of the ‘‘American Veterinary Association Panel on Euthanasia’’
and to the recommendations of the ‘‘Guide for the Care and Use of
Laboratory Animals of the National Institutes of Health of the
United States.’’ They were all approved by the local authorities
(Regierungspra ¨ sidium) and are also in full compliance with the
guidelines of the European community (EUVD 86/609/EEC) for the
care and use of laboratory animals. The brain of one of the animals
was used for vascular corrosion casts, 3 brains were used for
histochemical processing and 3 were stained for COX activity.
Vascular Corrosion Casts
The animal was perfused under deep sodium pentobarbital (120 mg/kg)
anesthesia with 0.9% warm heparinized 0.1 M phosphate buffered saline
(Polysciences Inc., Eppelheim, Germany) was injected. After full
polymerization overnight, the brain was carefully removed from the
skull.The monkey brains were frozen at –20 ?C and cut in cubesof about
15 3 15 3 15 mm using a dedicated saw. Brain tissue cubes were
macerated in 5% KOH. The maceration process, which included daily
distilledwaterand theregions ofinterest weretrimmed usinga cryostat,
yielding precise cuts without any damage of the fragile cast (see Figs 1
and 2). The specimens were then lyophilized overnight and sputter
coated with gold. Scanning electron microscopy at 10--20 kV (Hitachi S-
800) was then used to acquire high-resolution images of the vascular
corrosion casts. Large vessels penetrating the cortex from the surface
were individually imaged. On these high magnification images, each
vessel could be identified as either an artery or a vein on the basis of the
As described before, arteries show shallow and elliptical or lens-shaped
imprints. In contrast, the endothelial cell nuclei imprints on the venous
due to the lower blood pressure in the venous system. In total, we
analyzed 249 vessels that could be identified with sufficient certainty,
from which we could estimate the ratio between arteries and veins. The
shrinkage of the employed resin is known to be small (Lametschwandt-
ner et al. 1990) and was disregarded in this study.
Three animals were perfused transcardially under deep sodium
pentobarbital (120 mg/kg) anesthesia with 8 L of warm heparinized
0.1 M PBS, followed by 2 L of cold 1% paraformaldehyde (PFA) in 0.1 M
phosphate buffer (PB), then 4 L of 4% PFA in 0.1 M PB, and finally 1--2 L
of 10% sucrose in 0.1 M PB. The brain was removed from the skull and
stored in 4% PFA at 4 ?C. Before sectioning, cortical blocks were placed
in ascending concentrations of sucrose in 0.1 M PB (10%, 20%, and
30%) until they sank. Sixty-micrometer-thick horizontal frozen sections
were then cut serially on a sliding microtome (Microm HM 440E,
Walldorf, Germany) and stored at –20 ?C in cryoprotectant (30%
ethylene glycol and 10% sucrose in 0.05 M PB) until further processing.
Double fluorescence histochemical staining was carried out on every
tenth free-floating section. Sections were rinsed 3 times for 5 min each
in 0.1 M PBS before and after antigen retrieval (incubation overnight in
0.05 M TRIS buffer at 65 ?C). They were then blocked with TRIS saline
pH 7.4 (0.6% TRIS) containing 0.02% sodium azide, 5% dry milk, 1%
Triton X-100 (Sigma, Schnelldorf, Germany), and 1% goat serum for 1 h
at room temperature. Anticollagen has been successfully used to stain
all types of cerebral vessels (arteries, capillaries, and veins) with
excellent specificity (Hamann et al. 1995; Fukuda et al. 2004). The
primary antibody (monoclonal anticollagen type IV, clone col-94;
Sigma) was diluted 1:500 in TRIS saline containing 2% bovine serum
albumin (albumin bovine fraction V powder, Sigma), 0.05% thimerosal
(Sigma), and 1% Triton X-100 and the sections were incubated in this
solution overnight at 4? C. They were then washed again 3 times in 0.1
M PBS before being incubated overnight at 4 ?C in the dark in a solution
containing the secondary antibody (Cy-3--conjugated goat anti-mouse
IgG (H + L), Jackson ImmunoResearch, West Grove, PA, diluted 1:500 in
PBS containing 1% goat serum). In between 2 series of 3 rinses in 0.1 M
PBS the sections were incubated for 5 min in 0.4 3 10–3& DAPI (4#,6-
diamidino-2-phenylindole dihydrochloride; Sigma) in dH2O. Stained
sections were mounted on glass slides and cover-slipped with polyvinyl
alcohol (Mowiol 4-88; Hoechst, Frankfurt, Germany) containing 4% 1,4-
diazobicyclooctane (Merck, Darmstadt, Germany) as an antifading
reagent. Consecutive sections were stained for Nissl and myelin
(Werner’sche Markscheidenfa ¨ rbung). The tissue shrinkage due to the
perfusion and fixation was assumed to be minimal. The shrinkage
produced by subsequent tissue handling was measured according to
the protocol described in O’Kusky and Colonnier (1982), and was
found to be 5%. It is important to note that this value is rather small due
to the fact that the sections were not dry-mounted.
Cy-3 and DAPI images of identical fields of view were acquired using
a fluorescence microscope (Axiophot, 53 objective; Carl Zeiss,
Go ¨ ttingen, Germany) equipped with a CCD camera (Axiocam MRm,
controlled by Axiovision 4.3; Zeiss, Go ¨ ttingen, Germany).
Measurements were made on images extending from cortical layer I to
white matter (approximately 1 3 2 mm in size). The specific cortical
visual areas were identified with the help of an anatomic atlas (Saleem
and Logothetis 2006). Within a visual area, images were taken from
locations without obvious histological damage. All digital image
processing was performed using Matlab (Mathworks, Natick, MA).
The raw anticollagen images (Fig. 3C,E) were median filtered and
thresholded to yield binary images of the vessels (Fig. 3D,F). The total
volume fraction (%), length density (mm/mm3), surface density (mm2/
Figure 1. Scanning electron micrographs of a vascular corrosion cast from monkey
visual cortex (superior temporal gyrus). Casts were cut and trimmed to allow
a vertical view on the cortex. The gray--white matter demarcation line is shown as
dashed line. Note the continuous orderly distribution of large vessels oriented
perpendicularly to the cortical surface, their different length and branching patterns
and the rather homogeneous mesh size and density of the capillary bed. The
arrowheads in (A) and (B) exemplify a few vessel types, named according to the
different vessel classes introduced by Duvernoy et al. (1981) for the human brain.
According to this classification, a vessel of class 1 would for example feed/drain the
capillary bed in the vascular layer 1, whereas a class 6 artery would traverse the
cortex without any branching in gray matter. In Figure 1B, the larger penetrating
arteries are shaded red and the veins are shaded blue. (A 5 artery, V 5 vein, 1--6 5
category based on their cortical depth; scale bars 5 500 lm.)
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