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Developmental Brain Researc

Research report

Lack of experience-mediated differences in the immunohistochemical

expression of blood–brain barrier markers (EBA and GluT-1)

during the postnatal development of the rat visual cortex

Enrike G. ArgandonaT, Harkaitz Bengoetxea, Jose V. Lafuente

Laboratory of Clinical and Experimental Neuroscience (LaNCE), Department of Neuroscience, School of Medicine,

Euskal Herriko Unibertsitatea/University of the Basque Country, Leioako Campusa, Leioa E-48940, Spain

Accepted 15 February 2005

Available online 1 April 2005

Abstract

The development of the cortical vascular tree depends on functional development. External inputs are an essential requirement in the

modeling of the visual cortex, mainly during the critical period, when congruous blood supply is needed. The blood–brain barrier (BBB)

function regulates the passage of substances between the blood and the brain parenchyma, which is one of the main differential features of

central nervous system (CNS) microvessels. The endothelial barrier antigen (EBA) has been reported as a specific marker for the BBB

physiological function in rats. We studied the postnatal development of EBA expression in the visual cortex of rats reared under opposite

paradigms of visual experience, e.g., standard laboratory conditions, dark rearing, and enriched environment at 14, 21, 28, 35, 42, 49, 56, and

63 days postnatal (dpn). Parallel sections were immunohistochemically processed for endothelial barrier antigen (EBA) and glucose

transporter-1 (GluT-1). Total vasculature was quantified by Lycopersicon esculentum (LEA) lectin histochemistry. No differences in EBA

expression were found between groups, although quantitative differences were recorded paralleling differences in vascular density.

Paradoxically, there was no expression in certain cortical vessels which were GluT-1 immunopositive and positivity was consistent in non-

barrier areas such as the pineal gland. These findings were completely independent of age or experimental conditions. Therefore, the role of

the EBA antigen in the BBB remains unclear: it has been undeniably linked to vascular permeability, but its presence in non-barrier vessels

suggests another vascular function. Although visual experience modifies vascular density in the visual cortex, it has not been shown to have

an influence on the maturation of the BBB function.

D 2005 Elsevier B.V. All rights reserved.

Theme: Cellular and molecular biology

Topic: Blood–brain barrier

Keywords: BBB; Endothelial cell; Brain microvessels; Cerebral angioarchitecture; Pineal gland

1. Introduction

Postnatal development of the visual cortex is modulated

by experience. Extrinsic cues act as epigenetic factors in

concert with intrinsic developmental programs to shape

functional and structural cortical architecture [23]. Experi-

ence-mediated changes produce an increase in neuronal

0165-3806/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.devbrainres.2005.02.007

T Corresponding author. Fax: +34 94 4649511.

E-mail address: nfpguare@lg.ehu.es (E.G. Argandona).

URL: http://www.ehu.es/LaNCE/.

activity, which leads to increased metabolic demands [8,38]

involving the establishment of adaptive changes to meet new

requirements, such as changes in the vascular network [6,9].

Most of the changes induced by experience occur during a

defined time-window of postnatal life, called the critical

period; in rats, this period is between the 3rd and 5th weeks

postnatal, with the peak of maximum experience-induced

changes occurring between the 4th and 5th weeks [13].

The vasculature of the cerebral cortex is mainly

comprised of a capillary network which spreads throughout

the cortex [6], with differential characteristics as compared

h 156 (2005) 158 – 166

E.G. Argandona et al. / Developmental Brain Research 156 (2005) 158–166 159

to vasculature outside the brain, and even as compared to

some other brain regions such as the blood–brain barrier

(BBB). The BBB is the set of physical and metabolic

mechanisms regulating the passage of substances between

the blood fluid and the cortical parenchyma. Most of these

mechanisms are involved in the regulation of fluid perme-

ability and are based on endothelial cells.

Some of these enzymatic mechanisms have been used to

quantify vascular distribution, such as Butyryl cholinesterase

histochemistry [3], alkaline phosphatase histochemistry [14],

LEA lectin histochemistry [12,21,37], and immunohisto-

chemistry against antigens such as glucose transporter-1

(GluT-1) [18], PECAM [24], RECA-1 [26], and the

endothelial barrier antigen (EBA) [11,19,25].

The EBA has been described as a protein triplet of 23.5,

25, and 30 kDa located in the luminal membrane of

endothelial cells [35]. Although its function remains

unclear, some studies have related the transient disappear-

ance of its expression in several experimental models to

pathological conditions involving a permeability increase in

allergic encephalomyelitis [36], brain injury [18,20], and

toxic administration such as sarin [1,2] or Clostridium

perfringens toxin [39]. EBA expression is therefore restored

in tandem with the recovery of the barrier function after

those episodes. For this reason, the EBA has been proposed

as a valid marker to study the brain vascular network with

their BBB mature and physiologically fully functional.

The development of the cortical capillary network is

closely linked to the development of the cortex, as capillary

density parallels local function. In previous work, we have

reported the influence of visual inputs on the postnatal

development of the vasculature of the striate cortex, showing

a delay in the maturation of the microvascular pattern and a

decrease in vascular density in dark-reared rats [3,4].

However, some authors have reported that microvascular

development remains after the end of the critical period in rats

reared in an enriched environment, and have also quantified

an increase in vascular density in enriched environments

[30,34].

The present work compares the postnatal development of

EBA expression in rats deprived of visual inputs and rats

reared in an enriched environment to standard reared rats in

order to try to evaluate the influence of visual experience

during the critical period. These hypothetical differences in

EBA expression and/or distribution at a given age could

suggest different stages in the maturation of the BBB depend-

ing on experience. As it has been described, the immunopo-

sitivity of the EBA can be linked to a maturational stage when

the BBB acquires the adult function. This positivity also

appears after the pathological situations described above and

is a sign of the recovery of the physiological function.

The EBA labeling appears at the same time as another

BBB marker, s-laminin [11], at the end of the 2nd postnatal

week. Therefore, the development of the adult distribution

pattern is completely coincident with the beginning and the

end of the critical period for the visual cortex.

Quantification has been performed on cortical layer IV,

where experience-induced changes occur, as this layer is the

termination site of visual afferents coming from the

thalamus [28,33]. Some authors have previously found

important dark-rearing-induced synaptic [10,22,31,32],

astroglial [5], molecular [7], genetic [17], and vascular

[4,14] changes on this layer; therefore, experience-induced

changes in EBA expression signaling BBB changes should

be found on layer IV.

2. Materials and methods

2.1. Animals

Three series of pregnant Sprague–Dawley rats were

raised in one of three rearing conditions:

– Five pregnant rats were placed in a dark room at the

beginning of pregnancy. Litters were born in complete

darkness, weaned at 21 days postnatal (dpn), and eight rats

of both sexes were sacrificed at each of the following ages:

14 dpn, 21 dpn, 28 dpn, 35 dpn, 42 dpn, 49 dpn, 56 dpn, and

63 dpn.

– Five pregnant rats were placed in an enriched environ-

ment consisting of a large cage full of colorful toys and

complex objects that were changed every 2 days. Eight rats

of both sexes were sacrificed at each of the above-mentioned

ages.

– Eight rats of each age raised in standard conditions (12-h

light–dark cycle) were sampled as control.

Water and food were available ad libitum for the three

groups. Every effort was made to minimize animal suffering

and to reduce the number of animals used. All animal

experiments were performed in accordance with the Euro-

pean Community Council Directive of 24 November 1986

(86/609/EEC).

Animals were anaesthetized with 6% chloral hydrate

(performed under dim red light for the dark-reared group).

After anesthesia, the animals were transcardially perfused

with a fixative containing 4% paraformaldehyde in 0.1 M

phosphate buffer. Perfusion was carried out with a pump at a

constant pressure of 20 mm Hg. Following perfusion, brains

were stored overnight at 4 -C in fresh fixative. The following

day, a thick block of occipital cortex containing the visual

area was removed sagittally with a Rodent Brain Matrix

(Electron Microscopic Sciences, USA), rinsed in cold PBS

for 4 h, and embedded in paraffin. The block was serially cut

with a microtome into sections of 4 Am and mounted on

slides coated with APES (3-aminopropyltriethoxylane).

2.2. Histochemical procedures

Paraffin was removed from the tissue through xylene

immersion; the tissue was rehydrated with ethanol. Endo-

E.G. Argandona et al. / Developmental Brain Research 156 (2005) 158–166160

genous peroxidase activity was blocked by incubation in 4%

H2O2/methanol for 20 min. Sections were washed in Tris

buffer at pH 7.4 and incubated overnight with biotinylated

lectin from Lycopersicon esculentum (LEA 5 Ag/ml;

SIGMA L0651) at 4 -C. Sections were rinsed with a Tris

buffer and incubated with avidin biotin peroxidase complex

(Elite ABC kit, Vector laboratories, Burlingame, CA, USA).

Finally, the reaction product was detected using 3,3¶-

diaminobenzidine (DAB 0.25 mg/ml) and hydrogen per-

oxide solution (0.01%). Slides were lightly counterstained

with hematoxylin, dehydrated, and covered.

2.3. Immunohistochemical procedures

Paraffin was removed from the tissue through xylene

immersion; the tissue was rehydrated with ethanol. Endo-

genous peroxidase activity was blocked with 4% H2O2/

methanol for 20 min. Sections were kept in a Tris buffer at pH

7.4, then incubated for 30min at 37 -C in Trypsin 0.1%CaCl2for antigen retrieval. Sections were incubated overnight with

an anti-EBAmonoclonal antibody (SternbergerMonoclonals

SMI-71; working dilution: 1:2000) and anti GluT-1 poly-

clonal antibody (Chemicon AB 1340 working dilution:

1:1000). Biotinylated secondary antibodies were used (Vec-

tor, USA) and the immunohistochemical reaction was

revealed by the avidin-biotin complex using diaminobenzi-

dine as a chromogen. Sections were lightly counterstained

with hematoxylin and finally dehydrated and covered.

Some sections were used to co-localize EBA and GluT-1

antigens. These sections were incubated overnight with the

aforesaid primary antibodies and with the following secon-

dary antibodies: FITC conjugate anti-mouse IgG (Ref: F-

9137 Sigma; working dilution: 1:100) and TRITC conjugate

anti-rabbit IgG (Ref: F-0382 Sigma; working dilution:

1:100).

Also included in each staining run were negative controls

in which the primary antisera and the lectin were omitted.

Images were acquired for confocal fluorescence micro-

scopy with an Olympus Fluoview FV500 confocal micro-

scope using sequential acquisition to avoid overlapping of

fluorescent emission spectra.

2.4. Morphometric procedures

To quantify changes that occurred during development

and to compare the three situations (normal, enriched

environment EE, and dark rearing DR), a blind morphometric

study was performed. In this study, the individual who

measured the sections did not know the features of each case

(neither the age of the rats nor whether they belonged to DR,

EE, or control groups). All sections of each case were

incubated for immunohistochemistry and histochemistry, and

the LEA, the EBA and the GluT-1 positive density were

estimated counting the number of positive vascular profiles

per area of visual cortex in serial sections. This was selected

with the aid of the Paxinos andWatson atlas [27]. Layers were

differentiated using parallel sections stained with Nissl, and

with hematoxylin counterstaining as a reference.

To estimate the number of profiles per area, the number of

positive capillaries present in an area delimited by a grid fixed

in the eyepiece was counted at 20� optical magnification,

excluding those intersected by both the x and y axes. The grid

was a square 250 Am in length, and thus the total surface was

62.500 Am2. The grid was randomly placed between cortical

layers III and V, ensuring that layer IV was included.

Measurements of each slice of the cortex were taken in

both hemispheres for each of the 10 slices taken per animal

(i.e., 60 fields per animal) and the mean value per animal

was calculated. The average values per group (8 animals)

were compared at each age by statistical analysis (ANOVA)

on Statview IIi Abacus Concepts.

3. Results

3.1. Qualitative results

3.1.1. LEA expression

LEA histochemistry showed a consistent staining of the

vascular network, which was homogeneously present on all

cortical layers, from I to VI; however, vessels were more

densely packed on layer IV. Within the cerebral cortex, there

was no regional variability in labeling, showing strong

staining of all vessels. The same expression pattern and

distribution were present at all ages (Fig. 1).

3.1.2. EBA expression

EBA immunostaining was homogeneously present

throughout the cortex on blood vessels of all sizes, although

some capillaries remained unstained. No other structures but

vessels were positive, including the neuropil. The expression

pattern was comparable to that of the LEA, although age-

related quantitative differences were found at the first two

ages.

At 14 dpn, only a small proportion of microvessels were

positive. From 21 dpn onwards, positivity was present in

most brain microvessels, but some cortical capillaries re-

mained unstained (Fig. 1). All the EBA-unstained capillaries

were GluT-1 positive (Fig. 2). Apart from cortical expression,

positive vessels were found in regions without barrier

function, such as pial vessels and especially the pineal gland

(Fig. 1h). No differences in pineal EBA expression were

found between different ages or experimental conditions.

Pineal positivity was as consistent as cortical positivity.

3.1.3. GluT-1 expression

All cortical blood vessels were immunopositive for GluT-

1, with an expression pattern similar to that of the LEA and

the EBA. Nevertheless, immunostaining was positive at all

the ages studied, with no qualitative differences related to

age or experimental condition. By contrast, no positive

vessels were found in the pineal gland (Fig. 1g).

Fig. 1. (a–h) EBA, GluT-1, and LEA histochemical and immunohistochemical detection of vessels in the rat visual cortex at several ages of postnatal

development. Arrows show EBA-negative and LEA-positive vessels. (a) EBA immunohistochemistry and (b) LEA histochemistry at 14 dpn. (c) EBA and (d)

LEA positivity at 28 dpn. (e and f) EBA and LEA at 63 dpn. (g) GluT-1 and (h) EBA immunohistochemistry in the pineal gland at 56 dpn. All images

correspond to control animals, although they were qualitatively indistinguishable from other groups. Scale bar is 50 Am in all cases.

E.G. Argandona et al. / Developmental Brain Research 156 (2005) 158–166 161

3.2. Quantitative results

3.2.1. LEA

Vascular density underwent slight increases over the

course of postnatal development in the three experimental

groups, but quantitative differences were found from the

beginning of the critical period (Table 1 and Fig. 3b).

In dark-reared rats, vascular density was significantly

lower starting at the 5th week postnatal. Although at 14 dpn

there were 20% fewer vessels per area in dark-reared rats (23

vs. 29), differences disappeared at 21 dpn, when vascular

density was 8% lower in controls, although the difference

lacked statistical significance. At 28 dpn, density was (not

significantly) 5% lower in dark-reared rats; from 35 dpn

Fig. 2. (a–c) EBA and GluT-1 double labeling. Confocal fluorescent

images in c show co-localization (yellow) of both blood–brain barrier

markers in the visual cortex section of a 28-dpn control rat. (a) GluT-1

expression (red), (b) EBA expression showing the lack of staining in some

GluT-1-positive capillaries. (c) GluT-1 and EBA co-localization. Scale bar

is 50 Am.

E.G. Argandona et al. / Developmental Brain Research 156 (2005) 158–166162

onwards, differences were always significantly lower in dark-

reared rats: 9% at 35 dpn, 10% at 42 dpn, 8% at 49 dpn, 12%

at 56 dpn, and 7% at 63 dpn. In both conditions, the obtained

numbers were similar at all ages, ranging from 29 at 14 dpn to

36 at 63 and 49 dpn in controls, and from 23 at 14 dpn to 34 at

21 dpn in dark-reared rats.

Rats reared in an enriched visual environment showed

similar but opposite behavior, as vascular density was

higher than in controls. Greater differences were recorded

and also appeared earlier in the critical period, at the 4th

postnatal week.

At 14 dpn, environmentally-enriched rats showed 25%

fewer vessels per area than controls, a significant differ-

ence (22 vs. 29). As in the case of dark-reared rats,

numbers were similar at 21 dpn (mean value of 31.1 vs.

31.5), and from 28 dpn onwards, measurements were

always higher for the complex experience group: 29%

higher at 28 dpn; 11% higher at 35 dpn; 23% higher at 42

dpn; 19% higher at 49 dpn; 25% higher at 56 dpn; and

11% higher at 63 dpn. Although the numbers were again

similar for different ages within the same experimental

group, there was a major increase between 14 and 21 dpn

(21.6–31.1) and a lesser increase between 21 and 28 dpn

(31.1–37.7). Later, countings ranged from 37.7 at 28 dpn

to 42.4 at 49 dpn.

3.2.2. EBA

EBA cortical expression increased dramatically from 14

to 28 dpn, when it reached its adult level (Fig. 3a). Starting

at 21 dpn, significant quantitative differences were recorded

parallel to differences in vascular density.

Only 3.5 vessels per area were counted in controls at

14 dpn. However, there were already 23.1 by 21 dpn and

28.4 at 28 dpn. As of this age, EBA-positive numbers

remained close to those of the LEA (33 at 35 dpn; 32 at

42 dpn; 34 at 49 dpn; 31 at 56 dpn; and 35 at 63 dpn).

For rats reared in darkness, behavior was comparable to

controls, although with lower absolute values following the

measurements obtained with LEA. We recorded 29% fewer

vessels per area at 14 dpn, 22% more at 21 dpn (both values

were non-significant), and 11% lower vascular density at 28

dpn in dark-reared rats. This was 8.5% lower at 35 dpn;

13.6% lower at 42; 8% lower at 49 and 56 dpn; and 7%

lower at 63 dpn. Differences were significant from 28 dpn

onwards.

While EBA-positive vascular density was significantly

lower in animals reared in darkness from 28 dpn, it was

significantly higher in enriched experience-reared animals.

These results also corresponded to quantifications in total

vascular density measured with LEA histochemistry.

Numbers were similar to controls at 14 dpn in environ-

mentally-enriched rats, and they were 9% lower at 21 dpn.

From 28 dpn onwards, they were higher in controls: 23% at

28 dpn; 3.6% at 35 dpn; 12% at 42 dpn; 10% at 49 dpn;

28% at 56 dpn; and 10% at 63 dpn.

The percentage of negative vessels reached its max-

imum at 14 dpn when 88% of vessels remained unstained

in controls (89% in dark-reared rats and 84% in environ-

mentally-enriched rats) (Table 2). At 21 dpn, the percent-

age of positive vessels rose, and we recorded only 27% of

negative vessels in controls (17% in dark-reared rats and

32% in environmentally-enriched rats). As of 28 dpn, most

vessels were positive and the percentage of negatives was

less than 10% for all ages and experimental groups. At 28

dpn, negatives accounted for 3% in controls (C), 9% in

dark-reared rats (DR), and 7% in enriched environment

group (EE). At 35 dpn, it was 5% in C, 4% in DR, and

11% in EE. At 42 dpn, it was 2% in C, 6% in DR, and

11% in EE. At 49 dpn, it was 1% in C, 0.3% in DR, and

9% in EE. At 56 dpn, results showed 4% of unstained

vessels in C, 0.3% in DR, and 1.5% in EE. At the last

measured age, 63 dpn, it was 2.5% in C, 3% in DR, and

4% in EE (Fig. 3c).

Table 1

Positive vessel density along postnatal development (mean T SD)

Age EE DR Control DR vs. C (%) EE vs. C (%) p DR vs. C p EE vs. C

LEA

14 dpn 21.6 T 5.6 22.9 T 5.9 28.8 T 6.4 �20.5 �25 0.0001 0.0001

21 dpn 31.1 T 5.9 34.2 T 8 31.5 T 5.7 8.6 �1.3 0.06 0.75

28 dpn 37.7 T 6.7 27.9 T 6.6 29.2 T 3.7 �4.5 29.1 0.3 0.0001

35 dpn 38.3 T 5.3 31.5 T 6.7 34.6 T 5.9 �9 10.7 0.01 0.01

42 dpn 40.6 T 5.2 29.9 T 4.7 33.1 T 6.2 �9.7 22.7 0.004 0.0001

49 dpn 42.4 T 5.5 32.6 T 5.3 35.6 T 6.5 �8.4 19.1 0.01 0.0001

56 dpn 40.6 T 5.6 28.8 T 4.5 32.6 T 5.6 �11.7 24.5 0.0006 0.0001

63 dpn 39.8 T 9.2 33.2 T 4.6 35.7 T 6.5 �7 11.5 0.008 0.001

EBA

14 dpn 3.5 T 3.4 2.5 T 2.3 3.5 T 2.5 �28.6 0 0.04 0.9

21 dpn 21 T 6.4 28.3 T 8.9 23.1 T 4.9 22.5 �9.1 0.0008 0.1

28 dpn 35 T 8.5 25.3 T 4.2 28.4 T 4.3 �10.9 23.2 0.04 0.007

35 dpn 34.2 T 4.9 30.2 T 5.3 33 T 5.6 �8.5 3.6 0.02 0.4

42 dpn 36.3 T 8.6 28 T 5.8 32.4 T 3.6 �13.6 12 0.007 0.08

49 dpn 38.7 T 8.3 32.5 T 5.4 34.7 T 5.5 �7.7 9.9 0.04 0.04

56 dpn 40 T 5.1 28.7 T 3.1 31.2 T 4.8 �8 28.2 0.05 0.0001

63 dpn 38.2 T 5.7 32.3 T 5.4 34.8 T 7.4 �6.9 10.1 0.1 0.06

GluT-1

14 dpn 21.9 T 4.1 23 T 5.7 27.9 T 3.2 �17.6 �21.5 0.0001 0.0001

21 dpn 31 T 6.8 33 T 4.6 31.2 T 3.7 5.8 �0.6 0.0366 0.7594

28 dpn 37.8 T 5.3 27.9 T 4.5 29.1 T 4.4 �4.1 29.9 0.1827 0.0001

35 dpn 38.5 T 4.4 31.2 T 4.3 34.7 T 3.8 �10.1 11 0.0001 0.0001

42 dpn 40.1 T 5.2 30.4 T 5.2 33.9 T 3.8 �10.3 18.3 0.0005 0.0001

49 dpn 42.6 T 3.8 32.8 T 5.7 35.5 T 4.9 �7.6 20 0.0129 0.0001

56 dpn 41.9 T 3.3 29.4 T 4.4 33 T 4.4 �10.9 21.2 0.0001 0.0001

63 dpn 39.6 T 3.7 33.1 T 4.8 35.9 T 4.2 �7.8 10.3 0.0055 0.0001

Number of EBA-, LEA-, and GluT-1-positive vessels at various ages of development (dpn: days postnatal) for enriched (EE), dark-reared (DR), and control

groups (mean number of EBA- and LEA-positive vessels per 62,500 Am2 of visual cortex T standard deviation and statistical significance, P value).

E.G. Argandona et al. / Developmental Brain Research 156 (2005) 158–166 163

Despite the variability of differences among the ages and

experimental groups, these differences were not statistically

significant.

3.2.3. GluT-1

Numbers were almost identical to the LEA ones at all the

ages and conditions. They have been reproduced in Table 1.

Fig. 3. (a–c) Comparison of average measurements between environmentally-e

Horizontal axes show the age of the animals. Vertical axes show: (a) number of E

positive vessels per 62.500 Am2 of visual cortex, (c) percentage of EBA-negative

show EC control significance.

4. Discussion

The postnatal development of cortical vascularization

consists of the establishment of a dense capillary network

dependent on the local function. In standard conditions, it is

completed around the end of the 4th postnatal week [30]. On

other hand, in animals reared under different environmental

nriched, dark-reared, and control groups at each of the ages considered.

BA-positive vessels per 62.500 Am2 of visual cortex, (b) number of LEA-

vessels in 62.500 Am2 of visual cortex; + show DR control significance, *

Table 2

Percentage of EBA-unstained vessels

Age EE (%) DR (%) Control (%) p DR vs. C p EE vs. C

14 dpn 83.8 89.1 87.8 0.013 0.0001

21 dpn 32.5 17.3 26.7 0.38 0.3

28 dpn 7.2 9.3 2.7 0.22 0.23

35 dpn 10.7 4.1 4.6 0.5 0.32

42 dpn 10.6 6.4 2.1 0.35 0.08

49 dpn 8.7 0.3 1.1 0.78 0.14

56 dpn 1.5 0.3 4.3 0.37 0.08

63 dpn 4 2.7 2.5 0.9 0.02

Number of EBA-negative vessels for enriched (EE), dark-reared (DR), and

control groups per 62,500 Am2 of visual cortex and statistical significance,

P value.

E.G. Argandona et al. / Developmental Brain Research 156 (2005) 158–166164

conditions, vascular plasticity remains after this date to meet

increased or decreased requirements [3,4,34]. We found no

qualitative differences during postnatal development

(before, during, and after the critical period) nor among

different experimental conditions. The main increase in

EBA staining occurred from 14 to 21 dpn, which suggests

that the function of the EBA is not linked to capillary

plasticity related to experience, since its expression is

established before the critical period, when most changes

occur. In previous studies using enzymes belonging to the

BBB such as Butyryl cholinesterase, we have found that

BChE histochemistry stains the endothelial wall all over the

cortex [3], which indicates that the maturation of the BBB is

a complex process with different rhythms for each

component. In the same way, the GluT-1 immunohisto-

chemical expression is also present in all cortical vessels at

all stages of postnatal development.

The quantitative effects of both visual deprivation and

increase of visual experience have already been reported in

previous works [3,4,34]. Nevertheless, the study was

performed on parallel slices to correlate this with the

EBA quantifications, as there are great differences in the

thickness of the samples and in the way the tissue is

processed. Therefore, in order to avoid a technical bias, the

quantitative analysis has been performed on the same

material processed in exactly the same way. The marker

used for this purpose was the Lycopersicon esculentum

(LEA), which in the cerebral cortex is localized to all

microvascular profiles, at the luminal surface of the

plasmalemma of the endothelial cells [21,29]. As expected,

significant differences were obtained within the critical

period when, in line with the above-mentioned literature,

vascular density was higher in enriched environment

animals and lower in dark-reared animals.

However, one new result should be noted: the effects of

increased visual experience seem to have quantitative results

at an earlier age than the lack of visual experience,

appearing 1 week in advance. The increase in vascular

density also doubles the decrease in dark-reared animals.

This finding could suggest that enriched experience not only

has effects related to visual experience, but also that its

effects could also be more complex, affecting the visual

cortex in different ways including microvascular differ-

ences. The effects of the complex experience itself on the

development of the vascular pattern of the visual cortex

should be the subject of further study.

On the other hand, although quantitative results in EBA

expression were obtained among experimental groups, they

reflected only differences in vascular density due to the

influence of visual experience, as described before. This is

corroborated by the fact that no differences were found when

comparing the density of unstained vessels. As in the case of

entire vascular density, differences among experimental

groups were recorded only after the beginning of the critical

period.

Previous studies have posited the EBA as a specific

marker for the BBB in rats, linking its expression with the

functionality of the barrier [19,39], since a decrease in EBA

positivity has been reported in pathological conditions

where the administration of an anti-EBA antibody resulted

in increased permeability of brain microvessels [15,16].

These findings have led to the postulate that the EBA is a

useful tool for quantifying cortical microvascularization, as

opposed to commonly-used antigenic markers such as GluT-

1 and non-antigenic markers such as UEA and LEA lectin

[19]. Despite the studies that have been conducted, the

EBA’s role in brain vessels remains unclear.

Our results point to a wider range of speculation about

the biological significance of the EBA and even about the

nature of the BBB function itself, since a significant

percentage of cortical vessels lack EBA expression while

non-barrier endothelium shows EBA positivity. The first

finding could suggest a discontinuity of the function along

the brain microvascular tree, whereas the second could point

to barrier antigenic expression in brain vessels without

barrier function. This is linked to brain vessels, since we

found no positivity in vessels outside the brain (in the liver

or kidneys, for example). We also examined the expression

of another component of the BBB, the Glucose transporter

GluT-1, which has been proven to remain positive even

when pathological alterations of the barrier promote changes

in EBA expression [18]. As was expected, no GluT-1-

positive vessel was found in the pineal gland whereas all the

cortical vessels were positive and the quantitative results

were identical to the LEA results.

Both these facts call into question the promotion of the

EBA as a marker for quantitative microvascular studies.

However, they also open up a new horizon for qualitative

studies on the role of the EBA, since it has been linked with

certainty to the endothelial function but its relationship with

the barrier function remains unknown. Some authors have

suggested a link between EBA expression and the influence

of the astroglial ensheathment, which shows the role of the

astroglia in the induction and/or maintenance of the BBB

markers in the cortex [11]. Our previous results on the

distribution of S-100-positive astrocytes in the visual cortex

of rats reared under different visual conditions showed a

diminishing of the astroglial density, but with an absence of

E.G. Argandona et al. / Developmental Brain Research 156 (2005) 158–166 165

qualitative differences. Therefore, these results agree with

both findings, as the influence of experience results in

quantitative rather than qualitative changes.

Therefore, further studies are required to correlate EBA

expression and elucidate its endothelial function.

Acknowledgments

This work was supported by the Basque Country

University Research Project UPV 078.352-EA 8042 and 9/

212.327-15887 Mr. H. Bengoetxea is supported by a Basque

Country University grant. Confocal microscopy analysis was

performed at the ‘‘Servicio de Microscopıa Analıtica y de

Alta Resolucion en Biomedicina’’ (High Resolution Ana-

lytical Microscopy Service for Biomedicine) at the Univer-

sity of the Basque Country.

Appendix A. Supplementary data

Supplementary data associated with this article can be

found, in the online version, at doi:10.1016/j.devbrainres.

2005.02.007.

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