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International Journal of Developmental Neuroscience 69 (2018) 97–105
Contents lists available at ScienceDirect
International Journal of Developmental Neuroscience
journal homepage: www.elsevier.com/locate/ijdevneu
Disappearance of cerebrovascular laminin immunoreactivity as related to
the maturation of astroglia in rat brain
T
⁎
Mihály Kálmána, , Erzsébet Oszwalda, Károly Pócsaia,1, Zsolt Bagyuraa,2, István Adorjána,b
a
b
Department of Anatomy, Histology, Embryology, Semmelweis University, Budapest, Hungary
Department of Physiology, Anatomy, Genetics, Univ. of Oxford, UK
A R T I C LE I N FO
A B S T R A C T
Keywords:
brain development
GFAP
glio-vascular
glutamine synthetase
laminin
S100
The present paper provides novel findings on the temporo-spatial correlation of perivascular laminin immunoreactivity with the early postnatal astrocyte development. The cerebrovascular laminin immunoreactivity
gradually disappears during development. The fusion of the glial and vascular basal laminae during development
makes the laminin epitopes inaccessible for antibody molecules (Krum et al., 1991, Exp Neurol 111:151). The
fusion is supposed to correlate with the maturation of the glio-vascular connections. Glial development was
followed by immunostaining for GFAP (glial fibrillary acidic protein), S100 protein, glutamine synthetase as glial
markers and for nestin to visualize the immature glial structures. Our investigation focused on the period from
postnatal day (P)2 to P16, on the dorso-parietal pallium. In the wall of the telencephalon the laminin immunoreactivity disappeared between P5 and P10; in subcortical structures it persisted to P12 or even to P16. Its
disappearance overlapped the period when GFAP-immunopositive astrocytes were taking the place of radial glia.
Despite the parallel time courses, however, the spatial patterns of the two processes were just the opposite:
disappearance of the laminin immunoreactivity progressed from the middle zone whereas the appearance of
GFAP from the pial surface and the corpus callosum. Rather, the regression of the vascular laminin immunoreactivity followed the progression of the immunoreactivities of glutamine synthetase and S100 protein.
Therefore, the regression really correlates with a ‘maturation’ of astrocytes which, however, affects other astrocyte functions rather than cytoskeleton.
1. Introduction
the disappearance of laminin immunoreactivity (until P11, Krum et al.,
1991).
Since not all the astrocytes can be detected by immunohistochemical reaction against GFAP, it was also necessary to use
other astroglial markers, such as glutamine synthetase and S100 protein
(Ghandour et al., 1981; Ludwin et al., 1976; Norenberg, 1979). To visualize the immature glia we used immunostaining for nestin
(Hockfield and McKay, 1985; Zerlin et al., 1995; Kálmán and Ajtai,
2001).
The investigation period lasted from P2 to P16, i.e. it finished after
that the radial glia disappeared (Kálmán and Ajtai, 2001; Pixley and de
Vellis, 1984; Stichel et al., 1991) and the vascular laminin immunoreactivity became undetectable (Krum et al., 1991). The investigation was focused on the dorso-parietal pallium, so the data refer
to this area unless specified otherwise.
Laminin is a ubiquitous major component of basal laminae, however, the vessels of adult brains are not visualized by immunostaining
for laminin (Jucker et al., 1992; Krum et al., 1991). It is supposed that
the fusion of the glial and vascular basal laminae makes the laminin
epitopes inaccessible for the antibody molecules (Krum et al., 1991).
This fusion occurs during development and it is considered as a ‘maturation’ of the glio-vascular connections (Bär, 1980; Caley and
Maxwell, 1970; Marin-Padilla, 1985). Recent papers (Franciosi et al.,
2007; Gama Sosa et al., 2014) also mention this correlation.
An important mark of the astrocyte maturation is the appearance of
GFAP, the major cytoskeletal element of astrocytes (Bignami et al.,
1980; Pixley and de Vellis, 1984), which occurs gradually during the
first two postnatal weeks. It approximately coincides with the period of
⁎
Corresponding author at: Department of Anatomy, Histology, Embryology, Semmelweis University, H-1094, Tuzolto 58, Budapest, Hungary.
E-mail addresses: kalmanprof@gmail.com (M. Kálmán), oszwald.erzsebet@med.semmelweis-univ.hu (E. Oszwald), pocsai.karoly@gmail.com (K. Pócsai),
bagyura@zortal.hu (Z. Bagyura), adorist@freemail.hu (I. Adorján).
1
Present address: 2nd Dept. of Internal Medicine, Semmelweis University, Budapest, Hungary
2
Present address: Heart, Vascular Center, Semmelweis Univ., Budapest, Hungary
https://doi.org/10.1016/j.ijdevneu.2018.07.003
Received 27 February 2018; Received in revised form 24 May 2018; Accepted 9 July 2018
0736-5748/ © 2018 ISDN. Published by Elsevier Ltd. All rights reserved.
International Journal of Developmental Neuroscience 69 (2018) 97–105
M. Kálmán et al.
and white figures.
Our description includes the whole telencephalic wall not only the
cortex. Since the cortical layer system alters during the period studied,
the term ‘layer’ was avoided and ‘zone’ was used instead, which did not
relate to any conventional layer either in the mature cortex (e.g. external granular, etc.) or in the developing one (e.g. the cortical plate).
2.4. Pre-embedding electron microscopic immunohistochemistry
In this case 0.5% glutaraldehyde was added to the perfusion solution for a better fixation. The immunohistochemical reaction against
laminin was performed on vibratome sections according to the avidinbiotinylated peroxidase (ABC) method. Endogenous peroxidase was
inactivated with 3% H2O2 in PBS (5 min at room temperature) followed
by an intense rinse in PBS (30 min at room temperature). Incubations
with 20% normal goat serum and anti-laminin (see in Table 1) were
carried out as above except for that Triton X-100 detergent was reduced
to 0.1% to decrease tissue destruction. The procedure continued by
applying biotinylated anti-rabbit serum (Vector Laboratories, CA, USA,
1:100) followed by the avidin-biotinylated peroxidase complex (Vector
Laboratories, CA, USA). Both incubations lasted for 90 min at room
temperature and were followed by intense rinses in PBS (30 min, at
room temperature). To visualize the immunohistochemical reaction
product 0.05% 3,3’ diaminobenzidine-tetrahydrochloride (DAB), 0.01%
H2O2 in Tris-HCl buffer (0.05 M, pH 7.4, at room temperature) were
used. The peroxidase-reaction was stopped at visual color control by
replacing the solution with PBS.
2. Materials, Methods
2.1. Animals
Postnatal rats were used at the age of P2, P4, P5, P6, P7, P8, P10,
P12, P14, and P16, from 3 litters, 4-4 pups of either sex at each age.
Experiments were performed according to the Committee on the Care,
Use of Laboratory Animals of the Council on Animal Care at the
Semmelweis University of Budapest, Hungary (22.1/3491/003/2008)
in accordance with the guidelines of European Union Directive (EU
Directive 2010/63/EU). Animals were deeply anesthetized with ketamine and xylazine (80 and 20 mg/kg, respectively, intramuscularly)
and perfused through the aorta with 0.9% sodium chloride followed by
4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), After perfusion brains were post-fixed in the same fixative for 4 days at 4 °C.
Then the brains were embedded into agarose, serial coronal sections of
50 μm were cut with a vibration microtome (Leica VT 1000S) and
rinsed in phosphate buffered saline (PBS, Sigma) overnight.
2.5. Electron microscopic investigation
2.2. Fluorescent immunohistochemistry
Following the immunohistochemical reactions tissue areas were
selected under light microscope and cut from the vibratome sections.
They were immersed for 30 min into a 1% osmium tetroxide solution
then rinsed in phosphate buffer and dehydrated through a graded series
up to absolute ethanol. Following immersion in propylene oxyde
(10 min) the tissue samples were embedded into epoxy resin
(Durcupan, Fluka). Semithin sections were cut with a Reichert Ultracut
S ultramicrotome, areas were selected under light microscope and the
samples were adequately trimmed. The ultrathin sections were prepared with the same ultramicrotome and mounted on grids. The photomicrographs were taken by a JEOL 100B elecron microscope
equipped with a Sys Morada digital camera.
Floating sections were pretreated with 20% normal goat serum diluted in PBS, for 90 min at room temperature, to block the non-specific
binding of antibodies. This and the following steps were followed by an
intense rinse in PBS (30 min at room temperature). The sections were
incubated with primary antibodies (Table 1) for 40 h at 4 °C. The primary antibodies were diluted as shown in Table 1 in PBS containing
0.5% Triton X-100, 0.01% sodium azide. Fluorescent secondary antibodies (Table 2) were applied at room temperature for 3 h. The sections
were finally rinsed in PBS (1 h at room temperature), mounted onto
microscope slides, cover-slipped in a mixture of glycerol and distilled
water (1:1) and sealed with lacquer. Control sections were treated
identically, but the primary antibodies were omitted. No structurebound fluorescent labeling was observed in these specimens.
3. Results
3.1. The postnatal regression of the vascular laminin immunoreactivity
2.3. Fluorescent microscopy and digital imaging
At P2 laminin immunostaining delineated the vascular pattern in
the whole thickness of the brain wall (Fig. 1a). During the following
days up to P10, however, the vascular laminin immunoreactivity
withdrew from the vessels gradually but almost completely. In the
telencephalic wall this process started in the middle zone of its thickness at P5 (Fig. 1b) and extended toward the superficial and deeper
parts. By P7 this zone widened (Fig. 1c), by P10 the laminin immunoreactivity was confined to the entering (subpial) segments of
vessels and to the deepest part of the cortex (Fig. 1d, e). Corresponding
High-resolution microphotographs were taken with a Radiance2100 (BioRad, Hercules, CA) confocal laser scanning microscope
whereas low-power ones with a DP50 digital camera mounted on an
Olympus BX-51 microscope (both from Olympus Optical Co. Ltd,
Tokyo, Japan). Digital images were processed using Photoshop 9.2
software (Adobe Systems, Mountain View, CA) with some adjustments
for brightness and contrast. The confocal photomicrographs of doublelabeled specimens were presented in color figures, the others in black
Table 1
The primary antibodies applied in the study.
*
Against
Host
Company
Code Nr.
Dilu-tion
final con-centration
(μg/mL)
GFAP
GFAP
Glutamine synthetase
Glutamine synthetase
Mouse*
Rabbit**
Mouse*
Rabbit**
ga5
Z0334
610518
NB110-41404
1:100
1:500
1:100
1:500
100
5.6
2.5
Laminin 1
Nestin
Rabbit**
Mouse*
Novocastra, Newcastle, United Kingdom
DAKO, Galstrup, Denmark
Transduction Laboratories, Erembodegem, Belgium
Novus Biologicals
Littleton, Co, USA
Sigma, San Louis, MO, USA
Millipore, Temecula, CA, USA
1:100
1:1000
5
1
S100-protein
Rabbit**
Sigma, San Louis, MO, USA
l 9393
MAB353
s-2644
1:100
81
- monoclonal,
**
- polyclonal,
***
- the original concentration is not given by the firm,
98
***
International Journal of Developmental Neuroscience 69 (2018) 97–105
M. Kálmán et al.
Table 2
The secondary antibodies applied in the study.
Conjugated with
Against
Host
Absorbed/ emitted light (nm)
Company
Code Nr.
Dilution
Final cc.
(μg/mL)
Alexa Fluor 488
Rabbit
Donkey
488/519
A21206
1:500
4
Alexa Fluor 555
Mouse
Donkey
555/565
A31570
1:500
4
Biotin
Rabbit
Horse
———————————
InvitrogenCarlsbad Ca
USA
InvitrogenCarlsbad Ca
USA
Vector
Burlingame, CA, USA
BA 2000
1:100
0.05
Fig. 1. The postnatal regression of the vascular
laminin immunoreactivity.
a) At P(postnatal day)2, the laminin immunoreactivity (the original color was green)
marks the vessels (arrowheads) in the full
thickness of pallium. CC – corpus callosum, P –
pial surface.
b) At P5 the vascular laminin immunoreactivity
(arrowheads) is almost missing in a middle
zone. Around the asterisks: laminin-immunoreactive cells probably neurons (see
Discussion). P – pial surface.
c) By P7 the withdrawing of the vascular laminin immunoreactivity (arrowheads) has
continued. P – pial surface. Around the asterisks: laminin-immunoreactive cells probably
neurons.
d) By P10 the vascular laminin immunoreactivity (arrowhead) has been confined
to the subpial zone (P – pial surface)…
e) … and the deepest part of the telencephalic
wall (CC - corpus callosum). Around the asterisks: laminin-immunoreactive cells probably
neurons.
f) The vascular laminin immunoreactivity is
prolonged subcortically, e.g. in the septum
(Sp), P12. LV - lateral ventricle.
g) Mesencephalic aqueduct (AC), P12 (note the
alteration of the vascular laminin immunoreactivity with the distance from the
aqueduct).
h) A detail of the pons, P16.
Scale bars: a to d): 120 μm, e to h): 60 μm.
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International Journal of Developmental Neuroscience 69 (2018) 97–105
M. Kálmán et al.
immunoreactivity (started from P5, see Fig. 1b) preceded that of the
glial nestin (started from P7, see also Kálmán and Ajtai, 2001).
In the telencephalic wall numerous GFAP-immunoreactive astrocytes appeared first subjacent to the meningeal surface (Fig. 3c) and in
the corpus callosum (Fig. 3d). In these zones, however, laminin immunoreactivity persisted in spite of the numerous GFAP-immunoreactive astrocytes attached to the vessels (Fig. 3e to g).
3.4. Correlation with glutamine synthetase and S100 protein
immunoreactivities in astrocytes
When immunostaining for glutamine synthetase or S100 protein
was performed at P2, labeled cell bodies were found mainly in the
middle zone of the telencephalic wall (Fig. 4a, b). At this age processes
were only poorly visible (Fig. 4c, d), but by P4 they became detectable
well (Fig. 4e, f).
The zone that was colonized by glutamine synthetase and S100
protein-immunoreactive cells widened gradually from P2 to P10 when
it reached the pial surface (Fig. 4g–k). When laminin immunoreactivity
started to withdraw (P5), the vascular contacts of the glutamine synthetase-, S100 protein-immunoreactive astrocytes were already visible
(Fig. 5a, b). By P10 glutamine syntethase-immunoreactive end-feet
covered the vessels in the full thickness of the telencephalic wall, and
the laminin immunoreactivity had almost disappeared (Fig. 5c to e).
S100 protein immunoreactivity also revealed an extended system of
vascular end-feet (Fig. 5f).
When double-labeling was applied, glutamine syntethase and S100
protein were found to be co-localized (Fig. 5g, h). Following doublelabeling with nestin either S100 protein or glutamine syntethase immunoreactivity labeled mainly cell bodies whereas nestin was mainly
detected in the corresponding processes (Fig. 5i, j).
Fig. 2. Laminin immunoreactivity is confined to the perivascular space.
a) Laminin immunoreactivity (the original color was green) at the vessel segment subjacent to the pial surface (P8). Arrowheads: parenchymal basal lamina,
double arrowheads: vascular basal lamina. Double arrows point to the space
between them. In a short distance from their fusion the laminin immunoreactivity is not detectable (arrow).
Scale bar: 20 μm
b) Electron microscopy: laminin immunoreactivity at P8. The reaction product
is found in a narrow space around the vessel (double arrows). Where this space
closes, the immunoreactivity ceases (arrows). Compare it to the previous panel.
E – endothelial cell. Scale bar: 0.4 μm.
4. Discussion
4.1. Laminin immunoreactivity disappears, but laminin itself does not
Our light and electron microscopic immunohistochemical observations were congruent with the data of Krum et al. (1991) who found
that the vascular laminin immunoreactivity disappeared by P11 and
attributed it to the fusion of glial and vascular basal laminae, which
‘hides’ the laminin epitopes and makes them inaccessible for antibodies.
A similar masking mechanism has been published for the basal laminacomponent nidogen (Ae Seo et al., 2007). The disappearance of vascular laminin immunoreactivity coincides with the period when the
glio-vascular connections tighten and the perivascular clefts close (Bär,
1980; Caley and Maxwell, 1970; Marin-Padilla, 1985; Plate, 1999;
Robertson et al., 1985). EBA (endothelial blood-brain barrier antigen)
also appears around P3-7 and becomes generalized by P11 (Rosenstein
et al., 1992).
The fusion of basal laminae does not extend into the ‘Virchow-Robin
spaces’ and on the vessels of circumventricular organs. Here laminin
immunoreactivity persists in the mature brain. Following lesions the
glial and vascular basal laminae separate and the laminin immunoreactivity becomes detectable temporarily (see e.g. the review of
Hallmann et al., 2005). Freezing or enzyme (collagenase, matrix metalloprotease) digestion also unmask the laminin immunoreactivity in
the cerebral vessels (Eriksdotter-Nilsson et al., 1986; Mauro et al., 1984;
Franciosi et al., 2007). In a recent study we also explored by freezing
the laminin immunoreactivity of vessels in mature brain (Pócsai and
Kálmán, 2014). This proves that laminin was present only its immunoreactivity had become undetectable.
Laminins are heterotrimers composed of an α, β, and γ chain. To
date, 5 α, 3 β, 3 γ chains have been reported which can combine to form
up to 15 different laminin isoforms (for reviews see e.g. Hallmann et al.,
2005; Yurchenko, 2015). A polyclonal pan–laminin 1 antiserum (see
Table 1) which was applied by us reacts with either α1 or β1 or γ1 chain
to the so-called ‘Virchow-Robin spaces’ the vessels remained lamininimmunoreactive even in the mature brain.
These data refer to the dorsal pallium. In some other areas the laminin immunoreactivity persisted longer. At P12, e.g., there were still
laminin-immunoreactive vessels around the anterior commissure, in the
thalamus, pallidum, striatum, septum (Fig. 1f), hippocampus, and
around the cerebral aqueduct (Fig. 1g). In some parts of the pons,
medulla, and cerebellum there were laminin-immunoreactive vessels
even at P16 (Fig. 1h).
3.2. Laminin immunoreactivity is confined to perivascular spaces
Where the perivascular space was wide enough the glial and vascular basal laminae were seen separately following immunostaining for
laminin. After that point where the two basal laminae fused to a single
layer, the laminin immunoreactivity became undetectable (Fig. 2a).
Electron microscopic investigations demonstrated that the product of
the immunohistochemical reaction against laminin was found in
narrow spaces along the vessels. Where these spaces were closed the
immunoreactivity ceased as well (Fig. 2b).
3.3. Attaching of GFAP-immunopositive astrocytes: no effect on laminin
immunoreactivity
The most dense system of nestin-immunoreactive radial glia was
found below the pial surface (Fig. 3ab) where the vascular laminin
immunoreactivity persisted longest. On the other hand, in the middle
zone of the telencephalic wall the disappearance of the vascular laminin
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International Journal of Developmental Neuroscience 69 (2018) 97–105
M. Kálmán et al.
Fig. 3. Attaching of GFAP- immunoreactive astrocytes: no effect on laminin immunopositivity.
a) Double labeling of nestin (red) and laminin
(green), confocal microscopy, P2. The densest
system (double asterisks) of nestin-immunoreactive glial processes occurs mainly
below the pial surface (P). Vessels (arrowheads) show immunoreactivity to both nestin
and laminin (note yellow color, see enlarged
details in panel b). Laminin immunoreactivity
also labels cells, probably neurons (zona labeled by asterisks). Scale bar: 60 μm.
b) Enlarged details of a vessel (pointed by two
arrowheads in panel a). The yellow color refers
to the presence of laminin immunopositivity
beside that of nestin. Scale bar of panel a) here
labels 30 μm.
c) The subpial zone, double-labeling of GFAP
(red), laminin (green), confocal microscopy,
P8. P – pial surface. Yellow color refers to close
association of laminin with GFAP-immunoreactive end-feet. Note: the vessel wall is
green or yellow but not red. Scale bar: 20 μm.
d) Corpus callosum (CC), P8. GFAP-immunoreactive astrocytes (red) around vessels
(arrowheads) immunostained for laminin
(green). Double-labeling study, superposed
photomicrographs of the immunoreactivities of
GFAP and laminin. Laminin immunoreactivity
still persists despite the numerous GFAP-immunoreactive astrocytes, several of them are
associated closely to the vessels (long thin
arrow, note the yellow color), whereas around
the asterisk only cells probably neurons are
laminin-immunoreactive. Scale bar: 60 μm.
e to g) GFAP-immunoreactive astrocytes (red)
around vessels immunostained for laminin
(green). Double-labeling study, confocal fluorescent
microscopy.
The
laminin
immunoreactivity persists in the walls of vessels
in spite of the numerous GFAP-immunoreactive
astrocytes attached to them: their immunostaining remained green (laminin) or
turned to yellow (laminin plus GFAP) but did
not red (only GFAP). Scale bars: 10 μm
radial glia (Kálmán and Ajtai, 2001; Pixley and deVellis, 1984; Stichel
et al., 1991). However, the trends of the spatial courses were just the
opposite ones: GFAP immunoreactive astrocytes appeared first along
the pial surface and the corpus callosum, whereas the laminin immunoreactivity started to withdraw from the middle zone of the telencephalic wall in accordance with the observation of Krum et al.,
(1991). The middle zone of the cortex remains poor of GFAP-immunoreactive astrocytes even in the mature brain (Kálmán and Hajós,
1989; Ludwin et al., 1976; Zilles et al., 1991), whereas laminin immunoreactivity persisted just in the most superficial and deepest zones
i.e. in zones rich in GFAP-immunoreactive astrocytes. Therefore, the
maturation of cytoskeleton in the perivascular glia did not underlie the
disappearance of laminin immunoreactivity.
hence it recognizes any laminin type containing at least one of these
chains (Sixt et al., 2001). For a more detailed discussion of laminin
immunoreactivity see Szabó and Kálmán (2004).
Laminin immunoreactivity in neurons has already been mentioned
by several authors (e.g. Jucker et al., 1992; Powell and Kleinman, 1997;
Yamamoto et al., 1988) although its role has not been clarified yet. The
further investigation of this phenomenon, however, is out of the scope
of this study.
4.2. GFAP and laminin immunoreactivities: parallel changes in time but
opposite in space
The vascular connections of the radial glia cannot be responsible for
the disappearance of the vascular laminin immunoreactivity.
Concerning the nestin immunoreactivity of vessels, it has been demonstrated by several authors in both developing and mature vessels,
in both cerebral and extracerebral ones (see e.g. Mokry et al., 2004,
2008; Sugawara et al. 2002).
The period of the disappearance of the vascular laminin immunoreactivity overlapped the period when GFAP-immunoreactive
astrocytes were taking the place of the vimentin- and nestin-containing
4.3. Glutamine synthetase and S100 protein also mark maturation but that
of another function
Former investigations also found the first appearance of glutamine
synthetase (Patel et al., 1983) and S100 protein (Herschman et al.,
1971) at the first and second postnatal days, repectively. In our study
their progression in the cortex positively correlated with the
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International Journal of Developmental Neuroscience 69 (2018) 97–105
M. Kálmán et al.
Fig. 4. Glutamine synthetase and S100 protein
immunoreactivities: their progression.
a, b) Glutamine synthetase (the original color
was red) and S100 protein (the original color
was green) immunoreactivity, respectively.
Labeled cells occur in a middle zone (arrows)
of pallium, P2. Th- thalamus, VL – lateral
ventricle. Scale bars: 120 μm.
c, d) Glutamine synthetase- and S100 proteinimmunoreactive cells, respectively, at P2. Their
processes are hardly visible. Scale bars: 10 μm.
e, f) Glutamine synthetase and, S100 proteinimmunoreactive cells, respectively, at P4. Their
processes have appeared. Scale bars: 10 μm.
g, h) glutamine synthetase- (panel g, the original color was red) and GFAP- (panel h, the
original color was green) immunoreactivities,
double labeling, at P6. A wide middle zone
(arrows, only its upper part is visible) is colonized by glutamine synthetase protein immunoreactive cells whereas GFAP is confined
(arrow) to the subpial zone (P – pial surface).
Scale bars 20 μm.
i) Expansion of S100 protein-immunoreactive
cells (the original color was green) at P6 (arrows), an area similar to that seen in panel h
(P- pial surface). Scale bars 20 μm
j, k) Double labeling for glutamine synthetase
and laminin, at P8, displayed separately. The
glutamine synthetase-immunpositive cells
(panel j, the original color was red) extend near
the pial surface (P) and the laminin immunoreactivity of vessels (panel k, the original
color was green) has accordingly withdrawn
(arrowheads). In the panel j the cellular elements are laminin-immunolabeled cells probably neurons. Arrowheads label identical
points of both panels. Scale bars 20 μm
1991). Glutamine synthetase occurs in astrocytes mainly in areas rich in
glutamate- and GABA-ergic neurons (Norenberg, 1979; Patel et al.,
1985, Suarez et al., 2002) since astrocytes protect neurons against the
toxicity of ammonia and glutamate converting them into glutamine.
The preferred localization of GFAP is the ‘fibrous’ type of astrocytes
whereas that of glutamine synthetase is the’ protoplasmic’ astrocyte
(Didier et al., 1986; Ong et al., 1993; Patel et al., 1985) although the
difference is not exclusive (Linser, 1985; Connor and Berkowitz, 1985;
Ludwin et al., 1976). The glutamine synthetase activity and the GFAP
production are regulated by independent mechanisms (Condorelli et al.,
1991; Li and Bartlett, 1991). A recent study demonstrates that GFAP,
glutamine synthetase and S100 protein change differently with aging
disappearance of the laminin immunoreactivity. Their appearance
correlates with the maturation of astrocytes (Patel et al., 1983; Raponi
et al., 2007; Reichenbach and Wolburg, 2013). According to Kozlova
et al. (1993) the morphologic astrocyte differentiation corresponded to
the increase of glutamine synthetase activity in vitro. Endothelial cellderived extracellular matrix components (laminin and collagen) stimulated this process. At molecular level the correlation between the
presence of glutamine synthetase and the maturation of glio-vascular
connections remains to be clarified.
GFAP and glutamine synthetase participate in different astrocyte
functions. GFAP occurs mainly in the subpial and deepest levels of
cortex (Ludwin et al., 1976; Kálmán and Hajós, 1989; Zilles et al.,
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International Journal of Developmental Neuroscience 69 (2018) 97–105
M. Kálmán et al.
(caption on next page)
Our co-localization results supported that glutamine synthetase and
S100 protein immunoreactivities marked the same cell population,
which derived from nestin-immunoreactive precursors.
In general, the data indicate that in different astrocyte populations
different factors can be’ markers of maturation’ corresponding to the
local requirements.
(Rodriguez et al., 2014).
S100 protein inhibits the polymerization of GFAP (Bianchi et al.,
1995); it may explain that its appearance is opposite to that of GFAP
during development and corresponds rather to the appearance of glutamine synthetase see also the finding of Patel et al. (1983) mentioned
above. In adult rats, however, both types of astrocytes are already
immunopositive to S-100 protein (see also Didier et al., 1986; Ludwin
et al., 1976; Privat et al., 1995).
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M. Kálmán et al.
Fig. 5. Glutamine synthetase and S100 immunoreactivities: effect on laminin and colocalisations.
Confocal microscopy.
a, b) P4: Glutamine synthetase-immunoreactive astrocytes (red) around vessels (green: laminin of basal lamina). Only few vascular contacts have been formed yet
(arrow in Panel b). Scale bars: 10 μm.
c to e) P10: The glutamine synthetase-immunoreactive cells (red) cover the vessels with end-feet (arrows in panels c and e). The yellow segments which appear to
display co-localization may refer to close attachments. Where the vessel wall is red, there is no detectable laminin immunoreactivity. Note that by P10 glutamine
synthetase-immunoreactive cells have already colonized the cortex to the pia (P). Asterisks label identical vessels in panels d) and e). Scale bars: 10, 20, 10 μm,
respectively.
f) Double labeling of nestin and S100, P10. The S100 protein-immunoreactive cells (green) cover a nestin-immunoreactive vessel (red) with end-feet (arrowheads).
Yellow color refers to close association between the end-feet and the vessel (arrowheads). Scale bar: 10 μm.
g, h) Double laberling of glutamine synthetase (red), S100 protein (green), P2, P6, respectively. Co-localization (yellow color) is found in astrocytes. Scale bars:
10 μm.
i, j) Double labeling of nestin (red), S100 protein (panel i, green) or glutamine synthetase (panel j, green!). P8. Nestin is found mainly in processes (arrows), whereas
S100 protein or glutamine synthetase immunoreactivity labels mainly in cell bodies. Scale bars: 10 μm.
k, l) Z-stacks for Panels i, j, respectively. Scale bars: 10 μm.
4.4. Why from the middle zone of the telencephalic wall?
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The different astrocyte functions ‘mature’ according to different
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Acknowledgements
We, authors, declare that there is no actual or potential conflict of
interest including any financial, personal or other relationships with
other people or organizations. This study was supported by the scientific budget of the Dept. of Anatomy, Histology, Embryology,
Semmelweis University. This research did not receive any specific grant
from funding agencies in the public, commercial, or not-for-profit sectors. This article does not contain any studies with human participants.
All procedures involving animals were in accordance with the
Committee on the Care, Use of Laboratory Animals of the Council on
Animal Care at the Semmelweis University of Budapest, Hungary (22.1/
3491/003/2008), with the guidelines of European Union Directive (EU
Directive 2010/63/EU). The participation of the authors: M. Kalman
(senior): team leader, experimental design, evaluations, writing the
manuscript; Z. Bagyura, K. Pocsai: immunohistochemistry; I. Adorjan,
confocal microscopic investigations, writing the manuscript, E.
Oszwald: electron microscopic investigations. Technical assistance of A.
Őz, S. Deák, Z. Gróti is highly appreciated.
104
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