Redefining the concept of reactive astrocytes as cells that remain within their unique domains upon reaction to injury

Article (PDF Available)inProceedings of the National Academy of Sciences 103(46):17513-8 · December 2006with30 Reads
DOI: 10.1073/pnas.0602841103 · Source: PubMed
Abstract
Reactive astrocytes in neurotrauma, stroke, or neurodegeneration are thought to undergo cellular hypertrophy, based on their morphological appearance revealed by immunohistochemical detection of glial fibrillary acidic protein, vimentin, or nestin, all of them forming intermediate filaments, a part of the cytoskeleton. Here, we used a recently established dye-filling method to reveal the full three-dimensional shape of astrocytes assessing the morphology of reactive astrocytes in two neurotrauma models. Both in the denervated hippocampal region and the lesioned cerebral cortex, reactive astrocytes increased the thickness of their main cellular processes but did not extend to occupy a greater volume of tissue than nonreactive astrocytes. Despite this hypertrophy of glial fibrillary acidic protein-containing cellular processes, interdigitation between adjacent hippocampal astrocytes remained minimal. This work helps to redefine the century-old concept of hypertrophy of reactive astrocytes. • astrocyte domains • astrocyte hypertrophy

Figures

Redefining the concept of reactive astrocytes as
cells that remain within their unique domains
upon reaction to injury
Ulrika Wilhelmsson*, Eric A. Bushong
, Diana L. Price
, Benjamin L. Smarr
, Van Phung
, Masako Terada
,
Mark H. Ellisman
, and Milos Pekny*
*Department of Clinical Neuroscience and Rehabilitation, Institute of Neuroscience and Physiology, Sahlgrenska Academy, Go¨ teborg University, SE-405 30
Go¨ teborg, Sweden; and
National Center for Microscopy and Imaging Research, University of California at San Diego, La Jolla, CA 92093-0608
Edited by Pasko Rakic, Yale University School of Medicine, New Haven, CT, and approved September 15, 2006 (received for review April 7, 2006)
Reactive astrocytes in neurotrauma, stroke, or neurodegeneration
are thought to undergo cellular hypertrophy, based on their
morphological appearance revealed by immunohistochemical de-
tection of glial fibrillary acidic protein, vimentin, or nestin, all of
them forming intermediate filaments, a part of the cytoskeleton.
Here, we used a recently established dye-filling method to reveal
the full three-dimensional shape of astrocytes assessing the mor-
phology of reactive astrocytes in two neurotrauma models. Both in
the denervated hippocampal region and the lesioned cerebral
cortex, reactive astrocytes increased the thickness of their main
cellular processes but did not extend to occupy a greater volume
of tissue than nonreactive astrocytes. Despite this hypertrophy of
glial fibrillary acidic protein-containing cellular processes, interdig-
itation between adjacent hippocampal astrocytes remained mini-
mal. This work helps to redefine the century-old concept of
hypertrophy of reactive astrocytes.
astrocyte domains astrocyte hypertrophy
O
nce considered to be merely a cellular layer filling the inter-
neuronal space and gluing neurons together (hence the term
‘‘glia’’), astrocytes are receiving ever-increasing attention. The
rising recognition of their importance builds on knowledge of their
role in maintaining CNS homeostasis, providing nutrition for
neuronal cells, and neurotransmitter recycling. Recently, astrocytes
were shown to control the number and function of neuronal
synapses (1, 2) and blood flow in the brain (3, 4).
Astrocytes exhibit an intricate bushy or spongiform morphology,
and their very fine processes are in close contact with synapses and
other components of brain parenchyma (5–8). These fine terminal
processe s appear postnatally in the final stage of astrocyte matu-
ration. The subsequent elaboration of spongiform processes results
in the development of boundaries between neighboring astrocyte
domains, thus establishing exclusive territorie s for individual astro-
cytes, a phenomenon termed ‘‘tiling’’ (7, 9).
With earlier methods, including immunohistochemical detection
of astrocyte markers and impregnation techniques, the extent of
overlap between astrocyte territorie s was not amenable to investi-
gation. However, more recent staining methods, optical imaging
techniques, and 3D reconstruction paradigms using dye-filled as-
trocytes in semifixed tissue allowed the assessment of the bound-
aries of protoplasmic astrocyte territories in the CA1 area of the
uninjured rat hippocampus. Neighboring astrocytes invariably
touched each other but showed little interdigitation, basically tiling
to form unique domains (8).
Astrocytes react to many CNS challenges, and reactive astrocytes
are a hallmark of many neuropathologies. Reactive astrocytes are
characterized by high-level expression of glial fibrillary acidic
protein (GFAP), an intermediate filament protein, and by up-
regulation of intermediate filaments in the cytoplasm. Antibodies
against GFAP, the most frequently used astrocyte marker (10),
reveal the cytoskeletal structure but not the true cellular morphol-
ogy. Reactive astrocytes exhibit striking increases in GFAP immu-
noreactivity and in the number and length of GFAP-positive
processe s. These findings have been interpreted as cellular hyper-
trophy (Fig. 1) (11–17), which might be expected to increase the
extent of interdigitation between astrocytes, normally in close
contact in the absence of brain injury.
To determine how astrocyte activation affects both the volume of
tissue reached by individual cells and the interdigitation between
neighboring cells, we used a cell injection and 3D reconstruction
technique (7, 8, 18, 19) to assess the morphology of reactive and
nonreactive hippocampal and cortical astrocytes in mice. Contrary
to the widely accepted notion of hypertrophy of reactive astrocytes,
our findings suggest that the se cells remain within their unique
domains while at the same time increasing the thickness of their
main cellular processes.
Results
To assess the morphology of reactive astrocytes, we first subjected
adult mice to unilateral entorhinal cortex lesion. This injury partly
interrupts the perforant pathway, which innervates the molecular
layer in the ipsilateral dentate gyrus of the hippocampus (Fig. 1).
Axonal degeneration triggers astrocyte activation, 60% synaptic
loss, synapse remodeling in the molecular layer, and neurogenesis
in the dentate gyrus that is not directly affected by the trauma
(19–21). Astrocytes were visualized with antibodies against GFAP
4 days after the insult (Fig. 1), when astrocyte activation and
hypertrophy of cellular processes are maximal, as shown by GFAP
and glutamine synthase immunoreactivity (19, 22, 23). Astrocytes in
the uninjured contralateral dentate gyrus had slender GFAP-
positive cellular processe s resembling those in noninjured mice. On
the lesioned side, astrocyte reactivity was prominent in the outer
and middle molecular layers of the dentate gyrus, the area partly
denervated by the lesion. Reactive astrocytes appeared to have
more main cellular processes containing GFAP intermediate fila-
ments and more intermediate filament bundles than nonreactive
astrocytes (Fig. 1).
Next, individual astrocytes in the middle and outer molecular
layers of the dentate gyrus on the lesioned and contralateral side
were loaded with Lucifer yellow or Alexa Fluor 568. Both reactive
and nonreactive astrocytes had a bushy morphology with many fine
terminal processe s protruding from the main cellular processe s
(Fig. 2A), as did astrocytes in noninjured mice (data not shown).
Author contributions: U.W. and E.A.B. contributed equally to this work; U.W., E.A.B., D.L.P.,
M.H.E., and M.P. designed research; U.W., E.A.B., D.L.P., B.L.S., V.P., and M.T. performed
research; U.W., E.A.B., and M.P. analyzed data; and U.W., M.H.E., and M.P. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS direct submission.
Abbreviation: GFAP, glial fibrillary acidic protein.
To whom correspondence should be addressed at: Institute of Neuroscience and Physiol-
ogy, Department of Clinical Neuroscience and Rehabilitation, Go¨ teborg University, Box
440, SE-405 30 Go¨ teborg, Sweden. E-mail: milos.pekny@medkem.gu.se.
© 2006 by The National Academy of Sciences of the USA
www.pnas.orgcgidoi10.1073pnas.0602841103 PNAS
November 14, 2006
vol. 103
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Individual reactive and nonreactive astrocytes varied considerably
in shape and appearance. Many astrocytes had one or several
cellular processe s terminating in glial end-feet surrounding blood
vessels.
The main cellular processes appeared to be thicker in reactive
than in nonreactive astrocytes (Fig. 2A). In addition, we found
that the number of primary processes leaving the soma was
increased in reactive compared with nonreactive astrocytes
Fig. 1. Entorhinal cortex lesion triggers reactive gliosis in the hippocampus. Unilateral entorhinal cortex lesion triggers astrocyte activation in the outer and
middle molecular layer of the ipsilateral dentate gyrus of the hippocampus (Right, in gray). Antibodies against GFAP visualize bundles of intermediate filaments
predominantly found in the soma and the main cellular processes of astrocytes. Four days after unilateral entorhinal cortex lesioning, astrocytes in the molecular
layer of the dentate gyrus on the injured side were reactive and all showed greater GFAP immunoreactivity (Center) than the nonreactive astrocytes on the
contralateral side (Left). The square in Right denotes the area corresponding to the images in Left and Center. EC, entorhinal cortex. (Scale bar, 25
m.)
Fig. 2. Morphological assessment of reactive and nonreactive astrocytes in the hippocampus. (A) Maximum projections of dye-filled reactive and nonreactive
astrocytes in the molecular layer of the dentate gyrus 4 days after entorhinal cortex lesioning. Dye-filling revealed fine spongiform processes in reactive astrocytes
comparable with those of nonreactive astrocytes but a greater number of main processes extending from the cell soma of reactive astrocytes. (Scale bar, 25
m.)
(B and C) Quantification of main cellular processes leaving the soma (B) and processes visible 25
m from the cell soma (C). Thick processes were more numerous
in reactive astrocytes than in nonreactive astrocytes. Three-dimensional reconstruction (E) shows that reactive and nonreactive astrocytes access similar volumes
of tissue (D; unit y axis 10
3
m
3
). Error bars represent SEM.
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www.pnas.orgcgidoi10.1073pnas.0602841103 Wilhelmsson et al.
(6.1 0.2 versus 5.2 0.2; P 0.005; n 47 and 44 cells,
respectively) (Fig. 2B). On max imum projection images of
individual cells, reactive astrocytes also had more main processes
extending at least 25
m f rom the cell soma than nonreactive
astroc ytes (3.9 0.3 versus 2.1 0.3; P 0.0001; n 47 and
42 cells, respectively) (Fig. 2C).
To deter mine whether hypertrophy of cellular processes
af fects the volume of tissue reached by reactive astrocytes, we
c ompared the volume accessed by reactive astrocytes on the
injured side with that accessed by nonreactive astrocytes on
the uninjured side (Fig. 2 D and E). Single nonreactive and
reactive astrocy tes accessed similar volumes of tissue (43.4
1.4 versus 44.2 1.5 10
3
m
3
; P 0.71; range of 2669 and
30–70 10
3
m
3
; n 43 and 43 cells, respectively; w ith
0.05
and
0.2, a dif ference of 10% or higher would be detected)
(Fig. 2D). Thus, the increased thick ness of cellular processes
Fig. 3. Electrically induced lesion of the cerebral cortex triggers astrocyte activation in the surrounding tissue. Four days after injury, reactive astrocytes in the cerebral
cortex around the lesion show highly increased GFAP immunoreactivity (A and B). (C) Schematic presentation of the injury with the rectangle corresponding to the area
shown in A. An asterisk indicates the necrotic area; the dotted line indicates the injury border. CC, corpus callosum. (Scale bar, 100
m.)
Fig. 4. Morphological assessment of reactive and nonreactive cortical astrocytes. (A) Maximum projections of dye-filled reactive and nonreactive astrocytes
in layer I of cerebral cortex 4 days after cortical lesioning. Dye-filling reveals fine spongiform processes in reactive astrocytes comparable to those of nonreactive
astrocytes but a greater number of main processes extending from the cell soma of reactive astrocytes. (Scale bar, 25
m.) (B and C) Quantification of main cellular
processes leaving the soma (B) and processes visible 15
m from the cell soma (C). Thick processes were more numerous in reactive astrocytes than in nonreactive
astrocytes. Three-dimensional reconstruction shows that reactive and nonreactive astrocytes access similar volumes of tissue (D; unit y axis 10
3
m
3
). Error bars
represent SEM.
Wilhelmsson et al. PNAS
November 14, 2006
vol. 103
no. 46
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NEUROSCIENCE
does not alter the action radius of reactive astroc ytes in the
hippocampus.
We next investigated whether the lack of cellular hypertrophy
seen in reactive astrocytes in the deafferented dent ate gyrus was
a response shared by astrocytes outside the hippocampus. We
assessed the morphological appearance of reactive astrocytes in
the cerebral c ortex after electrically induced lesioning (24). This
injury paradigm induces extensive neuronal death and strong
astroc yte activation (Fig. 3C). Four days after the lesion, we
studied the morphology of reactive astroc ytes in cortical layer I
by loading astrocytes 200800
m from the injury border with
Lucifer yellow. Reactive astrocytes showed strongly GFAP-
positive cellular processes in the region surrounding the necrotic
area (Fig. 3A), whereas the same region in un injured mice
showed only weakly GFAP-positive astrocytes (Fig. 3B). Exam-
ination of reactive astroc ytes filled with Lucifer yellow revealed
a bushy morphology with many fine terminal processes protrud-
ing from the main cellular processes, similar to astrocytes filled
in uninjured mice. The main cellular processes appeared to be
thicker in reactive than in nonreactive astrocytes (Fig. 4A). The
number of primary processes leaving the soma was increased in
reactive compared with nonreactive astrocytes (6.2 0.2 versus
5.1 0.3; P 0.005; n 25 and 18 cells, respectively) (Fig. 4B).
On maximum projection images of individual cells, reactive
astroc ytes had more main processes extending at least 15
m
f rom the cell soma than nonreactive astrocytes (6.6 0.6 versus
3.4 0.3; P 0.001; n 24 and 18 cells, respectively) (Fig. 4C).
As demonstrated above for hippocampal astroc ytes, single non-
reactive and reactive cortical astrocy tes accessed similar volumes
of tissue (26.1 2.9 versus 22.7 2.8 10
3
m
3
; P 0.41; range
of 9–50 and 10–56 10
3
m
3
; n 18 and 20 cells, respectively; with
0.05 and
0.2, a difference of 40% or higher would be
detected) (Fig. 4D).
Thus, the increased thick ness of astroc yte processes did not
increase the astrocyte domains in the deafferented dentate gyrus
of the hippocampus or in the electrically lesioned cortex.
In the adult mammalian brain, the access domain of individual
hippocampal astrocytes shows min imal overlap and interdigita-
tion of fine cellular processes between adjacent astrocytes (8,
25). To compare the extent of interdigitation of reactive and
nonreactive astrocytes, we injected Lucifer yellow and Alexa
Fluor 568 into adjacent astroc ytes in the dentate gyrus after
un ilateral entorhinal cortex lesion and evaluated the overlap
bet ween astrocy te territories on optical sections (Fig. 5). Inter-
digit ation was also assessed on 3D images of dye-filled neigh-
boring astrocytes, and the overlap was highlighted in a pseudo-
c olor (Fig. 6A and Movies 1 and 2, which are published as
supporting infor mation on the PNAS web site). Quantification
of the overlapping domains on series of optical sections showed
min imal interdigitation between reactive astrocytes and compa-
rable with that between nonreactive astrocytes (4.5 0.8 versus
3.1 0.6%; ranging from 0.06 to 12.9% for individual astrocytes;
n 20 and 18 cells, respectively). Thus, the hypertrophy of
cellular processes upon astrocyte activation in the hippocampus
did not af fect the extent of overlap between the unique domains
of individual astrocytes.
Discussion
This study shows that astrocytes in the denervated dentate gyrus
and lesioned cerebral cortex remain within their domains and
that the overlap between indiv idual astroc yte domains in the
denervated dentate gyrus remains minimal. Although the main
cellular processes of reactive astroc ytes containing GFAP inter-
mediate filaments become thicker, the overall size of these cells
was similar to that of nonreactive astrocytes. When visualized by
antibodies against intermediate filament protein GFAP, these
thicker processes appear longer, because they can be followed
over greater distances. However, the hypertrophy of cellular
processes did not affect the volume of tissue ac cessed by
individual astrocytes via their fine spongiform processes (Fig.
6B) and thus did not result in cellular hypertrophy.
Previously, the volume of tissue ac cessed by dye-filled non-
reactive astrocytes was measured in rat and mouse CA1 stratum
radiatum of the hippocampus (8, 25). Our findings suggest that
both reactive and nonreactive astrocytes in the molecular layer
of the dent ate gyrus of the mouse hippocampus, and even more
so astroc ytes in the cortical layer I, access a smaller volume of
tissue than astroc ytes in stratum radiatum. Overlap of individual
astroc yte domains of nonreactive astrocytes in the stratum
Fig. 5. Overlap between astrocyte territories assessed by dye-filling of
neighboring cells (Lucifer yellow and Alexa Fluor 568). Maximum projections
of astrocytes in the molecular layer of the dentate gyrus on the lesioned and
contralateral sides show adjacent astrocyte territories (domains) on three
optical sections 4
m apart. Insets show territories with overlapping areas in
yellow. The extent of interdigitation between neighboring astrocytes, both
reactive and nonreactive, was limited and most prominent around blood
vessels (arrowheads). (Scale bar 25
m.)
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www.pnas.orgcgidoi10.1073pnas.0602841103 Wilhelmsson et al.
radiatum was very limited. In the molecular layer of the dentate
gyrus, we found a comparably small extent of interdigitation
bet ween processes of neighboring astrocytes. Most importantly,
this territorial overlap was minimal in astrocytes reacting to an
injury.
Quantification of astrocyte cell density in rat cerebral cortex
(26) and calculations of average volume of cortical astrocytes
based on the length of GFAP-positive processes (27) indicated
a substantial overlap between neighboring astroc yte domains in
the rat cortex (28). Our experimental data on cortical astrocytes
reacting to electrically induced lesions showed that the volume
of tissue accessed by reactive and nonreactive astroc ytes was
similar, suggesting that the amount of territorial overlap between
reactive astrocytes is comparable w ith that of nonreactive cor-
tical astrocytes. The extent to which this applies to other injuries
in the brain or in the spinal cord remains to be established,
because the morphological and functional responses of astro-
c ytes may depend on the type of insult.
In the neonatal CNS, the main proce sse s of neighboring imma-
ture astrocytes interdigitate extensively; gradually these cells de-
velop fine spongiform processe s and assume their mature territories
with only a limited overlap (7). Given their similarities with
immature astrocytes, reactive astrocytes might be expected to form
more extensive interdigitations between the domains of individual
astrocytes. However, our data indicate that this is not the case. The
limited overlap between both reactive and nonreactive astrocytes
suggests that the fine spongiform processe s are distributed to access
tissue evenly and efficiently. The best way to achieve this may be
through contact spacing and pruning of interdigitating processe s to
establish exclusive astrocyte territories.
Suf ficiently stable cellular territories might be necessary for
the morphological and functional connection within the astro-
c yte syncytium and in situations such as axonal degeneration or
a direct neurotrauma can be essential for the astrocyte network
to carry out functions such as c ommunication across gap junc-
tions or spacial buffering.
In conclusion, our findings suggest that the term ‘‘cellular
hypertrophy’’ frequently used to describe morphological changes
in reactive astrocytes may be misleading. Instead, reactive as-
troc ytes show hypertrophy of their intermediate filament-rich
main cellular processes but seem to remain within their unique
‘‘tiled’’ territories.
Methods
Surgical Procedures. Unilateral entorhinal cortex lesioning was
performed as described (29) in six 5-mo-old females. Electrically
induced injury of the cerebral c ortex was performed as described
(24) in six 10-mo-old female mice. All mice were on a mixed
genetic background (C57BL6, 129Sv, 129Ola) and were main-
t ained in a barrier animal facility.
A nesthetized mice were placed in a stereotactic frame, and a
hole was drilled through the skull. For entorhinal cortex lesion,
a retractable wire knife (Kopf Instruments, Tujunga, CA) was
lowered 1 mm down from the dura 3.6 mm laterally and 0.2
mm posterior to lambda. The wire knife was expanded 2 mm
horizont ally and then lowered 2 mm twice at 30° and 135° to
avoid the hippocampal formation. For electrically induced lesion
of the cerebral cortex, a fine-needle electrode was inserted
through the skull 2.25 mm laterally at the level of bregma and
lowered 1.0 mm (measured from the meningeal level) into the
c ortex of the right hemisphere. A sec ond electrode was att ached
to the root of the tail. By using Lesion Maker (Ugo Basile,
Comerio, Italy), a direct current of 5 mA was applied for 10 sec.
The mice were kept in heated cages until they recovered from
anesthesia.
Dye-Filling of Astrocytes. Cells in fixed tissue were filled w ith dye
by methods adapted f rom described protocols (30, 31). Four days
af ter lesion ing, the mice were deeply anesthetized with Nem-
but al (10 mg100 g of body weight) and transcardially perfused
with oxygenated Ringer’s solution (37°C) (0.79% NaCl0.038%
KCl0.02% MgCl
2
6H
2
O0.018% Na
2
HPO
4
0.125% NaHCO
3
0.03% CaCl
2
2H
2
O0.2% dextrose0.02% xylocaine) and then
with 4% paraformaldehyde in PBS (pH 7.4, 37°C) for 10 min.
The brain was extracted and postfixed in ice-cold 4% parafor-
maldehyde for 1 h and cut with a vibratome into 75- to
100-
m-thick slices. The slices were stored in PBS at 4°C until
analysis.
The slices were placed under a 60 water objective (N.A. of
1.4) and observed with an Olympus (Center Valley, PA)
BX50WI microscope with infrared differential interference con-
trast optics. Astrocytes were identified by the shape and size of
their somata. Glass micropipettes (o.d., 1.00 mm; i.d., 0.58 mm;
resistance, 100400 M) were pulled on a vertical puller (Kopf
Instr uments) and backfilled with 5% aqueous Lucifer yellow
(Sigma-A ldrich, St. Louis, MO) or 10 mM Alexa Fluor 568
(Molecular Probes, Eugene, OR) in 200 mM KCl. Astrocytes in
the outer and middle molecular layer of the dentate gyrus of the
hippocampus or in layer I of cerebral cortex were impaled and
iontophoretically injected with the respective dye by using 1-sec
pulses of negative current (0.5 Hz) for 1–2 min. Af ter several
cells were filled, the slices were placed in ice-cold 4% parafor-
maldehyde overnight and then coverslipped in Gelvatol (32).
Immunohistochemistry. The slices were placed in ice-cold 4%
parafor maldehyde overnight at 4°C. The next day, the slices were
repeatedly washed in PBS and per meabilized for1hatroom
temperature in PBS c ontaining 1% BSA, 0.25% Triton X-100,
and 3% nor mal donkey serum, followed by incubation with
guinea pig antibodies against GFAP (Sigma-Aldrich; 1:100) for
48 h at 4°C in PBS containing 1% BSA, 0.1% Triton X-100, and
0.3% normal donkey serum. After several washes in PBS, the
Fig. 6. The domains of nonreactive and reactive astrocytes—a concept. (A)
Interdigitation of fine cellular processes in a 3D reconstruction of astrocytes in
the dentate gyrus. The yellow zone shows the border area where cellular
processes of two adjacent astrocytes interdigitate. (B) Reactive astrocytes stay
within their domains, but their main cellular processes get thicker, making
them visible over a greater distance (illustrated here by the circles).
Wilhelmsson et al. PNAS
November 14, 2006
vol. 103
no. 46
17517
NEUROSCIENCE
slices were incubated overnight at 4°C with donkey anti-guinea
pig antibodies c onjugated with RRX (1:300; Jackson Immu-
noResearch, West Grove, PA) and mounted in Gelvatol.
Image Acquisition and Analysis. Confocal z-series were acquired
with a Radiance 2000 laser-scanning confocal microscope system
(Bio-Rad, Hercules, CA) attached to a Nikon (Kanagawa,
Japan) E600FN microscope and an Olympus FluoView 1000
laser-scann ing confocal microscope system attached to an Olym-
pus IX81 microscope, both equipped with a 60 oil-immersion
objective (Plan Apo N.A. of 1.4 and 1.42, respectively). Images
were visualized and analyzed with Imaris 4.0.4 (Bitplane, Zurich,
Switzerland) and ImageJ (National Institutes of Health, Be-
thesda, MD). The number of cellular processes leaving the soma
was assessed on the stack of optical sections from individual
astroc ytes. Main cellular processes extending more than 25 or 15
m from the soma (see Fig. 2C), for hippocampal or cortical
astroc ytes, respectively, were counted on maximum projections
of dye-filled astrocytes. The volume of tissue reached by dye-
filled astrocytes was measured on 3D rec onstructions of the
astroc ytes by thresholding the images and then creating a volume
of the thresholded voxels (see Fig. 2E). Interdigit ation between
adjacent hippocampal astrocytes was evaluated on optical sec-
tions through adjacent astrocytes filled with different dyes
(Lucifer yellow and Alexa Fluor 568). For quantification of
overlapping astrocyte domains, each astrocyte territory was
manually delineated and pseudocolored, the areas of overlap-
ping territories on optical sections were measured and ex pressed
as a percentage of the total astrocyte area. Interdigit ation was
also visualized on 3D reconstructed images of dye-filled astro-
c ytes by adding a Gaussian blur filter. The double-colored voxels,
indicating areas with neighboring astrocyte processes in close
proximity, were then selected and highlighted on the original 3D
image.
U.W. was supported by Swedish Medical Societ y Grant 16850, Wilhelm
och Martina Lundgrens stif telsen, and Hja¨rnfonden and Åhle´n-
stiftelsen; M.P. was supported by Swedish Research Council Grant
11548, the Swedish Stroke Association, ALF Go¨teborg, Hja¨rnfonden,
Trygg-Hansa, and Torsten och Ragnar So¨derbergs stiftelser; and M.H.E.
was supported by National Institutes of Health Grants RR004050,
NS046068, NS014718, and CA4084314.
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www.pnas.orgcgidoi10.1073pnas.0602841103 Wilhelmsson et al.
    • "In other words, voluntary exercise did not induce proliferation of Olig2- lineage astrocytes, but stimulated the arborization of their fine processes. These astrocytes are different from so-called reactive astrocytes, which are characterized by hypertrophy of GFAP-containing main processes (Wilhelmsson et al., 2006), since GFAP expression by Olig2-lineage astrocytes in the GP was continuously weak during our running-rest paradigms. In addition, we did not observe an increase of the GFPlabeled area in the Runner group in the morphometric analyses (Figure 5E). "
    [Show abstract] [Hide abstract] ABSTRACT: Changes in astrocyte morphology are primarily attributed to the fine processes where intimate connections with neurons form the tripartite synapse and participate in neurotransmission. Recent evidence has shown that neurotransmission induces dynamic synaptic remodeling, suggesting that astrocytic fine processes may adapt their morphologies to the activity in their environment. To illustrate such a neuron-glia relationship in morphological detail, we employed a double transgenic Olig2 CreER/WT ; ROSA26-GAP43-EGFP mice, in which Olig2-lineage cells can be visualized and traced with membrane-targeted GFP. Although Olig2-lineage cells in the adult brain usually become mature oligodendrocytes or oligodendrocyte precursor cells with NG2-proteoglycan expression, we found a population of Olig2-lineage astrocytes with bushy morphology in several brain regions. The globus pallidus (GP) preferentially contains Olig2-lineage astrocytes. Since the GP exerts pivotal motor functions in the indirect pathway of the basal ganglionic circuit, we subjected the double transgenic mice to voluntary wheel running to activate the GP and examined morphological changes of Olig2-lineage astrocytes at both the light and electron microscopic levels. The double transgenic mice were divided into three groups: control group mice were kept in a cage with a locked running wheel for 3 weeks, Runner group were allowed free access to a running wheel for 3 weeks, and the Runner-Rest group took a sedentary 3-week rest after a 3-week running period. GFP immunofluorescence analysis and immunoelectron microscopy revealed that astrocytic fine processes elaborated complex arborization in the Runner mice, and reverted to simple morphology comparable to that of the Control group in the Runner-Rest group. Our results indicated that the fine processes of the Olig2-lineage astrocytes underwent plastic changes that correlated with overall running activities, suggesting that they actively participate in motor functions.
    Full-text · Article · Jul 2016
    • "Activation or inhibition of CAR occurring during the gestation period could promote developmental changes impacting basal functions in the adult brain. We found signs of constitutive cerebrovascular barrier dysfunctions and parenchymal changes possibly reflecting homeostatic modifications (Wilhelmsson et al., 2006). The latter is clinically relevant as the association between cerebrovascular permeability, the immune system and neuronal dysfunction is gaining momentum (Friedman, 2011; Khandaker et al., 2015; Marchi et al., 2014 ). "
    [Show abstract] [Hide abstract] ABSTRACT: Nuclear receptors (NRs) are a group of transcription factors emerging as key players in normal and pathological CNS development. Clinically, an association between the constitutive androstane NR (CAR) and cognitive impairment was proposed, however never experimentally investigated. We wished to test the hypothesis that the impact of CAR on neurophysiology and behavior is underlined by cerebrovascular-neuronal modifications. We have used CAR−/− C57BL/6 and wild type mice and performed a battery of behavioral tests (recognition, memory, motor coordination, learning and anxiety) as well as longitudinal video-electroencephalographic recordings (EEG). Brain cell morphology was assessed using 2-photon or electron microscopy and fluorescent immunohistochemistry.
    Full-text · Article · May 2016
    • "Astrocytes were identified by their expression of GFAP and AQP4. Attention was given to the evidence of gliosis, characterized by astrocyte hypertrophy and domain organization (Oberheim et al., 2009; Oberheim et al., 2012; Verkhratsky and Butt, 2013; Wilhelmsson et al., 2006). Special attention was paid to prevalence of nerve cells in GFAP-positive patches for astrocyte membranes enclosing at least half of the circumference of nerve cell bodies sectioned through the nucleus, as suggestive of synaptic stripping, i.e. detachment of synapses from a nerve cell body (Blinzinger, 1968; Graeber et al., 1993; Hamberger et al., 1970; Kreutzberg, 1989). "
    [Show abstract] [Hide abstract] ABSTRACT: The syndrome idiopathic intracranial hypertension (IIH) includes symptoms and signs of raised intracranial pressure (ICP) and impaired vision, usually in overweight persons. The pathogenesis is unknown. In the present prospective observational study, we characterized the histopathological changes in biopsies from the frontal brain cortical parenchyma obtained from 18 IIH patients. Reference specimens were sampled from 13 patients who underwent brain surgery for epilepsy, tumors or acute vascular diseases. Overnight ICP monitoring revealed abnormal intracranial pressure wave amplitudes in 14/18 IIH patients, who underwent shunt surgery and all responded favorably. A remarkable histopathological observation in IIH patients was patchy astrogliosis defined as clusters of hypertrophic astrocytes enclosing a nest of nerve cells. Distinct astrocyte domains (i.e. no overlap between astrocyte processes) were lacking in most IIH biopsy specimens, in contrast to their prevalence in reference specimens. Evidence of astrogliosis in IIH was accompanied with significantly increased aquaporin-4 (AQP4) immunoreactivity over perivascular astrocytic endfeet, compared to the reference specimens, measured with densitometry. Scattered CD68 immunoreactive cells (activated microglia and macrophages) were recognized, indicative of some inflammation. No apoptotic cells were demonstrable. We conclude that the patchy astrogliosis is a major finding in patients with IIH. We propose that the astrogliosis impairs intracranial pressure-volume reserve capacity, i.e. intracranial compliance, and contributes to the IIH by restricting the outflow of fluid from the cranium. The increased perivascular AQP4 in IIH may represent a compensatory mechanism to enhance brain fluid drainage.
    Full-text · Article · May 2016
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