Structural plasticity of perisynaptic astrocyte processes involves ezrin and metabotropic glutamate receptors

Institut National de la Recherche Agronomique, Unité de Nutrition et Régulation Lipidique des Fonctions Cérébrales 909, 78352 Jouy-en-Josas, France.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 08/2011; 108(31):12915-9. DOI: 10.1073/pnas.1100957108
Source: PubMed
The peripheral astrocyte process (PAP) preferentially associates with the synapse. The PAP, which is not found around every synapse, extends to or withdraws from it in an activity-dependent manner. Although the pre- and postsynaptic elements have been described in great molecular detail, relatively little is known about the PAP because of its difficult access for electrophysiology or light microscopy, as they are smaller than microscopic resolution. We investigated possible stimuli and mechanisms of PAP plasticity. Immunocytochemistry on rat brain sections demonstrates that the actin-binding protein ezrin and the metabotropic glutamate receptors (mGluRs) 3 and 5 are compartmentalized to the PAP but not to the GFAP-containing stem process. Further experiments applying ezrin siRNA or dominant-negative ezrin in primary astrocytes indicate that filopodia formation and motility require ezrin in the membrane/cytoskeleton bound (i.e., T567-phosphorylated) form. Glial processes around synapses in situ consistently display this ezrin form. Possible motility stimuli of perisynaptic glial processes were studied in culture, based on their similarity with filopodia. Glutamate and glutamate analogues reveal that rapid (5 min), glutamate-induced filopodia motility is mediated by mGluRs 3 and 5. Ultrastructurally, these mGluR subtypes were also localized in astrocytes in the rat hippocampus, preferentially in their fine PAPs. In vivo, changes in glutamatergic circadian activity in the hamster suprachiasmatic nucleus are accompanied by changes of ezrin immunoreactivity in the suprachiasmatic nucleus, in line with transmitter-induced perisynaptic glial motility. The data suggest that (i) ezrin is required for the structural plasticity of PAPs and (ii) mGluRs can stimulate PAP plasticity.


Available from: Amin Derouiche
Structural plasticity of perisynaptic astrocyte processes
involves ezrin and metabotropic glutamate receptors
Monique Lavialle
, Georg Aumann
, Enrico Anlauf
, Felicitas Pröls
, Monique Arpin
, and Amin Derouiche
Institut National de la Recherche Agronomique, Unité de Nutrition et Régulation Lipidique des Fonctions Cérébrales 909, 78352 Jouy-en-Josas, France;
Institute of Anatomy, Technical University of Dresden, 01307 Dresden, Germany;
Institute of Anatomy I: Cellular Neurobiology, Universitätsklinikum
Eppendorf, 20246 Hamburg, Germany;
Morphogenèse et Signalisation Cellulaires, Unité Mixte de Recherche 144, Centre National de la Recherche
Scientique/Institut Curie, 75248 Paris 5, France;
Institute for Anatomy and Cell Biology, University of Freiburg, 79104 Freiburg, Germany;
Institute of
Cellular Neurosciences, University of Bonn, 53105 Bonn, Germany;
Institute of Anatomy II, University of Frankfurt, 60590 Frankfurt, Germany; and
Dr. Senckenbergisches, Chronomedizinisches Institut, University of Frankfurt, 60590 Frankfurt, Germany
Edited by Tomas G. M. Hökfelt, Karolinska Institutet, Stockholm, Sweden, and approved June 20, 2011 (received for review January 25, 2011)
The peripheral astrocyte process (PAP) preferentially associates
with the synapse. The PAP, which is not found around every
synapse, extends to or withdraws from it in an activity-dependent
manner. Although the pre- and postsynaptic elements have been
described in great molecular detail, relatively little is known about
the PAP because of its difcult access for electrophysiology or light
microscopy, as they are smaller than microscopic resolution. We
investigated possible stimuli and mechanisms of PAP plasticity.
Immunocytochemistry on rat brain sections demonstrates that
the actin-binding protein ezrin and the metabotropic glutamate
receptors (mGluRs) 3 and 5 are compartmentalized to the PAP but
not to the GFAP-containing stem process. Further experiments
applying ezrin siRNA or dominant-negative ezrin in primary as-
trocytes indicate that lopodia formation and motility require ezrin
in the membrane/cytoskeleton bound (i.e., T567-phosphorylated)
form. Glial processes around synapses in situ consistently display
this ezrin form. Possible motility stimuli of perisynaptic glial
processes were studied in culture, based on their similarity with
lopodia. Glutamate and glutamate analogues reveal that rapid (5
min), glutamate-induced lopodia motility is mediated by mGluRs 3
and 5. Ultrastructurally, these mGluR subtypes were also localized
in astrocytes in the rat hippocampus, preferentially in their ne
PAPs. In vivo, changes in glutamatergic circadian activity in the
hamster suprachiasmatic nucleus are accompanied by changes of
ezrin immunoreactivity in the suprachiasmatic nucleus, in line with
transmitter-induced perisynaptic glial motility. The data suggest
that (i ) ezrin is required for the structural plasticity of PAPs and (ii)
mGluRs can stimulate PAP plasticity.
ERM proteins
suprachiasmatic nucleus
circadian regulation
strocytes act as a third partner in synaptic signal processing
by responding to neurotransmitters and by releasing glio-
transmitters (1, 2). Structurally, the astrocyte functions mainly
through its peripheral processes, which constitute approximately
80% of the cells membrane (3). These peripheral astrocyte pro-
cesses (PAPs) are frequently extremely ne (<50 nm) and display
an extreme surface-to-volume ratio. They are rarely studied in live
tissue, as they are not directly accessible to electrophysiology,
cannot be isolated for biochemistry, and are smaller than micro-
scopic resolution. However, they also wrap synapses, but most
studies on gliasynaptic interaction can only indiscriminately refer
to them as the astrocyte. At the ultrastructural level, the PAPs,
although abundant in the neuropil, specically prefer contacting
synapses and dendrites versus axons (4). The synaptic wrapping
is highly dynamic (59) and also activity-dependent even in the
context of physiological functions, such as motor learning, daily
uctuations of the circadian clock, lactation, parturition, or de-
hydration (1015). Here, we asked two questions about the
structural basis of gliasynaptic interaction: What is the stimulus
for PAP plasticity, and what are the intracellular mechanisms
accomplishing the motility and the formation of the PAP? We
and others have shown that the membrane cytoskeleton linker
ezrin is present in astrocytes (1618). Ezrin regulates the structure
and the function of specic domains of the plasma membrane
(19, 20). It plays an important role in the assembly of epithelial
microvilli; we therefore hypothesized that it might be involved in
the formation of the PAP, which displays dimensions similar to
those of epithelial microvilli.
The PAPs are mostly submicroscopic structures. They appear
as a diffuse background even at high magnication, and have
thus been overlooked in a number of studies despite adequate
markers. We performed double immunostaining of GFAP and
ezrin in the CNS. We have applied subdiffraction microscopy by
combining 1.4-NA lenses (plan-apochromat oil objective, 63× or
100×; Zeiss) with deconvolution (calibration information is pro-
vided in Materials and Methods and Fig. S1). Ezrin immunore-
activity was not present in identied glial GFAP-positive pro-
cesses. Rather, GFAP and ezrin immunoreactivities were
mutually exclusive (Fig. 1A, 1), but the structures, when analyzed
as high-magnication 3D reconstructions, were continuous at
single points (Fig. 1A, 2, and Movies S1 and S2). Ezrin-positive
puncta were interconnected by minute structures, often at the
detection threshold of the system, and organized as bushy com-
plexes connected by a single stalk to the GFAP-positive glial
stem processes (Fig. 1A, 2, and Movies S1 and S2). We interpret
the ezrin-positive structures as abundant PAPs, as PAPs are
typically devoid of GFAP-positive glial laments (21). Further,
the expression of ezrin in brain is restricted to astrocytes (16) and
ependymal cells (22), and a comparable organization of bushy
complexes was observed in ultrastructural 3D reconstructions of
(Bergmann) glial cells (23).
Given the restricted localization of ezrin in the extremely ne
PAPs, we investigated the general signicance of ezrin for PAP
formation. When primary astrocytes were transfected with plasmids
expressing ezrin siRNA and the EGFP reporter, the EGFP-positive
cells displayed low ezrin immunoreactivity and signicantly fewer
lopodia in relation to neighboring, nontransfected cells or cells
transfected with a control plasmid (Fig. 1B, 1 and 2 and Fig. S2).
We then examined to which extent PAP formation requires
the regulated association of ezrin with the plasma membrane
and the actin cytoskeleton. Ezrin interacts with membrane pro-
teins through its N-terminal domain, and with F-actin through a
C-terminal actin-binding site (19). This interaction requires an
activation step that consists in the sequential binding of ezrin to
Author contributions: M.L., E.A., and A.D. designed research; M.L., G.A., F.P., and A.D.
performed research; F.P. and M.A. contributed new reagents/analytic tools; M.L., G.A.,
E.A., and A.D. analyzed data; and M.L., M.A., and A.D. wrote the paper.
The authors declare no conict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
To whom correspondence should be addressed. E-mail:
This article contains supporting information online at
1073/pnas.1100957108/-/DCSupplemental. PNAS
August 2, 2011
vol. 108
no. 31
Page 1
PIP2 and phosphorylation of Thr567 present in the F-actin
binding site (24, 25). Normal ezrinEGFP expressed in primary
astrocytes was present at the plasma membrane and in the nu-
merous lopodia (Fig. 1C, 1, and arrows in Fig. 1C, 25). In
contrast, astrocytes displayed signicantly less lopodia when
transfected with a C-terminally truncated form of ezrin (ezinΔ53
EGFP) that can no longer interact with the actin cytoskeleton (26)
(Fig. 1C, 6, and compare left and right in Fig. 1C, 15). This
truncated protein was homogeneously distributed at the plasma
membrane, yet it displayed a dominant-negative effect on lo-
podia formation (Fig. 1C, 1). Further, the role of ezrin in struc-
tural plasticity was studied by live microscopy. Cultured astrocytes
ezrin-EGFP ezin 53-EGFP
Δ Shape Factor / time point
ezrin ezin 53
shape filopodia/ time point
ezrin ezin 53
filopodia / cell
filopodia (% circumference)
** *
contr. ezrin siRNA
Fig. 1. Astrocytes in the CNS display two types of processes. (A) Double-labeled cryostat sections of rat hippocampus were investigated by epiuorescence
microscopy with subsequent deconvolution. 1, The GFAP-positive stem processes and discrete ezrin-positive puncta are mutually exclusive. 2, Selected pro-
cesses (boxed areas in 1) magnied to the limit of light microscopic resolution, and the rotation of 3D reconstructions (Movies S1 and S2) reveal the structural
continuity of the two structures (arrows mark points of continuity). (Scale bar: 1 μm.) (B) Ezrin is required for lopodia formation in primary astrocytes. 1,
Astrocytes transfected with an ezrin siRNA plasmid containing a CMV promotor-driven EGFP-cDNA (Inset, green; outlined in main gure) display diminished
ezrin immunoreactivity (red channel) compared with neighborin g, nontransfected cells. The processes and free cell boundaries of the transfected cells display
fewer ezrin-immunoreactive lopodia, in comparison with other boundaries running parallel (arrows in 1). Nuclei are stained blue. 2, The extent of lopodia-
covered cell circumference is signicantly reduced in the siRNA-transfected cells (n = 169) in relation to the control plasmids (n = 247 cells; P < 0,001, Students
t test; mean ± SD). Fig. S2 compares the two individual siRNAs and three control plasmids. (C) Filopodia dynamics in primary astrocytes requires the mem-
brane-to-cytoskeleton link by ezrin. 1, Left: EzrinEGFP in primary astrocytes is predominantly localized to lopodia and microspikes. 1, 2, and 6: Primary
astrocytes transf e cted with ezrinEGFP (Left, n = 18) display signicantly more lopodia (P < 0,05, MannWhitney test) than those transfecte d with dominant-
negative ezrinΔ53-EGFP (Right,
n = 13). 15 and 7: Thresholded live microscopy frames sampled at 30-min intervals show that cells expressing ezrinEGFP (n =
10) are more motile, displaying signicantly more (P < 0.05, Students t test) shape changes per time point (green arrows in 25 and 7) than cells expressing
ezrinΔ53-EGFP (n = 12), which are relatively stationary (Movie S3). (D) The phosphorylated form of ezrin is selectively localized in the PAPs. 1, Phospho-T567
ezrin, but not overall ezrin, is restricted to lopodia of xed, cultured astrocytes. 2, At higher magnication of hippocampal astrocytic processes in situ
(overview shown in Fig. S3A), phospho-T657-ezrinpositive puncta are always associated with glial, GS-positive structures. Phospho-T657-ezrin immunore-
activity is often found in PAPs ensheathing axon terminals (z-views of hairline crossing and arrows). Perisynaptic astrocyte processes in situ consistently display
activated ezrin. 3, In rat hippocampal specimens (stratum radiatum, CA1), synapses or phospho-T657-ezrin-containing PAPs were dened as objects. The
percentage of each obj ect class contributing to gliasynaptic contact was determined. White outlines are around synapses which contact PAPs positive for
phospho-T657-ezrin. In most cases, the contacts are obvious (arrows); if not, the corresponding PAP is in a different plane of section. Deconvolution, 0.1-μm
optical section. (Scale bars: A, 1 ,5μm; A, 2,1μm; B, 1,2μm, C, 15,15μm, D, 1,15μ
m; x/y/z arrows in D, 2 and 3,1μm.)
| Lavialle et al.
Page 2
can grow out short lopodia within less than 1 min (27). In astro-
cytes transfected with normal ezrinEGFP, the ezrin-containing
lopodia were highly dynamic (Fig. 1C, 15) and ezrin was asso-
ciated with membrane regions of high motility (Movie S3).
Grouped puncta appearing and disappearing in the cell represent
microspikes extending in the culture medium (Movie S3). In con-
trast, only small shape changes were observed in cells expressing
ezrinΔ53EGFP: they displayed very few short lopodia and mi-
crospikes (arrows in Fig. 1C, 15), and were also less motile (Fig.
1C, 7,andMovieS3).Transfections with ezrin siRNA or dominant-
negative ezrin thus indicate that ezrin and its linker function are
required for formation and motility of astrocytic lopodia.
As the phosphorylation of ezrin at T567 has been shown to
occur only when ezrin is associated with the subplasmalemmal
actin cytoskeleton (26), we examined the distribution of ezrin in
astrocytes. Cultured astrocytes were double-stained for ezrin and
phospho-T567 ezrin. The localization of ezrin phosphorylated at
T567 was absolutely restricted to lopodia and microspikes (Fig.
1D, 1). As an in vitro nding, substantial amounts of overall ezrin
could also be seen diffusely throughout the cytoplasm (Fig. 1 B, 1,
and D, 1), unlike astrocytes in situ, which are hardly recognizable
in ezrin-stained brain sections (16) (Fig. 1A, 1). The activated
form of ezrin is thus restricted to membrane extensions further
supporting a functional role of ezrin in lopodia formation. To
transfer this nding to the in vivo situation, the rat hippocampus
was triple-stained with antibodies directed against phospho-T567
ezrin, and glutamine synthetase (GS), an astrocytic marker that
also reveals the PAPs, particularly around synapses (28). An anti-
synaptophysin antibody was used to label the axon terminals.
Phospho-T567 ezrin in the neuropil showed a punctate distribu-
tion, which did not outline obvious cellular structures but was
clearly and consistently associated with identied, GS-positive
proles of astrocytes (Fig. S3A). Synaptophysin labeling did not
coincide with that of phospho-T567 ezrin; instead, it was fre-
quently juxtaposed to it (Fig. 1D, 2). Quantitative analysis shows
that nearly 58% of all synapses are touched by PAPs containing
phospho-T567 ezrin (Fig. 1D,
3, and Fig. S3B), which is compa-
rable to the proportion of hippocampal synapses with glial con-
tacts observed ultrastructurally (6070%) (29). The minor dis-
crepancy might be explained by limited detection of small
amounts of phospho-T567 ezrin in the nest PAPs.
Next we investigated the regulation of PAP motility. Applica-
tion of glutamate, the transmitter of most CNS synapses, to pri-
mary astrocytes seeded at low density induced elongation or
retraction of lopodia along all directions (Fig. 2A and Fig. S4),
as expected from previous studies (5, 6). The receptors mediating
this process were investigated by the use of glutamate analogues
(Fig. 2B). Trans-ACPD (40 or 100 μM), an agonist of group 1 and
group 2 metabotropic glutamate receptors (mGluRs 1/5 and 2/3,
respectively) (30), elicited responses analogous to those of glu-
tamate, and the same was induced by DCGIV (100 μM), specic
of group 2 mGluRs (30), and DHPG (100 μM), specic of group 1
mGluRs (30) (Fig. 2B). Combination of glutamate with MCCG I
(400 or 800 μM), an mGluR 2 antagonist (30), left the responses
unchanged. Taken together with previous molecular results that
had excluded the presence of mGluRs 1 and 2 in astrocytes (31),
our results suggest glutamate-induced motility of thin astrocyte
processes to be mediated by activation of mGluR 3 or 5.
mGluR 5
mGluR 2/3
con AC100 DCG MC
Fig. 2. Glutamate-induced lopodia motility in astrocytes is mediated by
mGluRs 3 and 5. Primaryastrocytes were incubated for 5 min with glutamate or
glutamate analogues, then xed and stained with anti-GFAP for cell identi-
cation, and with Oregon greenphalloidin for revealing the actin-containing
lopodia (Fig. S4A). (A) Mean values from experiments on glutamate-induced
lopodia dynamics show positive values for formation and negative ones for
retraction. Filled circles are signicantly different from control (P < 0,05, Stu-
dents t test). Each data point or bar in A or B includes lopodia measurements
from 80 to 170 astrocytes. (B) The bars show the absolute values for the mGluR
agonists and antagonists applied; all are signicantly higher than control (P <
0,05, Students t test). con, control; glu, glutamate; AC100, t-ACPD 100 μM;
AC40, t-ACPD 40 μM; DCG, DCG IV; MC, MCCG I. mGluRs 3 and 5 are prefer-
entially localized in the PAPs in vivo. (C and D) MGluR 2/3 and 5 labeling of
astrocytes in rat hippocampus appears faintly diffuse or as uffy patches
(boxed area in C), in addition to interneurons (arrows in D). (E and F) The
diffuse light microscopic staining (Fig. 2 C and D) is based on the abundance
of submicroscopic glial processes at the ultrastructural level. The silver grains
(silver-intensied DAB) can be seen in the extremely ne PAPs (<100 nm,
small arrows in E and F) ensheathing the pre- and/ or postsynaptic element
(spine head in E and F), sometimes sealing the synaptic cleft (bold arrow in
E). The systematic presence of both mGluRs in PAPs is shown in the overview
of this motif (Figs. S5 and S6). (Scale bars: C and D, 100 μm; E and F, 0.5 μm.)
pyr, stratum pyramidale; rad, radiatum; lm, lacunosum moleculare; fh, hip-
pocampal ssure; mol, molecular layer; gcl, granule cell layer; hil, hilus.
Lavialle et al. PNAS
August 2, 2011
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Expression of the latter receptors by glial cells in situ has not
been systematically established (32, 33). By using silver inten-
sication of DAB to detect the extremely small antigen quantities
in structures as small as the PAPs (28), we observed a very similar,
diffuse staining pattern for both mGluRs in the rat hippocampus
(Fig. 2 C and D). This is the pattern characteristic of the selective
labeling of the PAPs. At the ultrastructural level, the silver grains
were systematically present in the ne PAPs (Fig. 2 E and F and
Fig. S5 and S6), which collectively constitute the diffuse back-
ground (Fig. 2 C and D). The mGluR subtypes shown to mediate
lopodia motility in vitro might therefore stimulate rapid PAP
motility in the hippocampus as well, which is supported by the
preferential PAP localization of these mGluRs. Importantly, the
PAPs, although extremely thin, may also contain mitochondria
localized in bulgings or branching points (Fig. S7).
We further tested in the hypothalamus of the intact animal
whether a change in physiological, identied glutamatergic ac-
tivity is paralleled with concomitant PAP changes, a prediction of
glutamate-induced glial process motility. It is established that the
synapses of the retinal ganglion cells projecting to the hamster
suprachiasmatic nucleus (SCN) are glutamatergic and initiate
photic synchronization of the circadian clock (34), so that their
synaptic activity can be monitored over the light/dark (LD) cycle.
Also, diffuse mGluR5 staining characteristic of PAPs coincides
with the terminal eld of the retinal projection to the SCN (Fig.
S8). Anti-ezrin staining was applied to display the PAPs selec-
tively (shown at high resolution in Fig. S9). Ezrin immunoreac-
tivity in the SCN was compared in animals killed at two critical
time points around the LD transition, Zeitgeber time (ZT) 10
(light, n = 6) or ZT14 (dark, n = 7). The signicantly different
levels of ezrin staining at ZT10 and ZT14 (Fig. 3) are consistent
with rhythmic glutamate concentration in the SCN (35), in par-
ticular because ezrin staining was more intense at ZT14 when
glutamate levels peak (35), in line with glutamate-induced PAP
plasticity. Future biochemical and ultrastructural studies are,
however, required to clarify whether increased ezrin immuno-
reactivity reects ezrin protein synthesis at the onset of the night
or differential ezrin availability for immunoreactivity staining
based on sequestration or subcellular redistribution.
Altogether, the data show that the motility of astrocyte lopodia
in vitro requires the membrane-cytoskeleton linker ezrin and
that this motility can be induced by glutamate via mGluR3 and
mGluR5 activation. We assume that the ndings also apply
to the in vivo situation. (i) The relevant proteinsezrin and
mGluRs 3 and 5are present in and even preferentially targeted
to the PAPs (in situ). (ii) Although there are no synapses in the
cell culture studied, astrocyte lopodia and PAPs share some
features relevant to motility. They are comparable in size and
cytoskeletal equipment, being free from microtubules and in-
termediate laments (21). Also, as actin is their only cytoskeletal
component and only this can convey rapid (<1 min) shape
changes, motility of lopodium and the PAP must be based on
an actin mechanism (7), which is also rapid in vivo (6). (iii)As
the perisynaptic glial sheath also displays the activated form of
ezrin (Fig. 1D, 2 and 3), this forms the basis of its well known
plasticity. (iv) Activity changes of glutamatergic synapses in the
behaving animal are in synchrony with changes in PAPs (Fig. 3).
We suggest that synaptically released glutamate stimulates glial
mGluRs on the nearby PAP, which leads to PAP elongation or
retraction, a process involving intracellular mechanisms based
on actin and ezrin. This would also require constant, energy-
consuming actin remodeling. The presence of mitochondria in
PAPs (Fig. S7) (36) is thus important because mitochondrially
derived ATP helps to fuel actin assembly and disassembly, but
also to control sodium and glutamate homeostasis in a small
astrocytic compartment. In addition to providing ATP for GS
a key enzyme in the glutamateglutamine shuttleastrocytic
mitochondria in PAPs may also directly participate in degrada-
tion of glutamate (37). As an example, the present in vitro evi-
dence focuses on glutamate as the most abundant CNS transmitter,
and astrocytes cultured from cortex. We assume that, in vivo, other
transmitters are also operational, in addition to glutamate or in a
region-dependent manner, as astrocytes display receptors for most
transmitters. Also, a comparable inuence of, for example, growth
factors or cytokines on lopodia formation cannot be excluded.
The quantitative replication of glutamate effects by mGluR
ligands suggests that glutamate-induced lopodia motility is solely
mediated by mGluR activation. However, formation of the typi-
cally narrow and elongate PAP structure in Bergmann glia depends
on Ca
inux through AMPA receptors (8, 15) not identied in
hippocampal or cortical astrocytes (38). Here, mGluR-mediated
intracellular PAP motility mechanisms might similarly work by an
increase in intracellular Ca
concentration through release from
intracellular stores triggered by direct and indirect mGluR3 or 5
signaling (30). Regarding the possibility of indirect, glutamate-
rostro-caudal section level in SCN
Intensity of ezrin-ir
ZT 10h
ZT 14h
Fig. 3. Changes in the PAPs in vivo are in synchrony with changes in iden-
tied physiological glutamatergic activity. (A and B) Ezrin immunocyto-
chemistry in hamster brain sections at the level of the SCN (dashed line). (A)
Only the ependyma of the third ventricle (III.) and the midline tanycytes are
labeled at 2 h before onset of the animals nocturnal activity (ZT, 10 h). (B)
Two hours after start of nocturnal activity (ZT, 14 h) in the dark, the PAPs in
the SCN are selectively labeled in the characteristically diffuse pattern. (C)
This effect is signicant as quantied by densitometry (P < 0,002, paired
Students t test). Each data point represents the mean values from seven
(ZT14, lled squares) or six animals (ZT10, open circles). The entire experi-
ment was repeated twice with three or four animals per group, yielding
similar and signicant results. (Scale bars: 200 μm.)
| Lavialle et al.
Page 4
induced mechanisms, we can exclude factors released from neu-
rons, which are absent in primary astrocyte culture. Autocrine
effects cannot be strictly excluded, such as glutamate-induced
astrocytic release of, for example, growth factors, cytokines, or
small-molecule gliotransmitters (e.g., ATP). However, it appears
unlikely that glial release of ATP or other substances would lead
to effective medium concentrations (39, 40) in our model, in
which astrocytes are deliberately cultured at high interindividual
distance with 0.05 μL diffusion volume per cell.
The complementary staining pattern of GFAP and ezrin im-
munoreactivities in situ suggests that the glial stem processes
and PAPs represent distinct cellular compartments. The PAPs
particular morphofunctional properties are established by the
specic targeting of ezrin and mGluRs 3 and 5, among other
proteins, to the PAP (Figs. 1A, 1 and 2, and 2 and Figs. S5 and S6):
the PAP is extremely narrow (Fig. 2 E and F ) (21), and only the
PAP, but not the GFAP-positive stem process, is highly motile
in vivo (6). By responding to glutamatergic activity, the PAP can
also generate localized Ca
signals, which remain restricted to the
microdomain (23) and do not spread to the parent stem process.
However, it is unclear how these and other proteins are targeted to
the PAP, whether they are transported as supramolecular aggre-
gates, and how they are maintained within the PAP.
Materials and Methods
Primary astrocytes were prepared and enriched by the rotary shaker method
(41) and replated in appropriate dishes at densities for subsequent immu-
nostaining, transfection or lopodia measurements (SI Materials and
Methods). Brain sections were obtained from rats perfusion-xed with 4%
paraformaldehyde (PFA; for light microscopy) or with 2% PFA and 2% glu-
taraldehyde (for EM) in phosphate buffer (PB). Primary anti bodies applied in
this study were mouse anti-ezrin (42, 1:1,000, 2 h, clone 3C12; Sigma), anti-
GFAP linked to CY3 (1 h, 1:400, Sigma), rabbit antiphospho-ezrin/radixin/
moesin (for tissue sections; Cell Signaling Technology), mAb 297S (for cul-
tured cells) (43) (gift of S. Tsukita, Kyoto University, Kyoto, Japan), mouse
anti-GS (1:200; Chemicon), mouse anti-synaptophysin linked to oyster 656
(1:100; Synaptic Systems), rabbit-anti mGluR 2/3 (32) (1:500; Chemicon), and
rabbit anti-mGluR 5 (33) (1:100; Chemicon). The detailed protocols for
staining of cells and sections at the light microscopic or EM level are supplied
in SI Materials and Methods. For ultrastructural visualization of the very low
DAB signal even in the ne PAPs, which would normally not be detected, the
chromogen DAB was further silver-enhanced (28). Filopodia were quantied
by using the lopodia sensitive shape factor (FSSF ), which represents a
measure integrating length and number of the lopodia of a given cell in-
dependent of its overall shape and ramications (SI Materials and Methods).
For quantication of ezrin immunoreacti vity in the SCN, hamsters were
maintained under a 12/12 h LD cycle ( SI Materials and Methods), and image
processing and measurements were performed as previously described (44).
ACKNOWLEDGMENTS. We thank Dr. S. Tsukita for the generous gift of
mAb 297S (CPERM), Dr. R. Lamb for supplying the EGFP plasmids,
Dr. M. Thümmler for support in developing the automated lopodia meas-
urements, and Dr. J. Walter for kind support with cell culture facility. The
excellent technical assistance by S. Gaedicke, C. Papillon, B. Rost, and
T. Schwalm is gratefully acknowledged. This project was promoted by the
inspiring discussions with Dr. J. Wolff and Dr. M. Frotscher. This work was
supported by Deutsche Forschungsgemeinschaft Grant DE 676.
1. Haydon PG, Carmignoto G (2006) Astrocyte control of synaptic transmission and
neurovascular coupling. Physiol Rev 86:10091031.
2. Volterra A, Meldolesi J (2005) Astrocytes, from brain glue to communication ele-
ments: the revolution continues. Nat Rev Neurosci 6:626640.
3. Chao TI, Rickmann M, Wolff JR (2002) The Tripartite Synapse: Glia in Synaptic Trans-
mission, eds Volterra A, Magistretti P, Haydon P (Oxford Univ Press, Oxford), pp 323.
4. Eccles J, Ito M, Szentagothai J (1967) The Cerebellum as a Neuronal Machine
(Springer, Berlin).
5. Hirrlinger J, Hülsmann S, Kirchhoff F (2004) Astroglial processes show spontaneous
motility at active synaptic terminals in situ. Eur J Neurosci 20:22352239.
6. Haber M, Zhou L, Murai KK (2006) Cooperative astrocyte and dendritic spine dynamics
at hippocampal excitatory synapses. J Neurosci 26:88818891.
7. Nishida H, Okabe S (2007) Direct astrocytic contacts regulate local maturation of
dendritic spines. J Neurosci 27:331340.
8. Iino M, et al. (2001) Glia-synapse interaction through Ca
-permeable AMPA receptors
in Bergmann glia. Science 292:926929.
9. Reichenbach A, Derouiche A, Kirchhoff F (2010) Morphology and dynamics of peri-
synaptic glia. Brain Res Brain Res Rev 63:1125.
10. Lavialle M, Bègue A, Papillon C, Vilaplana J (2001) Modications of retinal afferent ac-
tivity induce changesin astroglial plasticityin the hamstercircadian clock. Glia 34:88100.
11. Oliet SHR, Piet R, Poulain DA, Theodosis DT (2004) Glial modulation of synaptic
transmission: Insights from the supraoptic nucleus of the hypothalamus. Glia 47:
12. Theodosis DT, Poulain DA (1993) Activity-dependent neuronal-glial and synaptic
plasticity in the adult mammalian hypothalamus. Neuroscience 57:501535.
13. Oliet SHR, Piet R, Poulain DA (2001) Control of glutamate clearance and synaptic
efcacy by glial coverage of neurons. Science 292:923926.
14. Theodosis DT, Poulain DA, Oliet SH (2008) Activity-dependent structural and func-
tional plasticity of astrocyte-neuron interactions. Physiol Rev 88:9831008.
15. Hoogland TM, et al. (2009) Radially expanding transglial calcium waves in the intact
cerebellum. Proc Natl Acad Sci USA 106:34963501.
16. Derouiche A, Frotscher M (2001) Peripheral astrocyte processes: Monitoring by se-
lective immunostaining for the actin-binding ERM proteins. Glia 36:330341.
17. Geiger KD, Stoldt P, Schlote W, Derouiche A (2000) Ezrin immunoreactivity is asso-
ciated with increasing malignancy of astrocytic tumors but is absent in oligoden-
drogliomas. Am J Pathol 157:17851793.
18. Grönholm M, et al. (2005) Characterization of the NF2 protein merlin and the ERM
protein ezrin in human, rat, and mouse central nervous system. Mol Cell Neurosci 28:
19. Bretscher A, Edwards K, Fehon RG (2002) ERM proteins and merlin: Integrators at the
cell cortex. Nat Rev Mol Cell Biol 3:586599.
20. Gautreau A, Louvard D, Arpin M (2002) ERM proteins and NF2 tumor suppressor: the
Yin and Yang of cortical actin organization and cell growth signaling. Curr Opin Cell
Biol 14:104109.
21. Peters A, Palay SL, Webster HdeF (1991) The Fine Structure of the Nervous System: The
Neurons and Supporting Cells. (Oxford Univ Press, Oxford), 3rd ed.
22. Berryman M, Franck Z, Bretscher A (1993) Ezrin is concentrated in the apical microvilli
of a wide variety of epithelial cells whereas moesin is found primarily in endothelial
cells. J Cell Sci 105:10251043.
23. Grosche J, et al. (1999) Microdomains for neuron-glia interaction: Parallel ber sig-
naling to Bergmann glial cells.
Nat Neurosci 2:139143.
24. Fievet BT, et al. (2004) Phosphoinositide binding and phosphorylation act sequentially
in the activation mechanism of ezrin. J Cell Biol 164:653659.
25. Tsukita S, Yonemura S, Tsukita S (1997) ERM proteins: Head-to-tail regulation of actin-
plasma membrane interaction. Trends Biochem Sci 22:5358.
26. Coscoy S, et al. (2002) Molecular analysis of microscopic ezrin dynamics by two-
photon FRAP. Proc Natl Acad Sci USA 99:1281312818.
27. Cornell-Bell AH, Thomas PG, Smith SJ (1990) The excitatory neurotransmitter gluta-
mate causes lopodia formation in cultured hippocampal astrocytes. Glia 3:322334.
28. Derouiche A, Frotscher M (1991) Astroglial processes around identied glutamatergic
synapses contain glutamine synthetase: Evidence for transmitter degradation. Brain
Res 552:346350.
29. Spacek J (1985) Three-dimensional analysis of dendritic spines. III. Glial sheath. Anat
Embryol (Berl) 171:245252.
30. Schoepp DD, Jane DE, Monn JA (1999) Pharmacological agents acting at subtypes of
metabotropic glutamate receptors. Neuropharmacology 38:14311476.
31. Condorelli DF, et al. (1999) Expression and functional analysis of glutamate receptors
in glial cells. The Functional Roles of Glial Cells in Health and Disease, eds Matsas R,
Tsacopoulos M (Kluwer, Dordrecht, The Netherlands), pp 4967.
32. Petralia RS, Wang YX, Niedzielski AS, Wenthold RJ (1996) The metabotropic gluta-
mate receptors, mGluR2 and mGluR3, show unique postsynaptic, presynaptic and
glial localizations. Neuroscience 71:949976.
33. Romano C, et al. (1995) Distribution of metabotropic glutamate receptor mGluR5
immunoreactivity in rat brain. J Comp Neurol 355:455469.
34. Ebling FJ (1996) The role of glutamate in the photic regulation of the suprachiasmatic
nucleus. Prog Neurobiol 50:109132.
35. Glass JD, Hauser UE, Randolph WW (1993)In vivo microdialysis of 5-hydroxyindoleacetic
acid and glutamic acid in the hamster suprachiasmatic nuclei. Am Zool 33:212218.
36. Lovatt D , et al. (2007) The transcriptome and metabolic gene signature of pro-
toplasmic astrocytes in the adult murine cortex. J Neurosci 27:1225512266.
37. Pardo B, et al. (2011) Brain glutamine synthesis requires neuronal-born aspartate as
amino donor for glial glutamate formation. J Cereb Blood Flow Metab 31:90101.
38. Seifert G, Steinhäuser C (2001) Ionotropic glutamate receptors in astrocytes. Prog
Brain Res 132:287299.
39. Gourine AV, et al. (2010) Astrocytes control breathing through pH-dependent release
of ATP. Science 329:571575.
40. Shigetomi E, Kracun S, Sofroniew MV, Khakh BS (2010) A genetically targeted optical
sensor to monitor calcium signals in astrocyte processes. Nat Neurosci 13:759766.
41. McCarthy KD, de Vellis J (1980) Preparation of separate astroglial and oligoden-
droglial cell cultures from rat cerebral tissue. J Cell Biol 85:890902.
42. Böhling T, et al. (1996) Ezrin expression in stromal cells of capillary hemangio-
blastoma. An immunohistochemical survey of brain tumors. Am J Pathol 148:367373.
43. Matsui T, et al. (1998) Rho-kinase phosphorylates COOH-terminal threonines of ezrin/
radixin/moesin (ERM) proteins and regulates their head-to-tail association. J Cell Biol
44. Vilaplana J, Lavialle M (1999) A method to quantify glial brillary acidic protein im-
munoreactivity on the suprachiasmatic nucleus. J Neurosci Methods 88:181187.
Lavialle et al. PNAS
August 2, 2011
vol. 108
no. 31
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    • "Eriksson et al. (1995) reported that sodium-dependent uptake of 100 mol/L glutamate stimulated astrocytic oxygen consumption by 55% within 30s, whereas this level of glutamate increased DG phosphorylation by only ~60% (Pellerin & Magistretti 1994), i.e., the rise in respiration will generate far more ATP than glycolysis. These findings are supported by many studies that have repeatedly demonstrated that glutamate is oxidized by astrocytes in increasing amounts as extracellular glutamate level rises (McKenna et al. 1996, Dienel 2013, McKenna 2013), consistent with the presence of mitochondria in astrocytic fine processes (Lovatt et al. 2007, Lavialle et al. 2011, Pardo et al. 2011, Derouiche et al. 2015). Recent studies identified macromolecular complexes that support astrocytic glutamate transport and metabolism and showed "
    [Show abstract] [Hide abstract] ABSTRACT: Aerobic glycolysis occurs during brain activation and is characterized by preferential upregulation of glucose utilization compared with oxygen consumption even though oxygen level and delivery are adequate. Aerobic glycolysis is a widespread phenomenon that underlies energetics of diverse brain activities, such as alerting, sensory processing, cognition, memory, and pathophysiological conditions, but specific cellular functions fulfilled by aerobic glycolysis are poorly understood. Evaluation of evidence derived from different disciplines reveals that aerobic glycolysis is a complex, regulated phenomenon that is prevented by propranolol, a non-specific β-adrenoceptor antagonist. The metabolic pathways that contribute to excess utilization of glucose compared with oxygen include glycolysis, the pentose-phosphate shunt pathway, the malate-aspartate shuttle, and astrocytic glycogen turnover. Increased lactate production by unidentified cells, and lactate dispersal from activated cells and lactate release from brain, both facilitated by astrocytes, are major factors underlying aerobic glycolysis in subjects with low blood lactate levels. Astrocyte-neuron lactate shuttling with local oxidation is minor. Blockade of aerobic glycolysis by propranolol implicates adrenergic regulatory processes including adrenal release of epinephrine, signaling to brain via the vagus nerve, and increased norepinephrine release from the locus coeruleus. Norepinephrine has a powerful influence on astrocytic metabolism and glycogen turnover that can stimulate carbohydrate utilization more than oxygen consumption, whereas β-receptor blockade 're-balances' the stoichiometry of oxygen-glucose or -carbohydrate metabolism by suppressing glucose and glycogen utilization more than oxygen consumption. This conceptual framework may be helpful for design of future studies to elucidate functional roles of preferential non-oxidative glucose utilization and glycogen turnover during brain activation. This article is protected by copyright. All rights reserved.
    Preview · Article · May 2016 · Journal of Neurochemistry
    • "The puncta did not outline obvious cellular structures but were clearly and consistently associated with EGFP-positive profiles of astrocytes in tissue obtained from transgenic GFAP-EGFP mice (Fig. 1D). For colocalization analysis, considering that both Homer1b/c and mGlu5 are expressed in spines and that astrocytic processes are intimately associated with synapses during the early postnatal developmental stage (Halassa et al. 2007; Clarke and Barres 2013) , we excluded the peripheral astrocytic processes (<100 nm in diameter/length; Lavialle et al. 2011) and instead focused on the main astrocytic processes (≥2 μm in diameter; Schubert et al. 2011 ). Colocalization was performed at very high magnification by inspecting the Homer1b/c and mGlu5 puncta located inside of a given astrocytic process (Fig. 1E–F, Supplementary Fig. 2 ). "
    [Show abstract] [Hide abstract] ABSTRACT: In astrocytes, the intracellular calcium (Ca(2+)) signaling mediated by activation of metabotropic glutamate receptor 5 (mGlu5) is crucially involved in the modulation of many aspects of brain physiology, including gliotransmission. Here, we find that the mGlu5-mediated Ca(2+)signaling leading to release of glutamate is governed by mGlu5 interaction with Homer1 scaffolding proteins. We show that the long splice variants Homer1b/c are expressed in astrocytic processes, where they cluster with mGlu5 at sites displaying intense local Ca(2+)activity. We show that the structural and functional significance of the Homer1b/c-mGlu5 interaction is to relocate endoplasmic reticulum (ER) to the proximity of the plasma membrane and to optimize Ca(2+)signaling and glutamate release. We also show that in reactive astrocytes the short dominant-negative splice variant Homer1a is upregulated. Homer1a, by precluding the mGlu5-ER interaction decreases the intensity of Ca(2+)signaling thus limiting the intensity and the duration of glutamate release by astrocytes. Hindering upregulation of Homer1a with a local injection of short interfering RNA in vivo restores mGlu5-mediated Ca(2+)signaling and glutamate release and sensitizes astrocytes to apoptosis. We propose that Homer1a may represent one of the cellular mechanisms by which inflammatory astrocytic reactions are beneficial for limiting brain injury.
    No preview · Article · Apr 2016 · Cerebral Cortex
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    • "All rights reserved. associated protein ezrin is a key component of peripheral astrocytic processes (PAP) associated with synapses and critical for motility of these fine processes and thereby the structural plasticity of PAPs (Lavialle et al. 2011). In conjunction with the down-regulation of aquaporin-4, which is generally positively correlated with astroglial volume and may regulate " adaptive swelling " of PAPs (Nagelhus et al. 2004), and increased levels of glutamine synthetase, reciprocal alterations in synaptic activity might be directly reflected on the level of the tripartite synapse (Clarke and Barres 2013). "
    [Show abstract] [Hide abstract] ABSTRACT: Learning and memory processes are accompanied by rearrangements of synaptic protein networks. While various studies have demonstrated the regulation of individual synaptic proteins during these processes, much less is known about the complex regulation of synaptic proteomes. Recently, we reported that auditory discrimination learning in mice is associated with a relative down-regulation of proteins involved in the structural organization of synapses in various brain regions. Aiming at the identification of biological processes and signaling pathways involved in auditory memory formation, here, a label-free quantification approach was utilized to identify regulated synaptic junctional proteins and phosphoproteins in the auditory cortex, frontal cortex, hippocampus and striatum of mice 24 h after the learning experiment. Twenty proteins, including postsynaptic scaffolds, actin-remodeling proteins and RNA-binding proteins, were regulated in at least three brain regions pointing to common, cross-regional mechanisms. Most of the detected synaptic proteome changes were, however, restricted to individual brain regions. For example, several members of the Septin family of cytoskeletal proteins were up-regulated only in the hippocampus, while Septin-9 was down-regulated in the hippocampus, the frontal cortex and the striatum. Meta analyses utilizing several databases were employed to identify underlying cellular functions and biological pathways. Data are available via ProteomeExchange with identifier PXD003089. This article is protected by copyright. All rights reserved.
    Full-text · Article · Apr 2016 · Journal of Neurochemistry
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