Structural plasticity of perisynaptic astrocyte processes
involves ezrin and metabotropic glutamate receptors
Monique Laviallea, Georg Aumannb, Enrico Anlaufb, Felicitas Prölsc, Monique Arpind, and Amin Derouicheb,e,f,g,h,1
aInstitut National de la Recherche Agronomique, Unité de Nutrition et Régulation Lipidique des Fonctions Cérébrales 909, 78352 Jouy-en-Josas, France;
bInstitute of Anatomy, Technical University of Dresden, 01307 Dresden, Germany;cInstitute of Anatomy I: Cellular Neurobiology, Universitätsklinikum
Eppendorf, 20246 Hamburg, Germany;dMorphogenèse et Signalisation Cellulaires, Unité Mixte de Recherche 144, Centre National de la Recherche
Scientifique/Institut Curie, 75248 Paris 5, France;eInstitute for Anatomy and Cell Biology, University of Freiburg, 79104 Freiburg, Germany;fInstitute of
Cellular Neurosciences, University of Bonn, 53105 Bonn, Germany;gInstitute of Anatomy II, University of Frankfurt, 60590 Frankfurt, Germany; and
hDr. 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 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 as-
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
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.
ERM proteins|suprachiasmatic nucleus|circadian regulation|actin
transmitters” (1, 2). Structurally, the astrocyte functions mainly
through its peripheral processes, which constitute approximately
80% of the cell’s membrane (3). These peripheral astrocyte pro-
cesses (PAPs) are frequently extremely fine (<50 nm) and display
anextremesurface-to-volumeratio.Theyarerarelystudied 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
to them as the astrocyte. At the ultrastructural level, the PAPs,
although abundant in the neuropil, specifically prefer contacting
synapses and dendrites versus axons (4). The synaptic wrapping
is highly dynamic (5–9) and also activity-dependent even in the
context of physiological functions, such as motor learning, daily
fluctuations of the circadian clock, lactation, parturition, or de-
hydration (10–15). Here, we asked two questions about the
structural basis of glia–synaptic 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
strocytes act as a third partner in synaptic signal processing
by responding to neurotransmitters and by releasing “glio-
ezrin is presentin astrocytes(16–18).Ezrinregulatesthe structure
and the function of specific 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 magnification, 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 identified glial GFAP-positive pro-
cesses. Rather, GFAP and ezrin immunoreactivities were
mutually exclusive (Fig. 1A, 1), but the structures, when analyzed
as high-magnification 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 filaments (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 fine
PAPs, we investigated the general significance 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 significantly fewer
filopodia 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 conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| August 2, 2011
| vol. 108
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PIP2 and phosphorylation of Thr567 present in the F-actin–
binding site (24, 25). Normal ezrin–EGFP expressed in primary
astrocytes was present at the plasma membrane and in the nu-
merous filopodia (Fig. 1C, 1, and arrows in Fig. 1C, 2–5). In
contrast, astrocytes displayed significantly less filopodia when
transfected with a C-terminally truncated form of ezrin (ezinΔ53–
(Fig. 1C, 6, and compare left and right in Fig. 1C, 1–5). This
truncated protein was homogeneously distributed at the plasma
membrane, yet it displayed a dominant-negative effect on filo-
podia formation (Fig. 1C, 1). Further, the role of ezrin in struc-
tural plasticity was studied by live microscopy. Cultured astrocytes
Δ Shape Factor / time point
∆ shape filopodia/ time point
ezrin ezin 53
filopodia / cell
filopodia (% circumference)
* * *
contr. ezrin siRNA
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) magnified 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 filopodia formation in primary astrocytes. 1,
Astrocytes transfected with an ezrin siRNA plasmid containing a CMV promotor-driven EGFP-cDNA (Inset, green; outlined in main figure) display diminished
ezrin immunoreactivity (red channel) compared with neighboring, nontransfected cells. The processes and free cell boundaries of the transfected cells display
fewer ezrin-immunoreactive filopodia, in comparison with other boundaries running parallel (arrows in 1). Nuclei are stained blue. 2, The extent of filopodia-
covered cell circumference is significantly reduced in the siRNA-transfected cells (n = 169) in relation to the control plasmids (n = 247 cells; P < 0,001, Student’s
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: Ezrin–EGFP in primary astrocytes is predominantly localized to filopodia and microspikes. 1, 2, and 6: Primary
astrocytes transfected with ezrin–EGFP (Left, n = 18) display significantly more filopodia (P < 0,05, Mann–Whitney test) than those transfected with dominant-
negative ezrinΔ53-EGFP (Right, n = 13). 1–5 and 7: Thresholded live microscopy frames sampled at 30-min intervals show that cells expressing ezrin–EGFP (n =
10) are more motile, displaying significantly more (P < 0.05, Student’s t test) shape changes per time point (green arrows in 2–5 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 filopodia of fixed, cultured astrocytes. 2, At higher magnification of hippocampal astrocytic processes in situ
(overview shown in Fig. S3A), phospho-T657-ezrin–positive 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 defined as objects. The
percentage of each object class contributing to glia–synaptic 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, 5 μm; A, 2, 1 μm; B, 1, 2 μm, C, 1–5, 15 μm, D, 1, 15 μm; x/y/z arrows in D, 2 and 3, 1 μm.)
Astrocytes in the CNS display two types of processes. (A) Double-labeled cryostat sections of rat hippocampus were investigated by epifluorescence
| www.pnas.org/cgi/doi/10.1073/pnas.1100957108Lavialle et al.
can grow out short filopodia within less than 1 min (27). In astro-
cytes transfected with normal ezrin–EGFP, the ezrin-containing
filopodia were highly dynamic (Fig. 1C, 1–5) 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
ezrinΔ53–EGFP: they displayed very few short filopodia and mi-
crospikes (arrows in Fig. 1C, 1–5), and were also less motile (Fig.
negative ezrin thus indicate that ezrin and its linker function are
required for formation and motility of astrocytic filopodia.
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 filopodia and microspikes (Fig.
1D, 1). As an in vitro finding, 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 filopodia formation. To
transfer this finding 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 identified, GS-positive
profiles 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 (60–70%) (29). The minor dis-
crepancy might be explained by limited detection of small
amounts of phospho-T567 ezrin in the finest 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 filopodia 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), specific
of group 2 mGluRs (30), and DHPG (100 μM), specific ofgroup 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.
con AC100 DCG MC
Glu AC40 DHPG
glutamate analogues, then fixed and stained with anti-GFAP for cell identifi-
cation, and with Oregon green–phalloidin for revealing the actin-containing
filopodia (Fig. S4A). (A) Mean values from experiments on glutamate-induced
filopodia dynamics show positive values for formation and negative ones for
retraction. Filled circles are significantly different from control (P < 0,05, Stu-
agonists and antagonists applied; all are significantly higher than control (P <
0,05, Student’s 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 fluffy patches
Glutamate-induced filopodia motility in astrocytes is mediated by
(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-intensified DAB) can be seen in the extremely fine 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 fissure; mol, molecular layer; gcl, granule cell layer; hil, hilus.
Lavialle et al. PNAS
| August 2, 2011
| vol. 108
| no. 31
Expression of the latter receptors by glial cells in situ has not
been systematically established (32, 33). By using silver inten-
sification 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 fine 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
filopodia 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, identified 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 field 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 significantly 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 reflects 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 filopodia
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 findings also apply
to the in vivo situation. (i) The relevant proteins—ezrin and
mGluRs 3 and 5—are present in and even preferentially targeted
to the PAPs (in situ). (ii) Although there are no synapses in the
cell culture studied, astrocyte filopodia and PAPs share some
features relevant to motility. They are comparable in size and
cytoskeletal equipment, being free from microtubules and in-
termediate filaments (21). Also, as actin is their only cytoskeletal
component and only this can convey rapid (<1 min) shape
changes, motility of filopodium 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 glutamate–glutamine shuttle—astrocytic
mitochondria in PAPs may also directly participate in degrada-
tion of glutamate (37). As an example, the present in vitro evi-
and astrocytescultured from cortex. Weassumethat, 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 influence of, for example, growth
factors or cytokines on filopodia formation cannot be excluded.
The quantitative replication of glutamate effects by mGluR
ligands suggests that glutamate-inducedfilopodiamotilityis solely
mediated by mGluR activation. However, formation of the typi-
on Ca2+influx through AMPA receptors (8, 15) not identified in
hippocampal or cortical astrocytes (38). Here, mGluR-mediated
intracellular PAP motility mechanisms might similarly work by an
increase in intracellular Ca2+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
tified 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 animal’s 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 significant as quantified by densitometry (P < 0,002, paired
Student’s t test). Each data point represents the mean values from seven
(ZT14, filled squares) or six animals (ZT10, open circles). The entire experi-
ment was repeated twice with three or four animals per group, yielding
similar and significant results. (Scale bars: 200 μm.)
Changes in the PAPs in vivo are in synchrony with changes in iden-
| www.pnas.org/cgi/doi/10.1073/pnas.1100957108Lavialle et al.
induced mechanisms, we can exclude factors released from neu- Download full-text
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 PAP’s
particular morphofunctional properties are established by the
specific targeting of ezrin and mGluRs 3 and 5, among other
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
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 filopodia measurements (SI Materials and
Methods). Brain sections were obtained from rats perfusion-fixed with 4%
paraformaldehyde (PFA; for light microscopy) or with 2% PFA and 2% glu-
taraldehyde (for EM) in phosphate buffer (PB). Primary antibodies 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 anti–phospho-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 fine PAPs, which would normally not be detected, the
chromogen DAB was further silver-enhanced (28). Filopodia were quantified
by using the filopodia sensitive shape factor (FSSF), which represents a
measure integrating length and number of the filopodia of a given cell in-
dependent of its overall shape and ramifications (SI Materials and Methods).
For quantification of ezrin immunoreactivity 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 filopodia 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 86:1009–1031.
2. Volterra A, Meldolesi J (2005) Astrocytes, from brain glue to communication ele-
ments: the revolution continues. Nat Rev Neurosci 6:626–640.
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 3–23.
4. Eccles J, Ito M, Szentagothai J (1967) The Cerebellum as a Neuronal Machine
5. Hirrlinger J, Hülsmann S, Kirchhoff F (2004) Astroglial processes show spontaneous
motility at active synaptic terminals in situ. Eur J Neurosci 20:2235–2239.
6. Haber M, Zhou L, Murai KK (2006) Cooperative astrocyte and dendritic spine dynamics
at hippocampal excitatory synapses. J Neurosci 26:8881–8891.
7. Nishida H, Okabe S (2007) Direct astrocytic contacts regulate local maturation of
dendritic spines. J Neurosci 27:331–340.
8. Iino M, et al. (2001) Glia-synapse interaction through Ca2+-permeable AMPA receptors
in Bergmann glia. Science 292:926–929.
9. Reichenbach A, Derouiche A, Kirchhoff F (2010) Morphology and dynamics of peri-
synaptic glia. Brain Res Brain Res Rev 63:11–25.
10. Lavialle M, Bègue A, Papillon C, Vilaplana J (2001) Modifications of retinal afferent ac-
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 57:501–535.
13. Oliet SHR, Piet R, Poulain DA (2001) Control of glutamate clearance and synaptic
efficacy by glial coverage of neurons. Science 292:923–926.
14. Theodosis DT, Poulain DA, Oliet SH (2008) Activity-dependent structural and func-
tional plasticity of astrocyte-neuron interactions. Physiol Rev 88:983–1008.
15. Hoogland TM, et al. (2009) Radially expanding transglial calcium waves in the intact
cerebellum. Proc Natl Acad Sci USA 106:3496–3501.
16. Derouiche A, Frotscher M (2001) Peripheral astrocyte processes: Monitoring by se-
lective immunostaining for the actin-binding ERM proteins. Glia 36:330–341.
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 157:1785–1793.
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 3:586–599.
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
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 105:1025–1043.
23. Grosche J, et al. (1999) Microdomains for neuron-glia interaction: Parallel fiber sig-
naling to Bergmann glial cells. Nat Neurosci 2:139–143.
24. Fievet BT, et al. (2004) Phosphoinositide binding and phosphorylation act sequentially
in the activation mechanism of ezrin. J Cell Biol 164:653–659.
25. Tsukita S, Yonemura S, Tsukita S (1997) ERM proteins: Head-to-tail regulation of actin-
plasma membrane interaction. Trends Biochem Sci 22:53–58.
26. Coscoy S, et al. (2002) Molecular analysis of microscopic ezrin dynamics by two-
photon FRAP. Proc Natl Acad Sci USA 99:12813–12818.
27. Cornell-Bell AH, Thomas PG, Smith SJ (1990) The excitatory neurotransmitter gluta-
mate causes filopodia formation in cultured hippocampal astrocytes. Glia 3:322–334.
28. Derouiche A, Frotscher M (1991) Astroglial processes around identified glutamatergic
synapses contain glutamine synthetase: Evidence for transmitter degradation. Brain
29. Spacek J (1985) Three-dimensional analysis of dendritic spines. III. Glial sheath. Anat
Embryol (Berl) 171:245–252.
30. Schoepp DD, Jane DE, Monn JA (1999) Pharmacological agents acting at subtypes of
metabotropic glutamate receptors. Neuropharmacology 38:1431–1476.
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 49–67.
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 71:949–976.
33. Romano C, et al. (1995) Distribution of metabotropic glutamate receptor mGluR5
immunoreactivity in rat brain. J Comp Neurol 355:455–469.
34. Ebling FJ (1996) The role of glutamate in the photic regulation of the suprachiasmatic
nucleus. Prog Neurobiol 50:109–132.
acid and glutamic acid in the hamster suprachiasmatic nuclei. Am Zool 33:212–218.
36. Lovatt D, et al. (2007) The transcriptome and metabolic gene signature of pro-
toplasmic astrocytes in the adult murine cortex. J Neurosci 27:12255–12266.
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 31:90–101.
38. Seifert G, Steinhäuser C (2001) Ionotropic glutamate receptors in astrocytes. Prog
Brain Res 132:287–299.
39. Gourine AV, et al. (2010) Astrocytes control breathing through pH-dependent release
of ATP. Science 329:571–575.
40. Shigetomi E, Kracun S, Sofroniew MV, Khakh BS (2010) A genetically targeted optical
sensor to monitor calcium signals in astrocyte processes. Nat Neurosci 13:759–766.
41. McCarthy KD, de Vellis J (1980) Preparation of separate astroglial and oligoden-
droglial cell cultures from rat cerebral tissue. J Cell Biol 85:890–902.
42. Böhling T, et al. (1996) Ezrin expression in stromal cells of capillary hemangio-
blastoma. An immunohistochemical survey of brain tumors. Am J Pathol 148:367–373.
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 fibrillary acidic protein im-
munoreactivity on the suprachiasmatic nucleus. J Neurosci Methods 88:181–187.
Lavialle et al. PNAS
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| vol. 108
| no. 31