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 PAP’s
particular morphofunctional properties are established by the
speciﬁc 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 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 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 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 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 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.
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August 2, 2011