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

Abstract

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.

Fig. 1.

Astrocytes in the CNS display two types of processes. (A) Double-labeled cryostat sections of rat hippocampus were investigated by epifluorescence microscopy with subsequent deconvolution. 1, The GFAP-positive stem processes and discrete ezrin-positive puncta are mutually exclusive. 2, Selected processes (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 membrane-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 immunoreactivity 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.)

Fig. 2.

Glutamate-induced filopodia motility in astrocytes is mediated by mGluRs 3 and 5. Primary astrocytes were incubated for 5 min with glutamate or glutamate analogues, then fixed and stained with anti-GFAP for cell identification, 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, Student's t test). Each data point or bar in A or B includes filopodia measurements from 80 to 170 astrocytes. (B) The bars show the absolute values for the mGluR 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 preferentially 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 (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, hippocampal fissure; mol, molecular layer; gcl, granule cell layer; hil, hilus.

Fig. 3.

Changes in the PAPs in vivo are in synchrony with changes in identified physiological glutamatergic activity. (A and B) Ezrin immunocytochemistry 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 experiment was repeated twice with three or four animals per group, yielding similar and significant results. (Scale bars: 200 μm.)