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.A strocytes act as a third partner in synaptic signal processing by responding to neurotransmitters and by releasing "gliotransmitters" (1, 2). Structurally, the astrocyte functions mainly through its peripheral processes, which constitute approximately 80% of the cell's membrane (3). These peripheral astrocyte processes (PAPs) are frequently extremely fine (<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 microscopic resolution. However, they also wrap synapses, but most studies on glia-synaptic interaction can only indiscriminately refer 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 dehydration (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 intra...

Morphological plasticity of astroglia: Understanding synaptic microenvironment

“…The GFAP‐positive main branches of an astrocyte make up only ∼15% of its total volume (Bushong et al, 2002), thus leaving the bulk of astroglial morphology largely beyond the diffraction limit of conventional optical microscopy. Structural changes in astroglial filopodia‐like protrusions can be routinely observed using time‐lapse light microscopy in cultured hippocampal or cortical astrocytes (Cornell‐Bell et al, 1990a; Lavialle et al, 2011; Molotkov et al, 2013). In acute brain slices some mobile astrocytic processes can be detected (Hirrlinger et al, 2004; Nishida and Okabe, 2007; Nixdorf‐Bergweiler et al, 1994).…”

“…( B ) Extremely fine PAPs (<100 nm, small arrows) ensheating pre‐ and/or postsynaptic elements (bold arrow in B1 pointing at synaptic cleft); mGluR2/3 (B1) and mGluR5 (B2) are present close to the synapse. Adapted from (Lavialle et al, 2011). ( C ) Double‐immunogold labeling of Kir4.1 (small particles, arrows) and aquaporin 4 (large particles) in vitreal perisynaptic Müller cell membranes (denoted M) surrounding photoreceptor terminals (Pt).…”

“…In cultured astrocytes, the ionotropic glutamate receptor agonists glutamate, kainate, and quisqualate evoke filopodia outgrowth, and so do agonists of mGluRs (Cornell‐Bell et al, 1990b; Lavialle et al, 2011). However, selective NMDAR agonists do not appear to trigger morphological changes whereas mGluR antagonists block them (Cornell‐Bell et al, 1990b).…”

“…Also the actin‐binding proteins alpha‐actinin, alpha‐adducin, and ezrin have been localized in PAPs (Fig. 2G) (Lavialle et al, 2011; Safavi‐Abbasi et al, 2001; Seidel et al, 1995). Ezrin connects the plasma membrane to the actin cytoskeleton and can be regulated through phosphorylation/dephosphorylation and hence does not require protein synthesis or breakdown (Reichenbach et al, 2010).…”

“…Ezrin connects the plasma membrane to the actin cytoskeleton and can be regulated through phosphorylation/dephosphorylation and hence does not require protein synthesis or breakdown (Reichenbach et al, 2010). Therefore, knockdown of ezrin interfered with astrocytic and neuronal filopodia growth and movement (Lavialle et al, 2011; Matsumoto et al, 2014). Clearly, the cytoskeletal machinery is essential to at least some forms of structural astroglial plasticity.…”