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...

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Astrocytes in vivo extend thin processes termed peripheral astrocyte processes (PAPs), in particular around synapses where they can mediate glia-neuronal communication. The relation of PAPs to synapses is not based on coincidence, but it is not clear which stimuli and mechanisms lead to their formation and are active during process extension/ retraction in response to neuronal activity. Also, the molecular basis of the extremely fine PAP morphology (often 50 to 100 nm) is not understood. These open questions can be best investigated under in vitro conditions studying glial filopodia. We have previously analyzed filopodial mechanisms (Lavialle et al. PNAS 108:12915) applying an automated method for filopodia morphometry, which is now described in greater detail. The Filopodia Specific Shape Factor (FSSF) developed integrates number and length of filopodia. It quantifies filopodia independent of overall astrocytic shape or size, which can be intricate in itself. The algorithm supplied here permits automated image processing and measurements using ImageJ. Cells have to be sampled in higher numbers to obtain significant results. We validate the FSSF, and characterize the systematic influence of thresholding and camera pixel grid on measurements. We provide exemplary results of substance-induced filopodia dynamics (glutamate, mGluR agonists, EGF), and show that filopodia formation is highly sensitive to medium pH (CO) and duration of cell culture. Although the FSSF was developed to study astrocyte filopodia with focus on the perisynaptic glial sheath, we expect that this parameter can also be applied to neuronal growth cones, non-neural cell types, or cell lines.