Peripheral astrocyte processes: monitoring by selective immunostaining for the actin-binding ERM proteins

Abstract

Astrocytes extend thin lamellate processes in the neuropil, in particular around synapses, where they can modulate synaptic function or mediate glial-neuronal communication. Previous studies have shown that these lamellate perisynaptic processes change their shape in response to neuronal activity, but the underlying mechanisms have remained unclear. Similarly, the molecular composition of these fine, sheet-like astrocytic processes (often 50-100 nm wide) is not understood but has to be related to their dynamic properties. To this end, we have studied the presence of ezrin, radixin, and moesin (ERM proteins) in the rat hippocampus and in primary cultured astrocytes, applying immunoperoxidase, immunofluorescence, and immunogold techniques. These three ERM proteins are known as actin-binding proteins that link the cell membrane to the actin cytoskeleton, particularly in microvillus-bearing epithelial cells. In cell culture, anti-ezrin and antiradixin, but not antimoesin, antibodies were specific for astrocytes, which often displayed selective staining of filopodia and microvilli. Nonoverlapping visualization of astrocytic peripheral and stem processes was obtained by immunocytochemical double labeling for ezrin and GFAP, respectively. In sections of rat hippocampus, homogeneous labeling of the neuropil, but not of cell layers, resulted from immunostaining of fine, peripheral astrocyte processes, as confirmed ultrastructurally. Our data show that the fine peripheral processes of astrocytes, which also constitute the perisynaptic glial sheath, are specialized in that they contain characteristic actin-associated molecules, likely to contribute to their dynamic properties. Applying anti-ezrin and anti-radixin as selective markers, plasticity of these perisynaptic glial processes can be analyzed.