Studying subcellular detail in fixed astrocytes: dissociation of morphologically intact glial cells (DIMIGs)

Julia Haseleu,Enrico Anlauf,Sandra Blaess,Elmar Endl,Amin Derouiche

doi:10.3389/fncel.2013.00054

Abstract

Studying the distribution of astrocytic antigens is particularly hard when they are localized in their fine, peripheral astrocyte processes (PAPs), since these processes often have a diameter comparable to vesicles and small organelles. The most appropriate technique is immunoelectron microscopy, which is, however, a time-consuming procedure. Even in high resolution light microscopy, antigen localization is difficult to detect due to the small dimensions of these processes, and overlay from antigen in surrounding non-glial cells. Yet, PAPs frequently display antigens related to motility and glia-synaptic interaction. Here, we describe the dissociation of morphologically intact glial cells (DIMIGs), permitting unambiguous antigen localization using epifluorescence microscopy. Astrocytes are dissociated from juvenile (p13–15) mouse cortex by applying papain treatment and cytospin centrifugation to attach the cells to a slide. The cells and their complete processes including the PAPs is thus projected in 2D. The entire procedure takes 2.5–3 h. We show by morphometry that the diameter of DIMIGs, including the PAPs is similar to that of astrocytes in situ. In contrast to cell culture, results derived from this procedure allow for direct conclusions relating to (1) the presence of an antigen in cortical astrocytes, (2) subcellular antigen distribution, in particular when localized in the PAPs. The detailed resolution is shown in an exemplary study of the organization of the astrocytic cytoskeleton components actin, ezrin, tubulin, and GFAP. The distribution of connexin 43 in relation to a single astrocyte's process tree is also investigated.

Highlights

The role of astrocytes for CNS synaptic transmission, including their own active signaling has become increasingly clear over the past years (Haydon and Carmignoto, 2006)
The astrocyte approaches the synaptic cleft, blood capillaries, the surface of neuronal somata and neuronal compartments (Reichenbach et al, 2004) mostly through these processes. These processes have been termed peripheral astrocyte processes (PAPs; Derouiche et al, 2002), and have been shown to preferentially display proteins and organelles involved in mediating glial interactions with neurons or vessels (e.g., Danbolt et al, 1998; Derouiche and Frotscher, 2001; Rouach et al, 2008)
The present paper suggests a technique facilitating the study of these processes, in particular antigen localization, by light microscopic immunocytochemistry in astrocytes

Summary

Introduction

The role of astrocytes for CNS synaptic transmission, including their own active signaling has become increasingly clear over the past years (Haydon and Carmignoto, 2006). Astrocytes are key players in the coupling of metabolism and blood flow to neuronal activity (Carmignoto and Gómez-Gonzalo, 2010; Giaume et al, 2010). In addition to their soma and GFAP-positive main processes, astrocytes—in particular in gray matter—display a high amount of extremely fine processes. The PAPs, emanating from the main processes, are best regarded as sponge-like structures rather than highly branched processes, based on electron microscopic 3D reconstructions (Ventura and Harris, 1999; Witcher et al, 2007) They fully engulf or embed neuronal structures, insulating them, and creating a micro-environment (Reichenbach et al, 2010)

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