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

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.

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

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.

Astrocytic exocytosis vesicles and glutamate: a high-resolution immunofluorescence study.

Physiological evidence has demonstrated that cultured astrocytes can release glutamate via Ca2+-dependent mechanisms. Also, glutamate released from astrocytes in the hippocampal slice interferes with synaptic neurotransmission. Since these observations suggest vesicular glutamate release from astrocytes, the presence of glutamate-containing exocytosis vesicles was investigated. We applied immunofluorescence techniques combined with high-performance deconvolution microscopy, which yields a resolution of <200 nm and permits evaluation of double labeling in individual vesicles. Using a well-characterized anti-glutamate antiserum and parameters minimizing fixative-induced autofluorescence, glutamate-immunoreactive (ir) puncta were found all over the astrocyte but were conspicuously dense at the cell boundary and in filopodia. Images were very similar with antibodies against vesicular glutamate transporters (vGluT1 and vGluT2). Labeling for the exocytosis markers rab3, synaptophysin, or synaptobrevin was also punctate, particularly dense at the cell boundary, but disappearing toward the perinuclear region. Sections of the cell boundary were delineated by rab3 immunoreactivity. In double-labeled cells, vesicular colocalization of glutamate and any of the exocytosis markers was frequent in filopodia and at the cell boundary. Within the cell, single-labeled glutamate-ir vesicles prevailed; double-labeled vesicles were infrequently present. By resolving single vesicles, in cultured astrocytes we visualize glutamate-containing vesicles, vesicles displaying vGluT1 or vGluT2, and exocytosis vesicles displaying glutamate-ir. This may provide the morphological correlate of Ca2+-dependent glutamate release from astrocytes, possibly occurring at defined sections of the cell membrane and at filopodia. However, since vGluTs and exocytosis markers are classically restricted to nerve terminals in the CNS, glutamate release from astrocytes in the CNS remains to be studied.