An electronic key to the mysteries of astrocytes
Authors: Rogachevsky V. V. , Shishkova E. A. 
Published: May 2024

Perisynaptic astrocytic processes (PAP) involved in the tripartite synapse respond to local depolarization activation with calcium release from intracellular stores inside astrocytic process nodes, resulting in local and generalized calcium events [1, 2]. Electrophysiology and imaging experiments demonstrate the existence of Ca2+ stores in astrocytic peripheral processes. However, the initial electron microscopic studies suggested that terminal astrocytic processes (TAP), in contact with synapse and located near the axon-spine interface, lack organelles, including the main astrocytic Ca2+ store — the cisternae of the smooth endoplasmic reticulum (sER) [3]. The Ca2+-dependent release of gliotransmitters in vivo is highly doubtful. This is due to several factors. These include the weak electron contrast of sER cisternae which restricts their detection and analysis, the examination of astrocytic processes on single sections, and the limited optical resolution of the equipment used.
Here, we conducted the first comprehensive analysis of TAP in murine hippocampal and cortical synapses, using serial section transmission electron microscopy and 3D reconstructions. The use of alternative approaches to brain tissue fixation and ultrathin staining allowed to increase the contrast of subunit membranes, such as those in sER cisternae of neurons and astrocytes [4, 5]. However, this technique increased the contrast of individual glycogen granules, which obscured cross-sections of sER cisternae due to their similarity in appearance to glycogen granules. To overcome this, we used the rapid disassembly of glycogen during non-anesthetic euthanasia to reveal sER cisternae in astrocyte processes. The removal of glycogen granules from astrocyte sections allowed the clear observation of the long sER cisternae. These cisternae have a diameter ranging from 30 to 60 nm and exhibit similar electron contrast to that of sER cisternae found in neuronal dendrites, including their specialized form in dendritic spines (spine apparatus). In addition to the extended sER cisternae, the PAP cytoplasm contains cross-sections of contrast structures that measure between 10 and 30 nm in diameter. When observed at low magnification, with a final image resolution of 1.7 nm per pixel, these structures appear morphologically and dimensionally indistinguishable from glycogen granules. Analysis at higher magnifications, with a working image resolution of 0.67–0.34 nm/pixel — ten to fifteen times higher than standard scanning electron microscopy resolution — enabled clear identification of membrane-bound organelles.
Serial sections analysis reveals that PAP structures contain two distinct pools of organelles: short (~130–170 nm) “filiform” cisternae of sER with a diameter of 10–30 nm and microvesicles. Additionally, if PAPs with branchlet morphology feature two types of sER cisternae (short “filiform” and extended dilated cisternae) and microvesicles, then TAP membrane organelles are represented only by fragments of short “filiform” sER cisternae and microvesicles. Groups of these slender cisternae and small vesicles are commonly found in close proximity to the active zones of the most active synapses. The non-random distribution of sER cisternae and microvesicles indicates the presence of an active mechanism for directed transport of membrane organelles within highly flattened TAP lamellae. We analyzed our results alongside existing immunoelectron microscopy data from the literature that examines the location of the Ca2+-binding protein calreticulin, a specific sER marker, in PAP. The observed thin sER cisternae serve as an ultrastructural foundation and primary contributor to the development of spontaneous and induced Ca2+ events in the PAPs, as well as a requirement for Ca2+-dependent vesicular release of gliotransmitters located near active synapses.
Despite the well-established belief that organelles are absent in TAP, the data suggest a dynamic regulation of organelle composition and number within PAP lamellae, dependent on synapse activity. These findings open new avenues for investigating neuron-glia interaction and the functional role of astrocytic microenvironments in tripartite synapse plasticity and pathological processes in the brain.