The Ultrastructural Properties of the Endoplasmic Reticulum Govern Microdomain Signaling in Perisynaptic Astrocytic Processes

Astrocytes are now widely accepted as key regulators of brain function and behavior. Calcium (Ca²⁺) signals in perisynaptic astrocytic processes (PAPs) enable astrocytes to fine-tune neurotransmission at tripartite synapses. As most PAPs are below the diffraction limit, their content in Ca²⁺ stores and the contribution of the latter to astrocytic Ca²⁺ activity is unclear. Here, we reconstruct hippocampal tripartite synapses in 3D from a high-resolution electron microscopy (EM) dataset and find that 75% of PAPs contain some endoplasmic reticulum (ER), a major calcium store in astrocytes. The ER in PAPs displays strikingly diverse shapes and intracellular spatial distributions. To investigate the causal relationship between each of these geometrical properties and the spatiotemporal characteristics of Ca²⁺ signals, we implemented an algorithm that generates 3D PAP meshes by altering the distribution of the ER independently from ER and cell shape. Reaction-diffusion simulations in these meshes reveal that astrocyte activity is governed by a complex interplay between the location of Ca²⁺ channels, ER surface-volume ratio, and spatial distribution. In particular, our results suggest that ER-PM contact sites can act as local signal amplifiers if equipped with IP₃R clusters but attenuate PAP Ca²⁺ activity in the absence of clustering. This study sheds new light on the ultrastructural basis of the diverse astrocytic Ca²⁺ microdomain signals and on the mechanisms that regulate neuron-astrocyte signal transmission at tripartite synapses.