DISTINCT ORGANIZATION OF ASTROCYTIC PERISYNAPTIC PROCESSES AND THEIR MITOCHONDRIA AT STABLE VERSUS NEWLY FORMED SPINES DURING MOTOR LEARNING

Motor learning induces synaptic remodeling in the primary motor cortex (M1), characterized by distinctive spine turnover in the apical tuft dendrites of layer 5 pyramidal neurons and involving both cortico-cortical and thalamo-cortical inputs (Sohn et al., Science Advances, 2022). However, how astrocytes—particularly their perisynaptic processes (PAPs)—respond to learning-related synaptic remodeling remains poorly understood.
In this study, we examined how PAPs, including their mitochondrial organization, interact with dynamically remodeled dendritic spines to elucidate astrocytic contributions to synaptic regulation as part of the tripartite synapse. Using Thy1-GFP mice, dendritic spines in the apical tuft of M1 layer 5 pyramidal neurons were longitudinally monitored during 8 days of motor training by in vivo two-photon microscopy through a cranial window. After fixation, the same cortical regions were analyzed by laser confocal microscopy and large-volume electron microscopy, enabling correlative light and electron microscopy (CLEM).
We reconstructed tripartite synapse structures in three dimensions and quantitatively analyzed PAP coverage using an in-house distance-dependent algorithm. PAP fractional volume was significantly higher within ~0.7 µm of synaptic interfaces compared to more distal regions. Moreover, PAP coverage was greater around stable spines than around newly formed spines. Large mitochondria within PAPs were preferentially located near stable spines, whereas only small mitochondria were observed beyond 0.4 µm from newly formed spines.
These findings suggest that PAPs and their mitochondrial distribution are selectively associated with stable synapses and may play a key role in fine-tuning synaptic transmission during motor learning–induced spine remodeling.