UNIQUE SPATIAL ORGANIZATION OF PERISYNAPTIC ASTROCYTIC PROCESSES IN LAYER 1 OF THE PRIMARY MOTOR CORTEX AFTER MOTOR SKILL LEARNING

Astrocytes play a key role in regulating synaptic transmission as part of the tripartite synapse. Each astrocyte typically occupies a distinct, non-overlapping domain. However, the plasticity of these domains—especially during learning-related synaptic remodeling—remains largely unknown. Using two-photon imaging in Thy1-GFP mice trained for 8 days on a forelimb seed-reaching task, we first identified dendritic segments of the apical tuft of a layer 5 pyramidal neuron that exhibited high spine turnover (Sohn et al., Science Advances, 2022). Correlative light and electron microscopy using large-scale volume EM data collected from these mice with automated tape-collecting ultramicrotome and scanning electron microscopy revealed that these active dendritic segments were contacted by perisynaptic astrocytic processes (PAPs) originating from 3–6 distinct astrocytes, which was analyzed quantitatively using our in house developed measurement methods. Notably, these astrocytic processes extended directly and specifically toward each active dendritic segment. Despite the convergence of processes from multiple astrocytes at the level of dendritic segment, individual dendritic spines were typically contacted by PAPs from only a single astrocyte, indicating highly organized astrocyte-synapse interactions. In addition, astrocyte cell bodies in layer 1 were arranged in a tiled pattern beneath the pia mater, forming a sheet-like organization. This suggests that layer 1 astrocytes exhibit compartment-specific structural organization.
Together, our findings suggest that astrocytic processes dynamically reorganize in an experience-dependent manner and may contribute to synapse-specific modulation during motor learning. Our EM-based analysis provides new insights into the fine-scale morphology of layer 1 astrocytes and their precise interactions with neurons.