Astrocytic Cholesterol Fine-Tunes the Balance of Different Modes of Synaptic Exo- and Endocytosis

Cholesterol is essential for organization of neurotransmitter release machinery, yet how it regulates the balance among different forms of synaptic exo- and endocytosis remains poorly understood. Moreover, which pre-synaptic processes rely on neuronal vs astrocyte-derived cholesterol is unknown. Using nanoscale-precision imaging of single-vesicle release in hippocampal synapses we demonstrate that astrocytic cholesterol is a critical determinant of both temporal and spatial aspects of presynaptic dynamics by differentially modulating the two main forms of synchronous release, uni-vesicular (UVR) and multi-vesicular (MVR), effectively fine-tuning their balance. Disruption of astrocytic cholesterol trafficking to neurons combined with its re-supplementation demonstrated that astrocyte-derived cholesterol is necessary and sufficient to determine the UVR/MVR balance. Moreover, astrocytic cholesterol determines the spatial distribution of vesicle release by modulating utilization of different release sites across the active zone. Astrocytic cholesterol also regulates the balance of the two main forms of single-vesicle endocytosis, fast and ultra-fast. These findings suggest that astrocytic cholesterol supply is a critical modulator of synaptic strength that fine-tunes the balance of different forms of synaptic vesicle exo- and endocytosis.