Fluorescence lifetime-based biosensor for monitoring compartmentalized autophagy dynamics in the intact mammalian brain

Abstract

Synaptic architecture underlies neuronal computation, with autophagy serving as a key regulator of synaptic plasticity, function and local metabolism. Yet, autophagy dynamics and regulation within intact neuronal circuits remains poorly understood. Here, we developed a pH sensitive fluorescence lifetime-based imaging method which allows to monitor autophagy dynamics at the synaptic level with a second-scale temporal resolution in the living mouse brain. This approach allowed us to uncover compartmentalized autophagic activity across soma, dendrites, and axons of layer 2/3 cortical neurons. Notably, we observe experience-dependent modulation of autophagy dynamics with pronounced mobility of autophagic vesicles in apical distal dendrites compared to basal proximal dendritic branches. We identified cell-type specific basal autophagy levels in excitatory, inhibitory, and astrocytic cells in the mouse cortex. Finally, combining this approach with disease associated genetic perturbation uncovers novel in vivo autophagy related phenotypes. Altogether, this approach highlights the spatial and functional complexity of autophagy in the intact mammalian brain and establishes a framework for investigating its role in synaptic regulation during development, plasticity, and aging.