In vivo imaging of autophagy dynamics in the intact mammalian brain using a fluorescence lifetime-based sensor

Abstract

Autophagy regulates neuronal development, plasticity, and metabolism, yet our ability to monitor its spatial and temporal organization within intact mammalian neural circuits remains limited. We developed an imaging approach combining a pH-sensitive fluorescence lifetime readout with independent morphological labeling to quantify autophagic vesicle state, abundance, and trafficking in vivo. Following pharmacological, genetic, and ultrastructural validations, we resolved compartment-specific autophagy levels across somata, dendrites, and axons of layer 2/3 cortical excitatory neurons in the intact mouse brain. We uncovered distinct patterns of dendritic autophagy in vivo, revealing faster autophagic vesicle trafficking in distal than proximal dendrites, that were preferentially sensitive to sensory experience. Using a newly generated transgenic mouse line, we found distinct cell-type-specific basal autophagy in excitatory neurons, interneurons, astrocytes, and oligodendrocytes. These findings reveal the spatial, cell-type-specific, and activity-dependent organization of autophagy in the brain and establish a framework for investigating its regulation during development, plasticity, and disease.