Activity-dependent control of axonal amphisome trafficking governs norepinephrine release in vivo

The postmitotic nature, exceptional longevity, and elaborate cytoarchitecture of neurons exert extraordinary demands on proteostasis and autophagy regulation. Amphisomes are organelles of the autophagy pathway that result from the fusion of autophagosomes with late endosomes. Previous work suggests that neuronal amphisomes also serve as signalling platforms, though their physiological relevance in vivo remains largely unexplored. Here, we demonstrate dynamic trafficking of amphisomes within the long-range, highly branched axons of locus coeruleus norepinephrine (LC-NE) neurons. Using in vivo photoconversion, we show that amphisomes originating in distal axons can traverse the entire axonal length to reach the soma. Two-photon imaging of LC-NE projections to the prefrontal cortex revealed that velocity and directionality of trafficking are tightly regulated by LC activity states, behavioural context, and autocrine norepinephrine signalling. Activation of Gi-coupled receptor signalling unifies directionality of transport, enhances somatic cargo delivery, whereas prolonged distal immobilization correlates with increased norepinephrine release, consistent with a signalling function. Together, these findings establish LC amphisomes as dual-function organelles that integrate degradative transport with activity-dependent signalling in vivo.