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We established simultaneous dual color 2- photon microscopy to visualize the trafficking of Neuropeptide Y-, LAMP1- and RAB7- tagged organelles in thalamocortical axons imaged in mouse cortex in vivo. This revealed that LAMP1- and RAB7-tagged organelles move significantly faster than NPY tagged organelles in both anterograde and retrograde direction. NPY travelled more selectively in anterograde direction than LAMP1 and RAB7. By using a synapse marker and a calcium sensor we further investigated the transport dynamics of NPY-tagged organelles. We found that these organelles slow down and pause at synapses. In contrast to previous in vitro studies, a significant increase of transport speed was observed after spontaneous activity and elevated calcium levels in vivo as well as electrically stimulated activity in acute brain slices. 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