Nassal, J. P. | bioRxiv Science

bioRxiv2022

Differential axonal trafficking of Neuropeptide Y-, LAMP1- and RAB7-tagged organelles in vivo

Different organelles traveling through neurons exhibit distinct properties in vitro, but this has not been investigated in the intact brain. 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. Together, we show a remarkable diversity in speeds and properties of three axonal organelle marker in vivo that differ from properties previously observed in vitro.

neuroscience↗

bioRxiv2018

Secretory vesicle trafficking in awake and anesthetized mice: differential speeds in axons versus synapses

Neuronal dense core vesicles (DCVs) transport many cargo molecules like neuropeptides and neurotrophins to their release sites in dendrites or axons. The transport properties of DCVs in axons of the intact mammalian brain are unknown. We used viral expression of a DCV cargo reporter (NPY-Venus/Cherry) in the thalamus and two-photon in vivo imaging to visualize axonal DCV trafficking in thalamo-cortical projections of anesthetized and awake mice. We found an average speed of 1 m/s, maximal speeds of up to 5 m/s and a pausing fraction of ~11%. Directionality of transport differed between anesthetized and awake mice. In vivo microtubule +-end extension imaging using Macf18-GFP revealed microtubular growth at 0.12 m/s and provided positive identification of antero- and retrograde axonal transport. Consistent with previous reports, anterograde transport was faster (~2.1 m/s) than retrograde transport (~1.4 m/s). In summary, DCVs are transported with faster maximal speeds and lower pausing fraction in vivo compared to previous results obtained in vitro. Finally, we found that DCVs slowed down upon presynaptic bouton approach. We propose that this mechanism promotes synaptic localization and cargo release.\n\nKey pointsO_LIDespite their immense physiological and pathophysiological importance, we know very little about the biology of dense core vesicle (DCV) trafficking in the intact mammalian brain.\nC_LIO_LIDCVs are transported at similar average speeds in the anesthetized and awake mouse brain compared to neurons in culture, yet maximal speed and pausing fraction of transport were higher.\nC_LIO_LIMicrotubule +-end extension imaging visualized microtubular growth at 0.12 m/s and revealed that DCVs were transported faster in the anterograde direction.\nC_LIO_LIDCV transport slowed down upon presynaptic bouton approach, possibly promoting synaptic localization and cargo release.\nC_LIO_LIOur work provides a basis to extrapolate DCV transport properties determined in cultured neurons to the intact mouse brain and reveal novel features such as slowing upon bouton approach and brain state-dependent trafficking directionality.\nC_LI

neuroscience↗