Differential axonal trafficking of Neuropeptide Y-, LAMP1-, and RAB7-tagged organelles in vivo
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Curated by eLife
eLife assessment
This is an important and fundamental, well-written and easily comprehended quantitative imaging study, analyzing the motion of endo-lysosomal compartments within axons in vivo using simultaneous multiphoton imaging in the mammalian brain. Taken together, this is a significant technical advance with interesting observations that substantively move the field forward.
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Abstract
Different organelles traveling through neurons exhibit distinct properties in vitro, but this has not been investigated in the intact mammalian brain. We established simultaneous dual color two-photon microscopy to visualize the trafficking of Neuropeptide Y (NPY)-, 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 traveled 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.
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Author Response
Reviewer #3 (Public Review):
The authors use two-photon imaging to visualize various axonal organelle populations that they have virally labeled with fluorescent proteins, including DCVs and late endosomes/ lysosomes. The latter topic is a bit contentious, as the authors use two labels that tag potentially overlapping and not highly specific markers so that the nature of the tagged organelle populations remains unclear. Notably, the authors also have previously published a detailed account of how DCVs traffic in vivo, so the novelty is mostly in comparing the behavior of different organelles and the potential influence of activity.
Overall, the reported results mostly corroborate the expectations from previous in vitro and in vivo work on these organelles and other cargoes, performed by the authors and their …
Author Response
Reviewer #3 (Public Review):
The authors use two-photon imaging to visualize various axonal organelle populations that they have virally labeled with fluorescent proteins, including DCVs and late endosomes/ lysosomes. The latter topic is a bit contentious, as the authors use two labels that tag potentially overlapping and not highly specific markers so that the nature of the tagged organelle populations remains unclear. Notably, the authors also have previously published a detailed account of how DCVs traffic in vivo, so the novelty is mostly in comparing the behavior of different organelles and the potential influence of activity.
Overall, the reported results mostly corroborate the expectations from previous in vitro and in vivo work on these organelles and other cargoes, performed by the authors and their collaborators, as well as in many other laboratories:
(i) Different organelles have different transport behaviors regarding speed, the ratio of anterograde to retrograde moving organelles, etc.
(ii) Organelles move in different ways when they pass specific anatomical landmarks in the axons, such as presynaptic terminals.
(iii) Activity of a neuron (here measured by calcium imaging) can impact the measured transport parameters, albeit in a subtle and mechanistically not well-defined manner. The chosen experimental design precludes a more detailed analysis, for example of the precise movement behavior (such as defining the exact pausing/movement behavior of organelles, which would require higher imaging speeds) or of a correlation of different organellar behavior at synaptic sites or during activity (which would require three-channel simultaneous imaging of two organelle classes plus a synaptic or activity marker).
In summary, this publication uses sophisticated in vivo labeling and imaging methods to corroborate and complement previous observations on how different axonal organelles move, and what influences their trafficking.
We thank the reviewer for the time dedicated to our manuscript. We are thankful for the critical and specific comments, which allowed us to further improve our manuscript. We agree that it would have been beneficial to have higher frame rates and there instead of two imaging channels. However, this would have further added technical complexity to an already complex experimental setup resolving fluorescent puncta with sizes below the resolution limit. And we are convinced that all our main conclusions are justified based on the imaging settings in the current data sets.
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eLife assessment
This is an important and fundamental, well-written and easily comprehended quantitative imaging study, analyzing the motion of endo-lysosomal compartments within axons in vivo using simultaneous multiphoton imaging in the mammalian brain. Taken together, this is a significant technical advance with interesting observations that substantively move the field forward.
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Reviewer #1 (Public Review):
This is an important, well-written and easily comprehended quantitative imaging study that analyzes the motion of endo-lysosomal compartments within axons in vivo using simultaneous multiphoton imaging in the mammalian brain. The simultaneous dual two-photon imaging is well-executed and represents a substantive advance in a field that relies heavily on in vitro neuronal culture preparations. This work opens the door to neurons that have aged appropriately and done so in the context of normal synaptic and neuromodulatory input, without an excess of added factors that occurs with in vitro cell culture. The authors solve an issue of cell polarity, providing strong support for their ability to determine directional movement (anterograde versus retrograde). In principle, this could become a generalized approach, …
Reviewer #1 (Public Review):
This is an important, well-written and easily comprehended quantitative imaging study that analyzes the motion of endo-lysosomal compartments within axons in vivo using simultaneous multiphoton imaging in the mammalian brain. The simultaneous dual two-photon imaging is well-executed and represents a substantive advance in a field that relies heavily on in vitro neuronal culture preparations. This work opens the door to neurons that have aged appropriately and done so in the context of normal synaptic and neuromodulatory input, without an excess of added factors that occurs with in vitro cell culture. The authors solve an issue of cell polarity, providing strong support for their ability to determine directional movement (anterograde versus retrograde). In principle, this could become a generalized approach, opening this type of experiment up to other investigators. Finally, interesting differences in motion are observed, including activity-dependent and calcium-dependent changes that differ from measurements made in vitro. This is a significant technical advance with interesting observations that substantively move the field forward.
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Reviewer #2 (Public Review):
Here the authors used viral expression and two-photon imaging, a very demanding approach, to explore the transport dynamics of three membrane markers (Neuropeptide Y-dense core vesicles, LAMP1-endolysosomes and RAB7-late endosomes) in vivo in the mouse brain. This allowed deciphering for the first time anterograde and retrograde velocities in vivo rather than in cultured neurons. The authors showed that the different vesicular compartments have different anterograde and retrograde velocities, pausing at synapses. They further used brain slices to explore the effect of increased calcium levels.
Major strengths reside in the novelty of the approach (in vivo!).
The main weakness relates to the lack of novel mechanisms and the difficulty of using such a sophisticated setup on a routine basis.
This is a technical …
Reviewer #2 (Public Review):
Here the authors used viral expression and two-photon imaging, a very demanding approach, to explore the transport dynamics of three membrane markers (Neuropeptide Y-dense core vesicles, LAMP1-endolysosomes and RAB7-late endosomes) in vivo in the mouse brain. This allowed deciphering for the first time anterograde and retrograde velocities in vivo rather than in cultured neurons. The authors showed that the different vesicular compartments have different anterograde and retrograde velocities, pausing at synapses. They further used brain slices to explore the effect of increased calcium levels.
Major strengths reside in the novelty of the approach (in vivo!).
The main weakness relates to the lack of novel mechanisms and the difficulty of using such a sophisticated setup on a routine basis.
This is a technical 'tour-de-force', a clear reference article for future studies addressing vesicular transport in vivo.
Of course, one would be curious to see many more markers studied in this setup. Also, the same study in mouse mutants would be extremely interesting.
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Reviewer #3 (Public Review):
The authors use two-photon imaging to visualize various axonal organelle populations that they have virally labeled with fluorescent proteins, including DCVs and late endosomes/ lysosomes. The latter topic is a bit contentious, as the authors use two labels that tag potentially overlapping and not highly specific markers so that the nature of the tagged organelle populations remains unclear. Notably, the authors also have previously published a detailed account of how DCVs traffic in vivo, so the novelty is mostly in comparing the behavior of different organelles and the potential influence of activity.
Overall, the reported results mostly corroborate the expectations from previous in vitro and in vivo work on these organelles and other cargoes, performed by the authors and their collaborators, as well as in …
Reviewer #3 (Public Review):
The authors use two-photon imaging to visualize various axonal organelle populations that they have virally labeled with fluorescent proteins, including DCVs and late endosomes/ lysosomes. The latter topic is a bit contentious, as the authors use two labels that tag potentially overlapping and not highly specific markers so that the nature of the tagged organelle populations remains unclear. Notably, the authors also have previously published a detailed account of how DCVs traffic in vivo, so the novelty is mostly in comparing the behavior of different organelles and the potential influence of activity.
Overall, the reported results mostly corroborate the expectations from previous in vitro and in vivo work on these organelles and other cargoes, performed by the authors and their collaborators, as well as in many other laboratories:
(i) Different organelles have different transport behaviors regarding speed, the ratio of anterograde to retrograde moving organelles, etc.
(ii) Organelles move in different ways when they pass specific anatomical landmarks in the axons, such as presynaptic terminals.
(iii) Activity of a neuron (here measured by calcium imaging) can impact the measured transport parameters, albeit in a subtle and mechanistically not well-defined manner. The chosen experimental design precludes a more detailed analysis, for example of the precise movement behavior (such as defining the exact pausing/movement behavior of organelles, which would require higher imaging speeds) or of a correlation of different organellar behavior at synaptic sites or during activity (which would require three-channel simultaneous imaging of two organelle classes plus a synaptic or activity marker).In summary, this publication uses sophisticated in vivo labeling and imaging methods to corroborate and complement previous observations on how different axonal organelles move, and what influences their trafficking.
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