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Multispectral labeling technique to map many neighboring axonal projections in the same tissue

Abstract

We describe a method to map the location of axonal arbors of many individual neurons simultaneously via the spectral properties of retrogradely transported dye-labeled vesicles. We inject overlapping regions of an axon target area with three or more different colored retrograde tracers. On the basis of the combinations and intensities of the colors in the individual vesicles transported to neuronal somata, we calculate the projection sites of each neuron's axon. This neuronal positioning system (NPS) enables mapping of many axons in a simple automated way. In our experiments, NPS combined with spectral (Brainbow) labeling of the input to autonomic ganglion cells showed that the locations of ganglion cell projections to a mouse salivary gland related to the identities of their preganglionic axonal innervation. NPS could also delineate projections of many axons simultaneously in the mouse central nervous system.

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Figure 1: Colors of retrogradely transported vesicles reflect a neuron's axonal projection.
Figure 2: The intensity of retrogradely labeled vesicles is a linear measure of the label intensity at axonal uptake sites.
Figure 3: A neural positioning system (NPS) for mapping axonal projections.
Figure 4: Ganglion cells innervated by the same preganglionic axon project to nearby regions in the gland.
Figure 5: NPS mapping of the thalamic projection to the barrel cortex.

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Acknowledgements

Support is gratefully acknowledged from the Humans Frontiers Science Foundation (S.T.), European Research Council (ERC) under the European Union's Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement no. 260914 (S.T., S.G. and A.M.B.), US National Institute of Mental Health Silvio Conte Center 1P50MH094271 (J.W.L.) and US National Institutes of Health grant NS076467 (J.W.L.). We thank R. Kafri for helping generating the Matlab code.

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Authors and Affiliations

Authors

Contributions

S.T. conceived of the NPS strategy, designed and conducted the experiments, analyzed the data and wrote the manuscript. S.G. conducted the experiments, analyzed the data and wrote the manuscript. R.W.D. created Mem-Brainbow mice and wrote the manuscript. A.M.B. designed the experiments, analyzed the data, supervised the project and wrote the manuscript. J.W.L. conceived of the study, designed the experiments, analyzed the data, supervised the project and wrote the manuscript.

Corresponding authors

Correspondence to Alexander M Binshtok or Jeff W Lichtman.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Preganglionic input, rather than ganglion cell body location, correlates with axonal arbor position

(a) The average distance between the axonal projections in the submandibular gland of pairs of ganglion cells that are innervated by the same preganglionic axon (red column) compared to pairs of neurons that are innervated by different inputs (white column). The difference is significant; P<0.001. (b) However, within the ganglion cell cluster, the average distance between the pairs of somata themselves is roughly the same for the neurons that share the same input (red column) versus those that don’t (white column). (c) Moreover when we looked at each of 515 pairs of neurons individually we found little correlation between the distances between somata and distances between axon projections pairs (r = 0.0092). Although pairs of ganglion cells that share common input (red circles) did tend to be closer to each other in the gland. (d) The separation of arbors within the gland is also affected by the degree to which two ganglion cells share the same axonal input. In cases where one ganglion cell of the pair is innervated by more than one preganglionic axon (gray column) the arbors are significantly more distant than ganglion cells pairs that have identical input (red column) but significantly closer than pairs that do not share any preganglionic input (white column). Data combined from P32 and P18 ganglia. Student t test; * = P<0.05; ** = P <0.01; *** = P <0.001; # = P >0.05.

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Supplementary Figure 1 and Supplementary Software (PDF 264 kb)

Three dimensional rendering of a confocal stack showing the location of all the vesicles in a ganglion cell

Shown are red and green vesicles following two injections to near but non-overlapping parts of the gland. Note that red and green vesicles are common but by rotation it can be seen that there are no yellow vesicles that share both colors. (AVI 46572 kb)

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Tsuriel, S., Gudes, S., Draft, R. et al. Multispectral labeling technique to map many neighboring axonal projections in the same tissue. Nat Methods 12, 547–552 (2015). https://doi.org/10.1038/nmeth.3367

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