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 based on the spectral properties of retrogradely transported dyelabeled vesicles. We inject overlapping regions of an axon target area with three or more different colored retrograde tracers. Based on 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. NPS combined with spectral (Brainbow) labeling of the input to autonomic ganglion cells show that the locations of ganglion cell projections to a mouse salivary gland relate to the identities of their preganglionic axonal innervation. We also show that NPS can delineate projections of many axons simultaneously in the mouse CNS.