Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes

Summary

We describe a simple and low cost technique for introducing high concentration of fluorescent and calcium-sensitive dyes into neurons or any neuronal tract using a polyethylene suction pipette.

Abstract

Retrograde labeling of neurons is a standard anatomical method^1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons^3-6. Generally, the dyes are applied as solid crystals or by local pressure injection using glass pipettes. However, this can result in dilution of the dye and reduced labeling intensity, particularly when several hours are required for dye diffusion. Here we demonstrate a simple and low-cost technique for introducing fluorescent and ion-sensitive dyes into neurons using a polyethylene suction pipette filled with the dye solution. This method offers a reliable way for maintaining a high concentration of the dye in contact with axons throughout the loading procedure.

Protocol

Fluorescent dextrans have been used as anatomical tools and for imaging neuronal activity^1-4. Fields et al., (2009)⁴ published a protocol for applying ion- and voltage-sensitive dyes to axonal tracts with a focus on the spinal cord as a model system. Here we describe a more detailed procedure for applying fluorescent and/or ion-sensitive dye to the cut ventral roots, dorsal roots or any neuronal tract of the spinal cord for in-vitro optophysiological and morphological studies.

1. Type-I and Type-II Pipettes

Start by pulling two short sections of polyethylene tubing (PE90, Cla....

Discussion

We describe here a simple and cost-effective protocol for introducing dyes into neurons, nerves and spinal tracts. This method involves exposing identified anatomical pathways to a highly concentrated dye solution for the duration of the loading process. This results in retrograde labeling of the target site with little background compared to microinjections, bath application and electroporation techniques. However, the method is limited to sites in the nervous system where a tract of axons can be isolated. Furthermore,.......