Labeling second-order sensory neurons in the posterior lateral-line system of zebrafish

The lateral line is a mechanosensory system that comprises a set of discrete sense organs called neuromasts, which are arranged in reproducible patterns on the surface of fish and amphibians. The posterior component of the system, the posterior lateral line (PLL), comprises the neuromasts on the body and tail and has its ganglion just posterior to the otic vesicle. The peripheral location of the PLL system makes it accessible and easily visualized by imaging methods. Neuromasts are innervated by a few afferent neurons (usually two, but sometimes more), which have their cell bodies clustered in cranial ganglia and project their central axons to the hindbrain, where they extend longitudinally along all rhombomeres. Positively charged lipophilic carbocyanine dyes, such as DiI, have traditionally been used to label neurons, as described here. This method is especially useful for the analysis of PLL innervation because injection of the dye into a neuromast leads to specific labeling of the afferent neurons. The method can also be used to follow the flow of PLL information to higher central nervous system levels by first labeling the central projection of chosen afferent neurons and then making a second injection of DiI within the synaptic field to label the second-order neurons that extend dendrites to this field.