An evaluation of retrograde tracing methods for the identification of chemically distinct cochlear efferent neurons

We have compared retrograde labelling of rat olivocochlear neurons after unilateral cochlear injections of wheatgerm agglutinin conjugated horseradish peroxidase (WGA-HRP) and free HRP. After cochlear injection of WGA-HRP, labelling of nerve cell bodies in the brainstem can be explained not only as conventional retrograde labelling resulting from uptake by efferent nerve terminals synapsing on or near hair cells, but also as spurious labelling originating from tracer leakage, through the periotic duct and over the eighth nerve sheaths, into the cerebral-spinal fluid. Depending on the length of survival time, spurious labelling can involve small portions of the nucleus of the trapezoid body or the entire auditory brainstem and other non-auditory centers. On the contrary, moderate amounts of free HRP delivered to the cochlea do not lead to spurious labelling. With free HRP as the tracer of choice, we found that cochlear efferent cells were located not only in the ipsilateral LSO body and bilaterally within MVPO and RPO as already described by White and Warr, but also surrounding the ipsilateral LSO and in the ipsilateral LVPO. The allocation of these newly described olivocochlear neurons to the medial large cell or lateral small cell system is uncertain because they are located laterally in the brainstem and project ipsilaterally but are large spherical to fusiform or multipolar cells. A zinc salicylate-formol fixative and a metal intensified DAB reaction were found to be effective in visualizing retrogradely transported HRP in neurons and allowed immunocytochemical staining of the same sections with antisera to glutamic acid decarboxylase and choline acetyltransferase. This double label protocol can be used to produce a neurochemical map of the OC systems.