Neuroanatomical technique for studying long axonal projections in the central nervous system: combined axonal staining and pre-labeling in parasagittal gerbil brain slices

A method is described for studying the morphological features of extensive axonal projections within the central nervous system of the gerbil, Meriones anguiculatus. Potentially long descending axonal projections between the auditory thalamus and lower brainstem were used as a model. The inferior colliculus (IC) in the tectum was injected in vivo with a fluorescent retrograde tracer, Fluoro-Gold, to label cells in the medial geniculate body (MGB) that had descending projections to the IC, and cells in the superior olivary complex (SOC) that had ascending projections to the IC. Another fluorescent retrograde tracer, fast blue, was injected into the cochlea to label olivocochlear (OC) cells in the SOC. Inferomedially curved parasagittal slices containing ipsilateral auditory cell groups from the thalamus to the brainstem were cut and descending axons of the pre-labeled MGB cells were traced anterogradely with Biocytin. After visualizing histologically the injected Biocytin, discretely labeled IC-projecting axons of the MGB cells were traced including their collaterals that extended further into the SOC. In the SOC, these axons terminated on pre-labeled cells including OC cells. The combination of anterograde and retrograde tracing in the slice preparations described here demonstrated extensive descending axonal projections from the thalamus to their targets in the lower brainstem that had known ascending/descending projections within the auditory system.