A major concern in the bulk implantation in the brainstem is the lateral spread of the tracer to the vestibular nerve root. In this case, the vestibular afferent fibers projecting to the cerebellum will be labeled from the tracer in the brainstem as well as from their respective tracer placed in the cerebellum, resulting in a false positive overlap. Since the cochlear nuclei lie lateral to and in the vicinity of the vestibular nerve root, all cochleas were examined to make sure that no retrogradely labeled cochlear afferents existed. Only cochlear efferents were labeled through the olivo-cochlear bundle from the brainstem. Preparations with labeled cochlear afferents were discarded from subsequent analysis.
Since this is the first report to use retrograde double labeling approach to study the peripheral distribution of the vestibular afferents, it is possible that the peripheral pattern could be due to difference in the physical properties of the two tracers employed. To control against this possibility, in one group, the same double labeling protocol was employed, except with reversed implantation sites. In this group, DiI was implanted in the cerebellum and Neuro Vue Maroon was implanted in the uvula and nodulus. A mirror-image reversal of colors would indicate a genuine anatomical pattern. Furthermore, a single DiI application in the brainstem in one group, and in the cerebellum in another, served as additional controls. At least 10 animals of successful tracer implantations and proper diffusions were used for analysis for each age group.
In nerve tracing experiments, the diffusion time is a crucial determinant of the outcome of the experiment. The optimal diffusion time for a given age group was determined based on a pilot experiment and our published observations (Maklad and Fritzsch 1999 , 2002 (link), 2003a (link)). However, to ensure that the distribution pattern of retrogradely labeled afferents from the brainstem and cerebellum does not change with different diffusion times, a group of P7 animals that received double implantation was examined after two different time points around our defined optimal diffusion times. In one subgroup, endorgans were examined after 3 weeks, a week earlier than our optimal diffusion time. A second subgroup was examined after 5 weeks (a week past the optimum). Furthermore, DiI and Neuro Vue have similar diffusion speeds. However, the primary vestibulo-cerebellar afferents travel a longer distance than those primary afferents projecting to the brainstem. To accommodate for the longer distance of the primary vestibulo-cerebellar afferents, in the double labeling experiment, Neuro-Vue Maroon was implanted 3 days ahead of DiI implantation in the brainstem. These 3 days of longer diffusion were determined from DiI single cerebellar implantation experiments.