Figure 6. Primary and secondary vestibular mossy fiber morphology.

(A) Expression of a fluorescent protein in primary vestibular afferents (green). Note central location in the granule cell layer (GCL). ML- molecular layer. (B) Expression of a fluorescent protein in secondary vestibular mossy fibers (magenta). Note distribution of mossy fibers throughout the width of the GCL. (C–D) In a separate set of experiments Glt25d2::ChR2-EYFP mice had AAV1-CAG-ChR2-mCherry injected into MV, which allowed labeling of both primary (green) and secondary (magenta) mossy fiber projections in the same brain sections. Note the intermingling of primary and secondary fibers in the brainstem vestibular nuclei. Primary afferents only projected to rostral lobe IX (IXc), whereas secondary fibers innervated the entire ventral leaflet (IXb and IXc). Careful observation revealed few colabeled mossy fibers, suggesting possible but unlikely confound of virus infected primary fibers. (E–F) Higher magnification of primary and secondary vestibular afferents in the same section. Note the larger diameter axon of the primary afferent (green). The apparent overlap of the mossy fibers in these maximum intensity projections is due to the position of one above the other in the z dimension. (G) The mean diameter of primary afferents was significantly thicker than that of secondary afferents. Data points are individual mossy fiber axons. ANOVA, p=0.0001, post hoc t-tests, p<0.0001, n = 206 axons. (H) The diameter of primary and secondary afferents that contacted UBCs that were recorded from and recovered. Data points with black outline are UBCs that had ChR2 expressing mossy fiber making major contact with brush. Data points without outline had ChR2 expressing mossy fiber making small contact with brush and the ChR2-evoked response was small. t-test, p<0.0001, n = 20 axons.