Figure 5.

Immunolocalization of granule cell ascending axon contacts with Golgi cells in the GL. Glutamatergic varicosities identified by immunostaining of the vesicular glutamate transporters vGluT1 (blue channel) and vGluT2 (red channel) in parasagittal sections obtained from GlyT2-EGFP mice (green channel: Golgi cell somata and processes). A, Large field of view (92 × 123 μm, projection depth 2.38 μm) showing the general organization of terminals in the cerebellar cortex. The GL is occupied by large mossy fiber rosettes displaying various ratios of vGluT1 and vGluT2 staining. There is absence of vGluT1-only rosettes in this region. vGluT1-only parallel fiber boutons are seen in the molecular layer (top left) together with rare vGluT2-only climbing fiber terminals. B1, B2, Enlargement of the region B, boxed in A, showing small vGluT1-only varicose profiles in the GL and their apposition to the cell body and dendrites of a GFP-positive GoC (projection depth 1.36 μm). vGluT1-only profiles most likely correspond to the boutons of GrC aa in the GL. In B2, the GFP channel was removed for better display of vGluT1-only profiles, which show weaker staining than the large mf rosettes. C1, C2, Same as B1, B2 for the region C (projection depth 1.36 μm), illustrating the dense innervation of the GFP-positive GoC neurites by vGluT1-only varicosities in the upper part of the GL. D, Relative intensity of vGluT1 and vGluT2 staining in punctuate profiles detected in the blue channel (vGluT1) in region C, both in mossy fiber rosettes and outside glomeruli. The vGluT2 intensity histogram (black curve) is bimodal, with a first population (nonglomerular puncta) showing background levels of immunoreactivity (vGluT1-only, blue dots). In contrast, glomerular puncta (red points) display fixed ratios of vGluT1 and vGluT2 staining, as evidenced by the linear correlation in their distribution. E, vGluT1-only puncta are displayed as blue dots on top of segmented GFP-positive GoC neurites, for the region displayed in C; distance of blue dots from the nearest GoC neurite is shown in F. By comparison with C2, all selected puncta in the GL are indeed extraglomerular. F, Blue curve represents the distribution of the distance of vGluT1-only puncta from the surface of GoC dendrites in the GL of the stack shown in A. Black curve represents randomized distance distribution obtained by rotating the GFP channel by 180°. G, Distance distribution (in blue) and randomized distance distribution (in black) for all the regions analyzed (6 confocal stacks, 2 animals). Gray curve represents the scaled randomized distance histogram accounting for the randomly located puncta in the blue distribution (see Results). The difference between the gray and blue curves is an estimate of the number of vGluT1-only puncta truly apposed to GFP-positive GoCs.