Immunohistochemical characterization of axon terminals of the adult rat cerebellar cortex

The study was carried out on adult rat using immunohistochemical techiques for demonstration of VGLUT1 and VGLUT2 and their co-localization with synaptophysin. In the molecular layer (ML): VGLUT1 terminals were distributed throughout the layer with a pattern resembling that of the parallel fiber terminals; VGLUT2 terminals were displaced along distinct spiral lines extending from deep to superficial layer, likely corresponding to terminals of climbing fibers. No colocalization of VGLUT1 and VGLUT2 was observed; on the contrary, terminals co-localizing GLUT1 and synaptophysin and VGLUT2 and synaptophysin, respectively, were detected, according to terminal nature of the immunolabelled elements. In the granular layer (GL): VGLUT1 and VGLUT2 terminals displayed similar distribution pattern, being clustered in restricted regions of the layer among granules (glomeruli). Most of them showed colocalization of VGLUT1 and VGLUT2, some showing positivity for VGLUT2 and negativity for VGLUT1, and all showing positivity for synaptophysin. Finally, some terminals were synaptophysin positive, but VGLUT1 and VGLUT2 negative. The results indicate that the glutamatergic terminals in the cerebellar cortex may be differentiated by combining immunohistochemistry for VGLUT1 and VGLUT2. Moreover, they identify different subpopulations of terminals of parallel, climbing and mossy fibers. In particular, a subpopulation of mossy fiber terminals, displaying positivity for VGLUT2 and negativity for VGLUT1, differently from the vast majority of mossy fiber terminals, displaying positivity for both VGLUT1 and VGLUT2, but similarily to climbing fiber terminals. It is intriguing to hypothesize that these mossy fibers may constitute a contingent of mossy fiber originated in the inferior olivary nuclear complex.