Immunocytochemical characterization of Lugaro cell transverse and sagittal axons in the molecular layer. Transverse (A–D) or sagittal (E) sections were double-stained with anti-calretinin antibody (green in A1, B1,C1) and anti-GAD65 (red inA2, B2) or anti-GlyT2 (redin C2) or with anti-GAD65 antibody (green in D1, E1) and anti-GlyT2 (red in D2,E2). A3, B3,C3, D3, and E3 are the superimposed images of A1 and A2,B1 and B2, C1 andC2, D1 and D2, andE1 and E2, respectively.A, Low magnification of a transverse section immunoreacted with calretinin (CR) and GAD65 is shown. Lugaro cell axons (arrowheads) run in the low molecular layer (low molec) of the cerebellar cortex, above the Purkinje cell bodies (P) that are surrounded by GABAergic terminals (arrows). Note the stronger background for the CR staining (asterisk) in the upper molecular layer. B, Varicosities of the calretinin-labeled transverse axons of the Lugaro cell show GAD65 immunoreactivity (arrowheads). Arrowsindicate GAD65-positive terminals devoid of calretinin.C, Transverse Lugaro cell axon stained for GlyT2 is shown. This is mainly visible in its varicosities (arrowheads). Thin fibers, probably parallel fibers, ascending in the molecular layer show faint calretinin immunoreactivity but are GlyT2 negative (arrows). D, GAD65 is also detected in GlyT2-immunoreactive axonal varicosities (arrowheads). In a few GlyT2-positive spots, GAD65 is not colocalized (double arrowheads). Because of their smaller diameter compared with double-stained varicosities, they most likely represent nonvaricose segments of Lugaro cell parasagittal axons cut transversally. E, Expression of GAD65 within varicosities stained for GlyT2 in the sagittal plane is shown. Note that in both transverse (D1) and sagittal (E1) sections, terminals positive for GAD65 only (arrows) outnumber varicosities coexpressing GABA and glycinergic markers (arrowheads), indicating the presence of numerous purely GABAergic boutons. Scale bars: A, 10 μm; B–E, 10 μm.