Fig. 4

Time dependent changes of cytosolic EU labelling. a–d Inverse grey scale confocal images (maximum projection of 9 images, 4-μm stacks) illustrating EU labelling in the somato-dendritic domain of Purkinje cells (b–d) and molecular layer interneurons (pink arrows in c′ and d′), and punctate labelling in the molecular layer (blue arrows in d′) presumably reflecting labelling in distal Purkinje cell dendrites. c″ and d″ are single confocal stack (1-μm optical section) of cells shown in c and d, respectively, illustrating cytoplasmic EU labelling in the perinuclear cytoplasm (yellow arrow). e–h Confocal images (1-μm optical section) illustrating co-localization of p62 immunoreactivity with cytoplasmic EU + particles (yellow arrows). Note, the prominent p62-immunoreativity in large particles in Purkinje cells after 4 days, and the difference in the size and frequency of cytoplasmic particles in 24 h Purkinje cells with intermediate (f) and high (g) EU labelling. Also note, single-labelled EU + (white arrows) and p62 + (arrow heads) particles (e–g). i 3D-reconstruction and confocal image of Purkinje cell after 4 days immunolabelled for p62 and LaminB1, illustrating the presence of EU + /p62 + bodies the within nuclear invaginations and in the peri-nuclear cytoplasm. j–l Triple staining for GABARAPL1 (GRL1), p62 and EU, showing the distribution of GABARAPL1 + autophagosomes in Purkinje cells after 4 days with no EU (j, d–n), intermediate (k, 4d–i) or high (l, 4d–h) EU levels. In EU-negative Purkinje cells p62 and GABARAPL1 generally co-localized (white arrows in j). In EU + cells GABARAPL1-immoreactivity was found in a subset of EU + /p62 + particles (cyan arrows in k), was absent in others (yellow arrows in k), and was consistently absent in large EU + /p62 + bodies (l). 0, 1.2 and 2.4 (in l) represent the z-position (in μm) of the individual images in the confocal stack. m, n Confocal images (maximum projection of 20 images, 9-μm stacks) and graph (n) illustrating the gradual disappearance of cytosolic EU + /p62 + bodies in Purkinje cells between 4 and 14 days. At 9 and 11 days, EU + /p62 + bodies were present in some Purkinje cells (yellow arrows), while absent in others, despite equal levels of nuclear EU labelling (white arrows); at 14 days all EU + /p62 + bodies had disappeared. Note, after 14 days, an example of a Purkinje cell filled with EU−/p62 + particles (arrow head). Values (in n) are derived from Purkinje cell counts in zones with high EU labelling and represent mean ± SE (n = 2 animals per time point). Scale bars: 5 μm (a, h, i); 10 μm (d′); 20 μm (m)