Figure 1.
Cellular anatomy of the cerebellum in NBH- and 22L-animals.
Calbindin-positive (green) Purkinje cells and nuclear stain (blue) of the cell-rich granule cell layer contrasts with the cell-sparse molecular layer illustrating the laminar cerebellar anatomy with the well defined arborizations of Purkinje cells penetrating deeply into the superficial molecular layer of a NBH control animal (18 weeks p.i.) at increasing magnification (A, D, G, J). Note the almost continuous layer of Purkinje cell bodies. Scale bars: 500 µm (A); 250 µm (D); 100 µm (G); 75 µm (J). In 22L-animals at ES (12 weeks) p.i. Purkinje cells and their dendrites appear relatively normal with calbindin immunoreactivity (green) relatively uniformly distributed within the cell (B, E, H). However, when observed with a 60× objective (K), fragmentation of the dendritic arborizations, revealed by a discontinuous labelling pattern, becomes apparent. Note that there are some gaps in the layer of the Purkinje cell bodies suggesting the early stages of Purkinje cell loss. Scale bars: 500 µm (B); 250 µm (E); 100 µm (H); 75 µm (K). At LS (18 weeks) p.i. in 22L-animals calbindin immunoreactivity (green) reveals a severe dendritic pathology in the molecular layer of the cerebellum (C, F, I, L). Some calbindin-positive arborizations appear to be disconnected from their neuronal somata (nuclear stain, blue) (F, I). The Purkinje cell layer, which in NBH-animals shows close, regularly spaced somata, reveals areas completely devoid of calbindin labelling, indicating a loss of neurons. The loss of the large DAPI-stained cell nuclei indicates that this is a loss of the Purkinje cell rather than a loss of the calbindin staining. Numerous calbindin-positive Purkinje cell somata appear to lack any primary dendritic process (L). Scale bars: 500 µm (C); 250 µm (F); 100 µm (I); 75 µm (L).
Figure 2.
Immunofluorescence of cerebellar sections at increasing magnifications illustrates the relationship between calbindin-positive (green) Purkinje cell bodies, their dendrites and VGLUT1-positive (red) parallel fibres and synaptic terminals in the molecular layer, NBH-animals at 18 weeks p.i.
The calbindin staining (green) reveals the Purkinje cells and the arborisation of their major processes in the molecular layer. The blue panel is DAPI staining of the cell bodies and the fourth panel shows the overlay of the three colours. VGLUT1 immunoreactivity (red) was abundantly present throughout the molecular layer and also in the granular layer associated with the cell somas of the granule cells (nuclear stain blue) that give rise to the parallel fibres. Scale bars: 400 µm (A); 112 µm (B); 57 µm (C).
Figure 3.
Calbindin and VGLUT1 staining in cerebellum of early stage 22L-animals highlights dominant dendritic pathology in early disease.
Co-labelling of Purkinje cells using the anti-calbindin antibody (green) and parallel fibers (VGLUT1 antibody, red) at increasing magnifications from panel A to C in 22L-animals at ES p.i. The VGLUT1 immunoreactivity remains preserved in the areas of dendritic disintegration as revealed by discontinuous outlines of Purkinje dendrites (B, C). There is no significant loss of neurons at this time (nuclear stain, blue). Scale bars: 405 µm (A); 88 µm (B); 61 µm (C).
Figure 4.
Calbindin staining in cerebellum of late stage 22L-animals highlights progressive dendritic pathology but limited presynaptic degeneration in VGLUT1 reactive parallel fibre synapses.
Calbindin immunoreactivity (green) illustrates the severe loss of Purkinje cells dendrites in the molecular layer of the cerebellum and a reduction in Purkinje cell number (nuclear stain, blue) at LS p.i. in 22L-animals. VGLUT1-positive (red) parallel fibers and synaptic terminals are uniformly distributed throughout the molecular layer and do not exhibit any detectable alterations as revealed from images at increasing magnifications from A to C. Scale bars: 385 µM (A); 110 µm (B); 58 µm (C).
Figure 5.
Quantification of VGLUT1 immunoreactivity in the molecular layer of the cerebellum from NBH, ES and LS 22L-animals.
The analysis revealed no decrease in the quantified immunostaining consistent with observation that the presynaptic compartment is resistant to degeneration. In one-way analysis of variance with Tukey’s multiple comparison test (**P<0.01) a significant increase in the captured fluorescence was detected in 22L-animals at LS p.i. This increased staining is attributed to the pathology induced shrinkage of the molecular layer associated with disease progression.
Figure 6.
Electron micrographs of the stratum radiatum of the hippocampus and of the cerebellar molecular layer illustrating typical neuropathological hallmarks of prion disease.
(A): A typical spongiform vacuole (arrow) within the pyramidal cell layer of CA1 region at ES p.i. (B): Numerous vacuoles containing whorled membrane fragments (arrows) within the stratum radiatum at LS p.i. (C): A large spongiform vacuole containing secondary vacuoles within the stratum radiatum at LS p.i. with ongoing synaptic pathology adjacent. (D): Proximal part of the molecular layer in the cerebellum at ES p.i. containing spongiform vacuole (arrow) with normal appearing synaptic structures in the vicinity. (E): Swollen neuronal terminal in the molecular layer of the cerebellum containing vacuole-like structure with numerous whorled membrane fragments in a 22L-animal at LS p.i. (F): Bergmann glia with bundles of fibrils (arrow) in the molecular layer of the cerebellum from a 22L-animal at ES p.i. (G, H): Numerous glial cells (arrows) within the stratum radiatum of the hippocampus with ongoing synaptic pathology at LS p.i. (I): A detail of a double-membrane enclosed autophagic vacuoles within the stratum radiatum of the hippocampus at LS p.i. (J): An autophagic vacuole (arrow) in the molecular layer of the cerebellum at LS p.i. in a 22L-animal. Scale bars: 10 µm (A, B); 5 µm (C, G); 1 µm (D, E, H, I); 2 µm (F, J).
Figure 7.
Pathology of synaptic terminals in the stratum radiatum and cerebellum of 22L-animals.
(A–C): Electron micrographs illustrating numerous synaptic profiles in the stratum radiatum with inwardly curved post-synaptic membranes and electron-dense pre-synaptic terminals (arrows) at ES p.i. (D): A detail of the synaptic profile arrowed in A. (E): A degenerating synaptic profile with visible synaptic vesicles enclosed in a dark terminal at LS p.i. in stratum radiatum of a 22L-animal. (F): A synaptic profile (arrow) appears completely engulfed by highly curved PSD of a dendritic spine in the stratum radiatum at LS p.i., note a dystrophic neuronal terminal (asterisk) in proximity to the synaptic bouton. (G, H) Electron micrograph illustrating synapses in the stratum radiatum of NBH-animals at ES p.i., H: a detail of the panel G. Pre-synaptic terminals are filled with small round vesicles (<40 nm in diameter) and appose PSDs within an intact neuropil. (I, J): Type I synaptic junctions in the molecular layer of the cerebellum at ES p.i. (I) and at LS p.i. (J) in 22L-animals. Normal appearing pre-synaptic terminals without any hallmarks of pathological process filled with small vesicles appose structurally intact post-synaptic membranes on spiny branchlets of Purkinje cells; post-synaptic compartments of every bouton are closely enveloped by a process of one Bergmann glia cell that appears almost completely translucent (asterisks). J: A degenerating synaptic terminal filled with swollen vesicles and electron-dense cytoplasm (arrow). Scale bars: 2 µm (A, G); 1 µm (B, C, F, H–J); 0.5 µm (D); 0.2 µm (E).
Figure 8.
Dendritic pathology in the cerebellum of 22L-animals.
Electron micrographs illustrating cerebellar ultrastructure (A, B). (A): Ultrastructure of the granule cell layer containing numerous granule cell bodies on the right and Purkinje cell layer with several cell bodies (asterisks) on the left side, NBH-animal 18 weeks p.i. (B): Molecular layer containing parallel and climbing fibers projections contacting Purkinje cells dendrites in 22L-animals at ES p.i. (C): A disintegrating dendritic segment of a Purkinje cell within the superficial molecular layer at ES p.i. filled with various lysosomal-like profiles (arrows), synapses in the proximity (asterisks) appear unaltered (a detail of the dendrite is shown in panel D). (E): A detail of disintegrating dendritic segment at LS p.i. in the molecular layer, note numerous electron-dense, dark profiles and vacuoles with whorled membrane fragments in the dendritic cytoplasm, general hallmarks of autophagic process. (F): A dystrophic Purkinje cell dendritic segment with mitochondria clustered against the plasma membrane (arrows), synaptic terminals in the proximity appear normal, molecular layer at LS p.i. Scale bars: 5 µm (A); 10 µm (B); 2 µm (C–F).