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. 2021 Apr 1;131(7):e140625. doi: 10.1172/JCI140625

Figure 3. Cerebellar abnormalities in RIMBP1-KO mice.

Figure 3

(A) Gross anatomy of the cerebellum. Left: Dorsal view of RIMBP1-WT and RIMBP1-KO brains. Middle: Confocal image of a parasagittal section stained with DAPI and anti-calbindin antibodies (green). Right: Summary plot of cerebellar volume (left) and of Purkinje cell (PC) number (right). Scale bars: 0.8 cm (left) and 20 μm (right). (B) Structure of PC dendrites in RIMBP1 control and mutant mice. Left: Low-magnification images of parasagittal sections stained with anti-calbindin antibodies. Middle: High-magnification images (single optical sections) of the areas indicated with the white boxes on the left displaying outer (0%–40%) and inner (40%–80%) PC dendritic domains. Right, top: Summary graphs of calbindin coverage (area) in different PC dendritic domains. Right, bottom: Summary graphs of calbindin intensity signal in the same PC dendritic domains. Scale bars: 1 mm (left) and 40 μm (right). (C) Organization of parallel fiber synapses. Left: Representative confocal images of cerebellar sections from a RIMBP1-WT mouse. Middle: Representative images from a littermate RIMBP1-KO mouse. Sections were stained for calbindin (green, to label PCs), for vGluT1 (red, to visualize parallel fibers), and with DAPI (blue, cell nuclei). Right: Summary graphs of vGluT1 intensity for the outermost and innermost PC dendritic domains in RIMBP1-WT mice and -KO littermates. Scale bar: 40 μm. (D and E) Same as in C but for climbing fiber synapses stained with anti-vGluT2 antibodies. In this case, the density (D, right) and the size (E) of vGluT2-containing puncta were compared. Scale bar: 40 μm. (F) Levels of key proteins in cerebellar lysates. Left: Representative blots. Right: Summary plots of band intensity values normalized to Tuj1. Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 by Student’s t test. NS, not significant. Number of experiments (mice/sections): (A) Left: 5 WT, 6 KO; Right: 6/62 WT, 6/47 KO; (B) Intensity: 6/59 WT, 6/64 KO; Area: 6/57 WT, 6/60 KO; (C) vGluT1: 6/59 WT, 6/56 KO; (D and E) vGluT2: 6/54 WT, 6/60 KO; (F) 10 RIMBP+/+, 10 RIMBP–/–.