Fig 1.
No apparent gross abnormality in the cerebellar lobules or layers, or in the densely-packed granule cell layer in Nissl-stained sections of v-KIND KO cerebella.
Sagittal sections of KO and WT mice cerebella (8-week-old) were analyzed by Nissl staining. (A) The lobular structure of the whole cerebellum. Scale bar, 1 mm. (B) The layer structure of the cerebellar cortex. Scale bar, 100 μm. (C) Magnified view of the granular layer indicated by the red square in (B). Cerebellar lobules II–X. ML, molecular layer; PCL, Purkinje cell layer; GL, granular layer; WM, white matter. (D) Number of Nissl-stained puncta/1,000 μm2 in the granule cell layer of WT and KO mice. 5~10 random areas per section, 2 sections per animal, and three mice for each genotype (N = 3) was statistically analyzed. Data are shown as mean ± SEM. Two sample Student’s t-test assuming equal variances showed no statistical significance (p > 0.5).
Fig 2.
Increased branches and terminals of cerebellar granule cell dendrites in v-KIND KO cerebella.
(A) Branched arborization pattern of granule cell dendrites in the granular layer of WT (left) and v-KIND KO (right) mouse cerebella at 8 weeks of age. Granule cells were visualized by DiI staining, and images were obtained by confocal microscopy. Scale bar, 10 μm. (B) Statistical analysis of the number of dendrites per cell (top left), number of branches per dendrite (top right), number of terminals per cell (bottom left) and average length of dendrites (bottom right) of cerebellar granule cells by Student’s t-test. Cells from four animals were analyzed for each genotype (N = 4) (WT: n = 13 cells; KO: n = 12 cells). Data are shown as mean ± SEM. Two sample Student’s t-test assuming equal variances; *p < 0.05, ***p < 0.001.
Fig 3.
Expression of presynaptic markers for mossy fibers and Golgi cell axons in cerebellar glomeruli of v-KIND KO mice.
(A) Immunohistochemistry of the presynaptic GAD65/67 protein (red) in Golgi cells and the presynaptic vGluT2 (green) in mossy fibers in the granule cell layers of WT and KO mice at 5 weeks of age. Panels a1 and b1 show representative images of co-immunostaining patterns in lobules IV–V of WT and KO mice, respectively. Panels a2 and b2 are magnified images of the cerebellar layer indicated by the white square in a1 and b1, respectively. Panels a3 and b3 are magnified images of the granular layer indicated by the red square in a2 and b2, respectively. Bars: 100 μm in a1 and b1, 10 μm in a2, a3, b2 and b3. Images of vGluT2 immunostaining of whole cerebellar sections are shown in S1 Fig. (B) Number of GAD65/67-immunopositive puncta (/1,000 μm2) was calculated by analyzing data from fifteen areas (7,182 μm2/area) (n = 15) of three different animals (N = 3) for each genotype. (C) Number of vGluT2-immunopositive puncta (/1,000 μm2) was calculated by analyzing 26 and 24 areas (2,500 μm2/area) in WT (n = 26) and KO (n = 24) mice, respectively, from five different animals (N = 5) for each genotype. Data are shown as mean ± SEM. Two sample Student’s t-test assuming equal variances (GAD65/67 N = 3; vGluT2 N = 5); ** p < 0.01 in (C).
Fig 4.
Increased number of postsynaptic densities in mossy fiber–granule cell synapses in v-KIND KO cerebellum.
(A) Representative electron microscopic images of cerebellar glomeruli in WT (left) and v-KIND KO (right) mice (8-week-old). Arrow heads indicate excitatory synapses between mossy fiber terminals (MFTs) and dendrites of cerebellar granule cells. High power views of each synaptic structure indicated by arrow heads are shown below. Scale bar, 1 μm. (B) Structural analysis of granule cell–mossy fiber synapses in the glomerulus. Left, the number of granule cell postsynaptic densities (PSDs) per MFT was increased in the KO compared with WT. Right, the perimeter of the MFT was not different between KO and WT animals. MFTs in two different electron microscopic images from 3 independent mice (N = 3) for each genotype were analyzed (number of MFTs analyzed: WT, n = 50; KO, n = 53). Data are shown as mean ± SEM. Two sample Student’s t-test assuming equal variances; **p < 0.01.
Fig 5.
v-KIND KO mice show increased grip strength but no difference in locomotion or wire hanging ability compared to their WT littermates.
Male mice (8–12 weeks of age) were tested. (A) Home cage activity (indicated by the number of crossings of the beam per 15 min) during the dark period for 6 days. WT: n = 9; KO: n = 8. There was no statistically significant difference between the two genotypes under dark (Fig 5A) or light (data not shown) conditions. (B) Distance (cm) traveled in an open field, illuminated at 50 lux, in 3 5-min bins. WT: n = 10; KO: n = 10. (C) Number of rearings in a period of 5 min. WT: n = 9; KO: n = 8. (D) Grip strength (in Newton [N]). WT: n = 6; KO: n = 10. (E) Latency to fall (s) in three trials of wire hanging is shown. WT: n = 6; KO: n = 10. Data are shown as mean ± SEM. Two sample Student’s t-test assuming equal variances; **p < 0.01 in (D).
Fig 6.
v-KIND KO mice have a tendency to display better motor performance than WT mice.
Balance beam test of WT and v-KIND KO mice. Left, number of slips while the mouse remained on the 9-mm or 6-mm beam. Right, time (s) the mouse crossed the 9-mm or 6-mm beam. Data are shown as mean ± SEM (WT: n = 6; KO: n = 10). *p < 0.05, **p < 0.01 (two-tailed, unequal variances Student’s t-test).