Article Figures & Data
Figures
- Figure 1.
Surgical transection of PFs. A, Illustration of the dorsal cerebellar view and slanted double incisions. B, C, Immunofluorescence for calbindin (green) and VGluT1 (red) in a coronal cerebellar section 7 d after PF transection. Both fluorescent signals are disrupted at the double incisions (arrows). Cerebellar islands between double incisions were used for analysis. C, An enlarged image of the boxed region in B. D, E, Injection of BDA into cerebellar islands or the corresponding cerebellar portions (yellow dots) in sham-operated control (D) and PF-transected (E) mice. Note that tracts of BDA-labeled PFs do not extend beyond the incision in PF-transected mice (E). Scale bars: B, D, E, 500 μm; C, 100 μm.
- Figure 2.
Changes in PFs and PF synapses after surgical transection. A–H, Electron micrographs of the superficial molecular layer in sham-operated control (A, E) and PF-transected mice on postlesion days 1 (B, F), 7 (C, G), and 30 (D, H) in wild-type (A–D) and GluD2-KO (E–H) genotypes. PF axons and terminals are pseudocolored in red and orange, respectively, whereas PC spines in contact with PF terminals or lacking synaptic contact (ie, free spines) are in blue or green, respectively. Pairs of arrowheads indicate the PSD. I–L, Bar graphs showing the density of PF axons per square micrometer of the molecular layer (I), the percentage of PC spines in contact with PF terminals (J), the size (short diameter) of PF terminals (K), and the contact ratio of PC spines per PF terminal (L; mean ± SD, n = 3 mice for each). Left and right bars represent scores in wild-type and GluD2-KO mice, respectively. Differences between sham-operated control and PF-transected mice in each genotype were assessed using ANOVA with Dunnett's test. **p < 0.01, *p < 0.05 (actual p value is indicated in the parenthesis). Scale bars, 500 nm.
- Figure 3.
Sprouting and elongation of PC collaterals. A–F, Phase-contrast light microscopic images of BDA-labeled PF trajectory in sham-operated control (A, D) and PF-transected mice on postlesion days 7 (B, E) and 30 (C, F) in wild-type (A–C) and GluD2-KO (D–F) genotypes. Arrowheads indicate varicosities or boutons along BDA-labeled PFs, while arrows indicate the branching point of PF collaterals. C, Inset, A different focal plane of the boxed region in the center, showing numerous varicosities along a long collateral of a BDA-labeled PF. G, Bar graphs showing the density of PF varicosities per mm of BDA-labeled PF (mean ± SD, n = 3 mice for each). No significant difference in density after PF lesioning was found in either wild-type (left) or GluD2-KO (right) mice. The density of PF varicosities, at ∼160/mm, is consistent with a previous study in the cat (Brand et al., 1976). Scale bars, 10 μm.
- Figure 4.
Changes in molecular layer thickness and CF reach after PF transection. A–H, Double-immunofluorescence for calbindin (green) and VGluT2 (red) in sham-operated control (A, E), and PF-transected mice on postlesion days 1 (B, F), 7 (C, G), and 30 (D, H) in wild-type (A–D) and GluD2-KO (E–H) genotypes. Arrowheads indicate the distal tips of VGluT2-labeled CF terminals. I–K, Bar graphs showing the vertical height to the tips of calbindin-positive PC dendrites (ie, molecular layer thickness; I) or to the tips of VGluT2-labeled CF terminals (ie, CF reach; J), and the CF reach relative to molecular layer thickness (K; mean ± SD, n = 3 mice for each). Differences between sham-operated control and PF-transected mice in each genotype were assessed using ANOVA with Dunnett's test, while those between wild-type and GluD2-KO mice at each time point were assessed using Student's t test. **p < 0.01, *p < 0.05 (actual p values are indicated in the parenthesis). Scale bars, 25 μm.
- Figure 5.
Ultrastructural reconstruction of dendritic innervation. A–D, Electron micrographs showing CF synapse labeled for VGluT2 by immunoperoxidase (A), PF synapse innervated by transverse axons on the transverse plane (B), free spines (fSP) and degenerating PF synapse 1 d after PF transection (C), and inhibitory synapse on PC spine labeled for VIAAT by silver-enhanced immunogold 1 d after PF transection (D). Compared with the well developed PSD in CF and PF synapses (pairs of arrowheads in A, B), the PSD in free spines is small and thin (pairs of arrowheads in C). E–L, Schematic illustrations showing the location of three kinds of spine-type synapses (PF synapse, red; CF synapse, yellow; inhibitory synapse, green) and free spines (pink) along reconstructed single dendritic tracts in sham-operated control (E, F) and PF-transected mice on postlesion days 1 (G, H), 7 (I, J), and 30 (K, L) in wild-type (E, G, I, K) and GluD2-KO (F, H, J, L) genotypes. We also illustrate the three distinct dendritic domains in different colors: PCD-I, blue; PCD-II, cyan; PCD-III, green. Scale bars, 500 nm.
- Figure 6.
Electron micrographs and 3D reconstructed images of PF synapses on distal dendrites in sham-operated control (A, B, I, J) and PF-transected mice on postlesion days 1 (C, D, K, L), 7 (E, F, M, N) and 30 (G, H, O, P) in wild-type (A–H) and GluD2-KO (I–P) genotypes. In electron micrographs, PC dendrites/spines and PF axons/terminals are pseudocolored in green and orange, respectively. Insets, Enlarged and nonpseudocolored images of PF–PC synapses in boxed regions of pseudocolored images. Pairs of arrowheads and asterisks indicate the PSD and free spines, respectively. Note the emergence of free spines at distal dendrites on postlesion day 1 in wild-type mice (C, D) and their very frequent occurrence on all postlesion days in GluD2-KO mice (K–P). Also, note the hypertrophic changes in PF–PC synapses on postlesion day 7 in wild-type mice (E, F). Scale bars, 500 nm.
- Figure 7.
Electron micrographs and 3D reconstructed images of CF synapses on proximal dendrites in sham-operated control (A, B, I, J) and PF-transected mice on postlesion days 1 (C, D, K, L), 7 (E, F, M, N), and 30 (G, H, O, P) in wild-type (A–H) and GluD2-KO (I–P) genotypes. In electron micrographs, PC dendrites/spines are pseudocolored in green, whereas CFs are labeled by dark precipitates for VGluT2. Insets, Enlarged images of PF–PC synapses in boxed regions. Pairs of arrowheads and asterisks indicate the PSD and free spines, respectively. Note the emergence of free spines at proximal dendrites on postlesion day 1 in wild-type mice (C, D) and their frequent occurrence on all postlesion days in GluD2-KO mice (K–P). Scale bars, 500 nm.
- Figure 8.
Changes after PF transection in the path length of innervation territories and in the number of PF synapses, CF synapses, free spines, and spine-type GABAergic synapses. A, The path length (μm) of whole single dendritic tracts and of PCD-I (blue), PCD-II (cyan), and PCD-III (green). B, The path length of the PF territory (ie, PCD-II + PCD-III). C, PF synapse number in the PF territory. D, The path length of the CF territory (ie, PCD-I + PCD-II). E, CF synapse number in the CF territory. F, The path length of dendrites studded with free spines. G, Free spine number. H, Spine-type GABAergic synapse number. Left and right bars represent scores in wild-type and GluD2-KO mice, respectively. All scores were measured from three mice at each time point for each analysis, and expressed as the mean ± SD. Differences between sham-operated control and PF-transected mice in each genotype were assessed using ANOVA with Dunnett's test. **p < 0.01, *p < 0.05 (actual p values are indicated in the parenthesis).
- Figure 9.
Summary schematic illustrating the three distinct phases of regenerative PF wiring in wild-type mice (top) and the arrested regeneration in GluD2-KO mice (bottom). Three dendritic domains are colored in blue for PCD-I, cyan for PCD-II, and green for PCD-III. In wild-type mice, PF axons and synapses massively degenerate after PF transection. In this degenerative phase, numerous free spines emerge and the PF territory expands aberrantly. Then, PF synapses enlarge, and there is an increase in the contact ratio with PC spines (to 1:2) in the hypertrophic phase. In this phase, the number and density of PF synapses nearly recover to normal levels, and free spines nearly completely disappear, but PF density remains low. In the remodeling phase, PF density increases, at least partly through sprouting and elongation of PF collaterals, and PF synapses reacquire a compact structure and a 1:1 contact ratio with PC spines. This suggests that competitive synaptic wiring by PFs and CFs onto PCs is regained in wild-type mice. In contrast, in the GluD2-KO mice, the degenerative phase persists, without a shift to the hypertrophic or remodeling phases of PF–PC regenerative rewiring.
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