GluRδ2 promotes an increase in PF inputs on the PC proximal dendrite of δ2/GFP-ho mice.

<p>(A–F) Immunostaining of PF innervations (blue) on the PC proximal domain of δ2/GFP-ho mice (A–D) and GFP-ho mice (E–F). (A) In the δ2/GFP-ho group, numerous spines (arrowheads) bearing GluRδ2 (red, B) appear in the proximal domain, and the PF contacts, labeled with VGluT1 antibody (blue, C and D), are more numerous relative to GFP-ho mice (E–F). The overlap between GluRδ2 and the PF synaptic terminals appears as fuchsia (D). (G) Histogram shows the mean percentage of spines overlapping with VGluT1. A significant increase is observed in the δ2/GFP-ho mice relative to the GFP-ho and δ2/GFP-ho CTR groups and also to GFP-wt mice. These results show that in presence of GluRδ2, indicated as the percentage of spines expressing GluRδ2 (black column), the PF input has a competitive advantage. ***p<0.001. Error bars indicate SE. Scale bar: A–F = 2 µm.</p

Difference in the distribution of PF contacts along the ascending domain of Golgi cell expressing GluRδ2.

<p>(A–D) Immunostaining of the ascending dendritic tract of a Golgi cell (green, A) characterized by differential localization of GluRδ2 (arrowheads) (red, B) and relative VGluT1 (blue, C) signals (D, merge). GluRδ2 expression gradually increases in the proximal domain (gl) at the level of the PC layer (pl), reaching high levels in the distal tract (ml). Although the expression of GluRδ2 is less prominent in the proximal domain, the area that is in contact with the PF inputs is significantly increased relative to both the control groups and the negative control. (E) Histogram shows the mean percentage of the GFP area that colocalizes with GluRδ2 in Golgi cell dendrites of GFP/δ2 mice. A significant reduction of GluRδ2 expression in the proximal tract is observed. (F) Histogram shows the mean percentage of the GFP area that colocalizes with VGluT1 in Golgi cells of GFP-wt, GFP-ho, and δ2/GFP-ho mice. The white columns represent the value obtained in the distal dendritic domain; the light gray columns are the value in the proximal dendritic tract; and the dark gray columns are the negative control value of colocalized GFP-VGluT1 in the rosette. The ectopic expression of GluRδ2 induces a significant increase in PF contacts in both layers. * p<0.05; ***p<0.001. Error bars indicate SE. Scale bar A–D: 10 µm.</p

GluRδ2 promotes formation of PF contacts in the PC distal domain of δ2/GFP-ho mice.

<p>(A–D) Immunostaining of PF innervations on PC distal dendrites of δ2/GFP-ho mice (A–B) and GFP-ho mice (C–D). GFP spines bearing GluRδ2 (red, A) are contacted by PF terminals labeled by VGluT1 antibody (blue) (B). (E–F) Histograms show the mean density of spines emerging from the distal dendritic domain and the percentage of spines contacted by the PFs in this compartment. (E) The mean spine density does not change between the experimental groups (p = 0.096). (F) The mean percentage of spines overlapping with VGluT1 is increased in δ2/GFP-ho mice relative to control ho groups (GFP-ho and δ2/GFP-ho CTR), while there is no significant difference between δ2/GFP-ho mice and the GFP-wt group. In the presence of GluRδ2, indicated as the percentage of spines expressing GluRδ2 (black column), the number of PF contacts reaches that of wild-type mice. *** p<0.001. Error bars indicate SE. Scale bars:  = 2 µm.</p

GluRδ2 induces spinogenesis in the PC proximal dendritic compartment of δ2/GFP-ho mice.

<p>(A–D) Immunostaining of PC proximal dendrites in δ2/GFP-ho (A–C) and GFP-ho mice (D). In δ2/GFP-ho mice, many new spines, expressing the GluRδ2 subunit (red) (B and C), appears in the proximal dendrite relative to GFP-ho mice (D). (E) Histogram shows the mean spine density in the proximal dendritic domain. In the presence of GluRδ2, the number of spines significantly increases relative to control groups (GFP-wt; GFP-ho and δ2/GFP-ho CTR). *** p<0.001. Error bars indicate SE. Scale bars: A–E = 2 µm.</p

GluRδ2 expressed by HEK293 promotes formation and differentiation of GC axonal contacts.

<p>(A–F) Merge of light microphotographs of GCs in coculture with fluorescent 293 cells expressing GFP and GluRδ2 (in red) (A) or GFP alone (E). The corresponding immunofluorescence images are magnified as a single optical section in B–F. The GluRδ2 labeling around the 293 cell perimeter is shown in (C). GluRδ2 expression induces an increase in synaptic contacts, as indicated by the corresponding VGluT1 labeling (B–D). No contacts are visible around the perimeter of the 293-GFP cells; the blue labeling indicates synaptic contacts on a GC cluster (F). (G) EM quantitative analysis of the GC axonal contacts on the 293 cell perimeter. The 293-GluRδ2 cells (black columns) are in contact with a higher number of GC round terminals relative to the control (white columns); in both groups, most of the round terminals contained vesicles. In the 293-GluRδ2 cells, more terminals with vesicles oriented toward the postsynaptic membrane were observed. (H–I) EM images of differentiation of the presynaptic GC terminals induced by 293-GluRδ2 cells. (H) Contact between 293-GFP cell and a round GC terminal containing homogeneously distributed vesicles. (I) A 293-GluRδ2 cell contacted by round GC terminal containing oriented vesicles; the arrow indicates the vesicle cluster. Scale bars: A and E = 20 µm. B-C-D-F = 10 µm. H and I = 0.25 µm. *** p<0.001; ** p<0.01.</p

GluRδ2 increases PF contacts on Golgi cell dendrites of δ2/GFP-ho mice.

<p>(A–C and J–K) Immunostaining of transfected Golgi cells in δ2/GFP-ho mice (A–C) and in control GFP-ho mice (J–K). The cell bodies (A, J) are in the granular layer (gl), and the ascending dendrites also are visible in the molecular layer (ml). In δ2/GFP-ho mice, GluRδ2 is ectopically expressed in the Golgi dendrites (red, B). (D–I and L–N) High magnification of two Golgi cell dendrites in the molecular layer of δ2/GFP-ho and GFP-ho mice, respectively. (D–I) In a Golgi cell expressing GluRδ2 (in red, E–G–H), the dendritic area that is in contact with the PF inputs is higher (blue, F–H–I) (arrowheads) relative to that (M–N) of the Golgi cell (L) in GFP-ho mice. Scale bars: A–E = 20 µm. F–N = 2 µm.</p

Morphological analysis of CF varicosities in hotfoot and wild type mice

<p>Mean values of major axis length (MA), minor axis length (ma), and ratio (MA/ma); one- way ANOVA; * post-hoc Holm-Sidack test, p<0.05.</p

GluRδ2 causes a reduction of CF inputs on the PC proximal dendrite in δ2/GFP-ho mice.

<p>(A–D) Immunostaining of CF varicosities (blue) on the PC proximal domain of δ2/GFP-ho mice (A–B) and GFP-ho mice (C–D). (A–B) In δ2/GFP-ho mice, numerous spines bearing GluRδ2 (red, A) appear in the proximal domain. The number of CF varicosities labeled with the VGluT2 antibody (blue, A–B) is reduced relative to GFP-ho mice (C–D). The arrowheads indicate the CF varicosities in the δ2/GFP-ho that are smaller relative to the control. (E) Histogram shows the mean percentage of spines overlapping with VGluT2. A significant reduction is observed in the δ2/GFP-ho mice relative to the GFP-ho and δ2/GFP-ho CTR groups and also to GFP-wt mice. These results show that in presence of GluRδ2, indicated as the percentage of spines expressing GluRδ2 (black column), the number of CF contacts is strongly reduced. *** p<0.001. Error bars indicate SEM. Scale bars in A–D = 2 µm.</p

TA is localized in V2-positive CFs, VGAT-positive BC and PC terminals at P14.

<p>Merge of TA (red) and V2 (cyan) staining and of the colocalization mask TA/V2 (mTA/V2 white). TA is localized in the V2-positive CF terminals which impinge on the PC proximal dendrite. <b>B</b>) The negative CTR (CTR-) is represented by the overlapping signal between the V2-positive CFs (cyan) contacting the Cb-positive PC dendrites (green). The panel on the right is the relative colocalization mask (mCb/V2, white). <b>C</b>) Quantitative colocalization analysis of TA in V2-positive terminals. The mean of the TA/V2 overlap coefficients was significantly different from the negative CTR mean value. The insets are high magnification of the white boxes in A and B and show V2 and the relative colocalization masks. <b>D</b>) Merge of TA (red) and VGAT (cyan) staining and of the colocalization mask TA/VGAT (mTA/VGAT white) in the PCL and ML. TA is localized in the VGAT-positive terminals of inhibitory neurons contacting PC-dendrites and bodies. <b>E</b>) The negative CTR (CTR-) is represented by the overlapping signal between the VGAT-positive terminals (cyan) and the Cb-positive PC bodies (green). VGAT-positive PC collaterals were excluded from the analysis. The panel on the right is the relative colocalization mask (mCb/VGAT, white). <b>F</b>) Quantitative colocalization analysis of TA in VGAT-positive terminals (gray column). The mean of the TA/VGAT overlap coefficients was significantly different from negative CTR mean value (white column). The insets are high magnification of the white boxes in D and E showing VGAT and the relative colocalization masks. <b>G</b>) Merge of TA (red) and VGAT (cyan) staining and of the colocalization mask TA/VGAT (mTA/VGAT DCN, white) in the DCN region. TA is localized in the VGAT-positive synaptic terminals of PCs which contact DCNs. <b>H</b>) The relative negative control was the overlapping signal of VGAT-positive terminals (cyan) impinging on SMI32-positive DCN bodies (green). The right panel is the relative colocalization mask (m SMI32/VGAT DCN, white). <b>I</b>) Quantitative colocalization analysis of TA in VGAT-positive terminals (gray column). The mean of the TA/VGAT overlap coefficients was significantly different from negative CTR mean value (white column). The insets are high magnification of the white boxes in G and H and show VGAT and the relative colocalization masks. *** p < 0.001. Data are represented as mean± SEM. Scale bars in A-B, D-E and G-H: 10 µm; in C–F–I: 1 µm.</p

TA expression in the mouse cerebellum.

<p><b>A</b>) Western blot of TA protein at various postnatal stages (P7, P14, P21, P60). The amount of TA was quantified relative to actin (ACT). The graph shows the quantitative analysis of TA levels at the various ages normalized to P60. TA was highly expressed during postnatal development (P7-P14) and significantly decreased in the adult age. <b>B</b>–<b>B</b>’) Double immunofluorescence of a cerebellar sagittal slice at P14 showing the distribution of TA immunoreactivity (red) and Cb which labels PCs (green). <b>B’</b> shows a diffuse distribution of TA immunoreactivity in all cerebellar layers (EGL, ML, PCL, IGL and WM) and also in the DCN. <b>C</b>–<b>E</b>) Specificity of the rabbit polyclonal anti-TA antibody by immunostaining. Serial cerebellar sections incubated either with the anti-TA antibody (red, C) or with the anti-TA antibody preincubated with the TA-antigen specific peptide (<b>D</b>–<b>D</b>’). Slices were counterstained with Dapi (white). Some slices were incubated only with the cy3-coniugated anti-rabbit secondary antibody (<b>E</b>). The absence of signal in D’ and E demonstrated the specificity of the anti-TA antibody. All scale bars: 100 µm. One-way ANOVA in <b>A</b>: ** p < 0.01 and *** p < 0.001.</p