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

Intensity correlation analysis of TA in PC-spine/PF synapses.

<p><b>A</b>–<b>C</b>) Representative ICA analysis of TA and Cb at PC-spine in the z-dimension. <b>A</b>) ICA plots of TA (left) and Cb (right) staining intensities against their respective PDM values (central section). <b>B</b>) Serial sections of PDM images showing positive pixels inside the spine. The image is pseudocolored and a PDM scale bar is shown. <b>C</b>) Positive ICQ values (sign test p < 0.001) at each serial section indicate a dependent distribution of TA and Cb in the PC spine. <b>D</b>–<b>F</b>) Representative ICA analysis of TA and V1 at PF terminals in the z-dimension. <b>D</b>) ICA plots of TA (left) and V1 (right) staining intensities against their respective PDM values (central section). <b>E</b>) Serial sections of PDM images showing positive pixels inside the PF terminal. <b>F</b>) Positive ICQ values (sign test p<0.001) at each serial section indicate a dependent distribution of TA and V1 in the PF. <b>G</b>–<b>I</b>) Representative ICA analysis of Cb-spine and V1-PF terminal in the z-dimension as control. <b>G</b>) ICA plots of Cb (left) and V1 (right) staining intensities against their respective PDM values (central section). <b>H</b>) Serial sections of PDM images showing negative pixels inside the spine. <b>I</b>) negative ICQ values (sign test p < 0.001) at each serial section indicate a segregated distribution of Cb and V1 in the spine. <b>J</b>) Statistical analysis of ICQ values based on multiple contacts. The ICQ values were consistently positive and highly significant not only in the central optical sections but also in the up and down sections for both spines and PF terminals. Accordingly, the ICQ values were consistently and significantly negative along the z-dimension (t-test, p <0.05 relative to 0) in CTR (spine/PF). Of note that the mean values of the three optical sections were not significantly different among them (one-way ANOVA, p < 0.05). Data are represented as mean ± SEM.</p

TA localization in the mouse cerebellar cortex at P14.

<p><b>A-A</b>’’and <b>B–B</b>’<b>’)</b> Cerebellar sagittal section immunostained for PV (green) and TA (red) and counterstained with Dapi (cyan). <b>A</b>) Merge image between Dapi-positive nuclei (cyan) and PV-positive PC dendrites (green) in the upper part of the ML and the EGL. <b>A</b>’) Shows the corresponding TA immunostaining (red). <b>A</b>’’<b>)</b> Merge image of all markers (PV-TA-Dapi) plus the colocalization mask TA/PV (mTA/PV in white) highlighting the expression of TA in PC dendrites and spines. <b>B</b>) Merge image of PV staining and Dapi-positive nuclei, highlighting the PV-positive PC bodies and dendrites and interneurons of the ML (green). The inset (white box) is an high magnification of a BC body. <b>B</b>’) TA protein (red) is abundantly present in PCs and BCs (inset), both in the cytoplasm and nuclei. Black spaces are TA-negative nuclei in the ML. <b>B</b>’’) merge image of all markers (PV-TA-Dapi) plus the colocalization mask TA/PV (mTA/PV in white). <b>C</b>) Merge image between DAPI-positive nuclei and V1-positive (green) rosettes to define the IGL. Most of the DAPI-positive nuclei in the IGL belong to granule cells <b>C</b>’) TA staining is diffuse also in this layer; granule cells (high magnification in the inset) and Golgi-like cells are intensively labeled. <b>C</b>’’) Merge image of all markers (TA-V1-Dapi) plus the colocalization mask TA/Dapi (mTA/Dapi in white). The absence of the white mask highlights no expression of TA in almost all cell nuclei. Few white dots in the inset indicate overlapping signal of TA around the nucleus. Scale bars in A, B and C 10 µm; in the insets 2 µm.</p

TA expression in glial cells of the cerebellar cortex at P14.

<p><b>A</b>, <b>B</b>, <b>E</b>, <b>F</b>) Merge images between DAPI-positive nuclei (cyan), TA immunostaining (red) and GFAP-positive glia cells (green) in cerebellar sagittal slices. <b>A</b>) The colocalization mask TA/GFAP (mTA/GFAP, white) highlights the expression of TA in Bergmann glia (BG) palisades and endfeets in the upper ML and EGL. <b>B</b>) The colocalization mask TA/GFAP (mTA/GFAP) shows TA localization in glia cells localized in the PCL and ML. <b>B</b>’) Serial sections of the inset in B highlights TA expression (red) inside the cell body of BG. <b>C</b>) Merge between Cb-positive PC dendrites (red), GFAP and the colocalization mask Cb-GFAP (mCb/GFAP) in the ML. The overlapping signal between these two markers represent the negative control for quantification analysis. <b>D</b>) Quantitative colocalization analysis of TA expressed in GFAP-positive glia cells in the ML (left panel) and IGL (right panel). The mean values of the r overlap coefficients of TA/GFAP (gray columns) were significantly different from the negative CTR Cb/GFAP (white column), indicating TA localization in glia cells. <b>E</b>–<b>F</b>) The colocalization masks TA/GFAP (mTA/GFAP) in E and F show a differential expression of TA in bushy glia cells localized in the IGL. <b>G</b>) Merge of Cb-positive PC axon (red), GFAP (green) and the colocalization mask Cb-GFAP (mCb/GFAP) in the IGL. The overlapping signal between these two markers represent the negative control for quantification analysis. *** p < 0.001. Data are represented ad mean ± SEM. Scale bars, 10 µm.</p