DTA expression by oligodendrocyte-specific AAVs deletes oligodendrocytes in the mouse cerebellum during the specific developmental period.

A, Diagram illustrating the injection of AAV-hMAG-mCherry and AAV-hMAG-DTA into the mouse cerebellum at early postnatal days (P6-7). B, Fluorescence microscopy image showing mCherry expression (red) in the section of cerebellum at P14 following AAV-hMAG-mCherry injection at P7. Scale bar, 300 µm. C, Specificity of mCherry expression (red) in ASPA-positive oligodendrocytes (green) at P14 by AAV-hMAG-mCherry injection at P7. Scale bar, 100 µm. D, Scatter plot graph depicting the percentage of RFP and ASPA double-positive cells among RFP-positive populations (from 2 mice). E, Sequential visualization of oligodendrocyte reduction over time, indicated by ASPA staining in cerebellar sections from control (AAV- hMAG-mCherry) and DTA-treated (AAV-hMAG-DTA) mice at P10, P14, P21, and P78. Scale bar, 100 µm. F, Quantification of ASPA-positive cell density at each stage (from 3 mice per group per each stage). Bars and dots indicate mean and data from individual fields of view, respectively. **** p < 0.0001 (Mann-Whitney U test).

Oligodendrocyte reduction by oligodendrocyte-specific AAVs reduces the synchrony of PC population activity during mouse cerebellar development.

A, Diagram illustrating the time course of AAV injection (P7) and calcium imaging (P13-P15) in control and DTA-expressing mice with jGCaMP7f expression in the PCs. B, Schema of in vivo calcium imaging and representative one frame of full-frame scan movie recorded in lobule Ⅵ/Ⅶ of the cerebellum of control mouse at P14. Scale bar = 0.5 mm. C-F, Decrease in correlation coefficients (CCs) indicating reduced synchrony of spontaneous activities among PC population at P13-15 following DTA-mediated oligodendrocyte ablation. C, Representative time-course of spontaneous calcium transients by extracting relative fluorescence changes by in vivo calcium imaging captured from 25 regions of interest (ROIs) in the cerebellum of control and DTA-expressing mice at P14. Scale bar = 20 s. Y-axis is ΔF/F0 = (F - F0)/(F0). D, Correlation coefficient matrices for spontaneous calcium transient activity across multiple ROIs in CTL (upper panel) and DTA (lower panel) mice, with the color scale indicating the strength of the correlation between pairs of ROIs. E, Mean correlation coefficients between calcium traces from pairs of ROIs plotted against the distance of separation between pairs of ROIs in control (n = 4 mice) and AAV-hMAG-DTA-injected (n = 4 mice) mice. Error bars indicate SEM. F, Scatter plot graph of CCs for ROI pairs, categorized by separation distances of 0–80 mm and 120–200 mm (analyzed from four mice per group). Lines and plots indicate mean and data from individual separation distances, respectively. G-I, Quantification of the frequency, amplitude, and integrated area of calcium transients in control (AAV-hMAG-mCherry) and DTA-treated (AAV-hMAG-DTA) mice at P13-15 (data from four mice per group). Bars and dots indicate mean and data from individual ROIs, respectively. **** p < 0.0001 (Mann- Whitney U test).

Oligodendrocyte ablation during postnatal development diminishes neuronal synchrony at adult stage.

A, Representative traces of spontaneous calcium transients from 25 regions of interest (ROIs) in control (CTL, top) and AAV-hMAG-DTA-injected (DTA, bottom) mice, illustrating changes in synchrony. B, Correlation matrices for spontaneous calcium transients comparing CTL (upper) and DTA (lower) mice, with correlation strength indicated by color intensity. C, Scatter plots of correlation coefficients for ROI pairs within short (0-80µm) and long (120-200µm) distances in control and DTA mice, showing significant reductions in the DTA group. D, Line graph showing the correlation coefficient as a function of inter-ROI distance, with CTL (orange) and DTA (cyan) mice displaying differing correlation coefficient profiles. E-G, Bar graphs comparing the mean of frequency (E), amplitude (F), and integrated area (G) of fluorescence transients in CTL and DTA mice. Each graph includes individual data points for each ROI. Statistical significance in panels e and g is indicated by asterisks (**** p < 0.0001, Mann- Whitney U tests).

Oligodendrocyte reduction during development leads to behavioral abnormalities.

A-C, Assessment of exploratory behavior and locomotor activity in control (CTL, orange) and DTA-injected (DTA, cyan) mice in a novel environment, with trajectories of mouse exploratory behavior (A), distance traveled (B), and time spent in center (C) for mice with the injection of AAVs into distinct compartments (left, center, right) of the cerebellum (LCH, CBV, RCH). Bars and dots indicate mean and data from individual mice, respectively. D-F, Representative images and quantitative analysis of sociality in CTL and DTA mice with the injection of AAVs into distinct compartments (LCH, CBV, RCH). Diagram in (D) shows the method of sociality test; (E) displays the heatmap of mouse exploratory behavior, and (F) shows the mean percentage of time spent around strange mouse with data points representing individual mice. G, Line graph showing mean latency to fall in the rotarod test over a three-day period for CTL and DTA mice, with separate graphs for mice with the injection of AAVs into distinct compartments (LCH, CBV, RCH). Error bars indicate SEM. Error bars indicate SEM. Statistical significance is indicated by asterisks (*p < 0.05, **p < 0.01, *** p < 0.001, **** p < 0.0001, Mann-Whitney U tests).

Oligodendrocyte reduction after 3 weeks old in mice does not affect PC activity synchrony and mouse behaviors except for motor learning.

A, B, Fluorescence microscopy images comparing ASPA expression in the cerebellum of control (CTL) and 3-week DTA-treated (3W_DTA) mice at 4 weeks (A) and 8 weeks (B) old. ASPA-positive oligodendrocytes appear in pink. Scale bar, 50µm. C, Quantification of the number of ASPA-positive cells per mm² in the cerebellum of CTL and 3W_DTA mice at 4 and 8 weeks old, with data points for each field of view overlaid on the bar graphs (mean). D, Representative calcium imaging traces from PCs in the cerebellar cortex of control (CTL) and 3-week DTA-treated (3W_DTA) mice at 4weeks old, showing 25 regions of interest (ROIs). E, Correlation coefficient matrices of calcium transients between ROIs from CTL and 3W_DTA mice, indicating the level of synchrony, with warmer colors representing higher correlation coefficients. F, Line graph depicting mean correlation coefficients with increasing inter-ROI distance for CTL and 3W_DTA mice. Error bars indicate SEM. G, Scatter plot comparing the correlation coefficients of calcium transients in PCs between adjacent ROIs in CTL and 3W_DTA mice. H-J, Bar graphs displaying mean frequency (H), amplitude (I), and area (J) of fluorescence calcium transients for CTL and 3W_DTA mice, with significant differences denoted by asterisks. Data points represent individual ROIs. K-M, Assessment of exploratory behavior and locomotor activity in control (CTL, orange) and 3 weeks DTA- injected (3W_DTA, cyan) mice in a novel environment, with trajectories of mouse exploratory behavior (F), distance traveled (G), and time spent in center (H). N, O, Representative images and quantitative analysis of sociality in CTL and 3W_DTA mice. N displays the heatmap of mouse exploratory behavior, and (O) shows the mean percentage of time spent around strange mouse with data points representing individual mice. P, Line graph showing mean latency to fall in the rotarod test over a three-day period for CTL and 3W_DTA mice. Error bars show SEM. Statistical significance is indicated by asterisks (**p < 0.01, **** p < 0.0001, Mann- Whitney U tests).

Kir2.1 expression in CFs reduces the synchrony of PC population spontaneous activity and leads to abnormal behaviors similar to the phenotype of mice with developmental oligodendrocyte reduction.

A, Fluorescent images of sagittal cerebellar sections showing Car8 immunoreactivity (Purkinje cells, magenta) and GFP expression (Climbing fibers (CFs), green). Scale bar, 200 µm. B, Higher magnification of the boxed area in A showing detailed GFP-labeled CF morphology. Scale bar, 100 µm. C, Scatter plot graph depicting the percentage of PCs with GFP-positive CFs among PC populations (17 sections from 10 mice). D, Schematic timeline of AAV injection at P7 and Ca2+ imaging with jGCaMP7 at P13-15 for the CTL and Kir2.1 groups. E, Representative time-course of spontaneous calcium transients from regions of interest (ROIs) in the mouse cerebellum at P13 showing spontaneous PC activity in control mice (CTL, top) and mice expressing Kir2.1 in a subset of CFs (kir2.1, bottom). Scale bar, 20 s. Y-axis is ΔF/F0 = (F - F0)/(F0). F, Correlation matrices for spontaneous PC activity between ROIs in control (top) and kir2.1-expressing (bottom) mice. The color scale indicates correlation strength. G, Graph displaying the correlation coefficient of Ca2+ transients among PCs as a function of distance between adjacent PCs in CTL and Kir2.1 groups. Data points indicate the mean ± SEM. H, Scatter plot comparing correlation coefficients for ROI pairs within 0-80μm and 120- 200μm separation distance ranges in CTL (orange) and kir2.1-expressing (cyan) mice at P13- 15, with lines representing average values. I-K, Bar graphs showing the comparison between CTL and Kir2.1-overexpressing CFs in terms of (I) frequency, (J) amplitude, and (K) area under the curve of Ca2+ transients. Data are presented as mean. Each graph includes individual data points for each ROI. L-N, Analysis of exploratory behavior and locomotor activity in CTL (orange) and Kir2.1-expressing (green) mice with distance traveled (L, M) and percentage of time spent in the center (N) in an open-field test. O, P, Representative images and quantitative analysis of sociality in CTL and Kir2.1-expressing mice. (O) displays the heatmap of mouse exploratory behavior, and (P) shows the percentage of time spent around strange mouse. Q, Rotarod test. The line graph shows the mean latency to fall across three days for CTL and Kir2.1-expressing mice. Error bars indicate SEM. Statistical significance is indicated by asterisks (**p < 0.01, *** p < 0.001, Mann-Whitney U tests).