Volume 21, Issue 8, Pages 2066-2073 (November 2017) Maturation of Cerebellar Purkinje Cell Population Activity during Postnatal Refinement of Climbing Fiber Network  Jean-Marc Good, Michael Mahoney, Taisuke Miyazaki, Kenji F. Tanaka, Kenji Sakimura, Masahiko Watanabe, Kazuo Kitamura, Masanobu Kano  Cell Reports  Volume 21, Issue 8, Pages 2066-2073 (November 2017) DOI: 10.1016/j.celrep.2017.10.101 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 21, 2066-2073DOI: (10.1016/j.celrep.2017.10.101) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 Developmental Decline in Correlation of PC Population Activity (A) Full-frame scan movies recorded at the PC layer (P5 to P10) or molecular layer (P11 and P22). ROIs (in black) corresponding to single-PC somata (P5 to P10) or dendrites (P11 and P22) were numbered (in white) following rostral (r) to caudal (c) and then medial (m) to lateral (l) order. (B) Spontaneous calcium transients by extracting relative fluorescence changes from each ROI. (C) Reconstructed CS firing rates from temporal deconvolution of calcium transient traces. (D) Matrices displaying CCs between the deconvolved calcium traces of all possible pairs. (E–H) Mean CCs between deconvolved calcium traces from pairs of PCs plotted against the m-l (E) and r-c (G) distance of separation between pairs of PCs. The r-c distance of separation is not taken into account in the plots for the m-l separation in (E), and vice versa for the r-c separation in (G). Averaged CCs are for m-l (F) and r-c (H) separations of 0–40 and 80–120 μm. P3–P5, 6 mice, 412 pairs; P6–P7, 12 mice, 599 pairs; P8, 5 mice, 848 pairs; P10–P12, 7 mice, 177 pairs (for m-l separation), and 5 mice, 124 pairs (for r-c separation); P21–P24, 8 mice, 153 pairs (for m-l separation). The r-c distance of separation could not be measured for P21–P24, because recordings could be obtained exclusively from dendrites in this age group. Data are represented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. ns, not significant. See also Figures S1–S3, Tables S1 and S2, and Movies S1 and S2. Cell Reports 2017 21, 2066-2073DOI: (10.1016/j.celrep.2017.10.101) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 Developmental Decline in Correlation of CF Terminal Population Activity (A) Full-frame scan movies recorded at the PC layer in Htr5B-tTA::tetO-YC-Nano50 mice at P5 and P9. ROIs (in black) corresponding to CF terminals around single-PC somata were numbered (in white) following rostral (r) to caudal (c) and then medial (m) to lateral (l) order. (B) Spontaneous calcium transients by extracting relative fluorescence changes from each ROI. (C) Color-coded matrices displaying CCs between ΔR/R traces of all possible pairs. (D–G) Mean CCs between pairs of ΔR/R traces obtained from CF terminals and between pairs of deconvolved calcium traces obtained from PCs (data from Figure 1), plotted against the m-l (D) and r-c (F) distance of separation between pairs of ROIs. CF: P5, 4 mice, 975 pairs; P8–P10, 5 mice, 1,132 pairs. PC: P3–P5, 6 mice, 412 pairs; P8, 5 mice, 848 pairs. Averaged CCs for pairs of ROIs with a m-l separation (E) or r-c separation (G) of 0–40 and 80–120 μm. Data are represented as mean ± SEM. ∗p < 0.05, ∗∗∗p < 0.001. ns, not significant. See also Figure S1. Cell Reports 2017 21, 2066-2073DOI: (10.1016/j.celrep.2017.10.101) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 Aberrant PC Population Activity and Pruning of CF Collaterals in PC-Cav2.1 KO Mice from P9 to P10 (A) Full-frame scan movies recorded in control (P9) and PC-Cav2.1 KO (P10) mice. ROIs were defined as in Figure 1A. (B) Corresponding calcium transients obtained as in Figure 1B. (C) Matrices displaying CCs between the deconvolved calcium traces of all possible pairs. (D–G) Mean CCs between pairs of deconvolved calcium transients plotted against the m-l (D) and r-c (F) separation in control and PC-Cav2.1 KO mice. Control: P4–P5, 5 mice, 185 pairs; P9–P10, 6 mice, 436 pairs. PC-Cav 2.1 KO: P4–P5, 5 mice, 190 pairs; P9–P10, 8 mice, 722 pairs. (E and G) Averaged CCs in control and PC-Cav2.1 KO mice for cell pairs with a m-l (E) or r-c (G) separation of 0–40 and 80–120 μm. (H and I) Triple fluorescence labeling for calbindin (gray) and VGluT2 (green), together with anterogradely labeled CFs by dextran Alexa 594 (DA-594, red), in parasagittal cerebellar sections from control (H) and PC-Cav2.1 KO (I) mice at P10. The left-right direction in these pictures corresponds to the r-c direction in the cerebellum. (H1 and I1) Arrows indicate representative DA-594-labeled CF collaterals that are visible from their bifurcation from the main branches to the terminal tips on the PC soma. (H2 and I2) VGluT2-positive CF terminals can be seen on PC somata and dendrites. Scale bar, 10 μm. (J) Mean horizontal length of CF collaterals in both axes and genotypes. (Left) r-c plane. Control: 13.3 ± 1.2 μm, n = 39. PC-Cav2.1 KO: 32.5 ± 2.2 μm, n = 55, p < 0.001, (right) m-l plane. Control: 10.6 ± 0.7 μm, n = 101. PC-Cav2.1 KO: 10.7 ± 0.5 μm, n = 97, p = 0.163. (K) Numbers of VGluT2-positive terminals per CF collateral. Control: 2.4 ± 0.3, n = 39. PC-Cav2.1 KO: 12.8 ± 1.1, n = 55, p < 0.001. Data are represented as mean ± SEM. ∗∗p < 0.01, ∗∗∗p < 0.001. ns, not significant. See also Figures S2 and S4 and Tables S1 and S2. Cell Reports 2017 21, 2066-2073DOI: (10.1016/j.celrep.2017.10.101) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 Aberrant PC Population Activity in GluD2 KO Mice from P14 to P15 (A) Full-frame scan movies recorded in control (P12) and GluD2 KO (P14) mice. ROIs were defined as in Figure 1A. (B) Corresponding calcium transients obtained as in Figure 1B. (C) Matrices displaying CCs between the deconvolved calcium traces of all possible pairs. (D) Mean CCs between pairs of deconvolved calcium transients plotted against the m-l separation in control and GluD2 KO mice. The r-c separations cannot be accurately measured at the dendritic level. Control: P8, 5 mice, 848 pairs; P10–P24, 15 mice, 330 pairs. GluD2 KO: P8, 5 mice, 203 pairs; P14–P15, 4 mice, 92 pairs. (E) Averaged CCs in control and in GluD2 KO mice for cell pairs with a m-l separation of 0–40 μm (left) and 80–120 μm (right). Data are represented as mean ± SEM. ∗∗p < 0.01, ∗∗∗p < 0.001. ns, not significant. See also Figure S2 and Tables S1 and S2. Cell Reports 2017 21, 2066-2073DOI: (10.1016/j.celrep.2017.10.101) Copyright © 2017 The Author(s) Terms and Conditions