Fig. 1*C* | Normal | RM one-way ANOVA | PI^{+}: *F*_{(2,52.2) }= 42.7, *p* < 0.0001 |

GCaMP-filled: *F*_{(2.58,67) }= 18.8, *p* < 0.0001 |

Fig. 1*D* (inset) | Normal | Unpaired *t* test (two-tailed) | *t* = 0.037, df = 32, *p* = 0.97 |

Fig. 1*F* | Nonparametric | Spearman's correlation | Spearman's *ρ* = 0.66, *p* < 0.0001 |

Extended Data Fig. 1-2*C* | Normal | Two-way RM ANOVA | Interaction: *F*_{(9,99) }= 4.025, *p* = 0.0002 |

Depth: *F*_{(9,99) }= 16.8, *p* < 0.0001 |

Treatment: *F*_{(1,11) }= 12.3, *p* = 0.0049 |

Extended Data Fig. 1-2*D* | Normal | Unpaired *t* test (two-tailed) | *t* = 3.51, df = 11, *p* = 0.005 |

Extended Data Fig. 1-2*E* | Normal | Unpaired *t* test (two-tailed) | *t* = 2.76, df = 11, *p* = 0.02 |

Extended Data Fig. 1-2*F* | Normal | Two-way RM ANOVA | Interaction: *F*_{(9,99) }= 4.15, *p* = 0.0001 |

Depth: *F*_{(9,99) }= 6.18, *p* < 0.0001 |

Treatment: *F*_{(1,11) }= 12.3, *p* = 0.062 |

Extended Data Fig. 1-2*G* | Normal | Two-way RM ANOVA | Interaction: *F*_{(9,90) }= 2.18, *p* = 0.034 |

Depth: *F*_{(9,90) }= 14.1, *p* < 0.0001 |

Treatment: *F*_{(1,10) }= 0.041, *p* = 0.93 |

Extended Data Fig. 1-3*C* | Normal | Two-way ANOVA | Interaction: *F*_{(6,87) }= 0.3, *p* = 0.936 |

Time point: *F*_{(3,87) }= 9.2, *p* < 0.0001 |

Layer: *F*_{(2,87) }= 2.42, *p* = 0.095 |

Extended Data Fig. 1-3*D* | Normal | Two-way ANOVA | Interaction: *F*_{(6,82) }= 0.06, *p* = 0.99 |

Time point: *F*_{(3,82) }= 0.95, *p* = 0.42 |

Layer: *F*_{(2,82) }= 0.78, *p* = 0.45 |

Extended Data Fig. 1-4*A* | Normal | Two-way RM ANOVA | Interaction: *F*_{(9,351) }= 2.04, *p* = 0.034 |

Depth: *F*_{(9,351) }= 129.4, *p* < 0.0001 |

Slice thickness: *F*_{(1,39) }= 1.74, *p* = 0.125 |

Extended Data Fig. 1-4*B* | Normal | Two-way RM ANOVA | Interaction: *F*_{(9,342) }= 2.04, *p* = 0.0007 |

Depth: *F*_{(9,342) }= 52.51, *p* < 0.0001 |

Slice thickness: *F*_{(1,38) }= 1.81, *p* = 0.186 |

Extended Data Fig. 1-4*C* | Normal | Two-way RM ANOVA | Interaction: *F*_{(9,216) }= 0.08, *p* = 0.99 |

Depth: *F*_{(9,216) }= 91.3, *p* < 0.0001 |

Age: *F*_{(1,24) }= 0.36, *p* = 0.56 |

Extended Data Fig. 1-4*D* | Normal | Two-way RM ANOVA | Interaction: *F*_{(9,306) }= 0.87, *p* = 0.56 |

Depth: *F*_{(9,306) }= 54.16, *p* < 0.0001 |

Age: *F*_{(1,34) }= 10.49, *p* = 0.0027 |

Fig. 2*B* | Normal | RM one-way ANOVA | PI^{+}: *F*_{(2,52.2) }= 42.7, *p* < 0.0001 |

Neuropil: *F*_{(2,52.2) }= 42.7, *p* < 0.0001 |

Fig. 2*C* | Nonparametric | Spearman's correlation | Spearman's *ρ* =0.68, *p* < 0.0001 |

Fig. 2*E* | Normal | Pearson's correlation | Pearson's *r* = 0.61, *p* < 0.0001 |

Fig. 3*B* | Normal | Two-way RM ANOVA | Interaction: *F*_{(27,198) }= 1.578, *p* = 0.043 |

Depth: *F*_{(9,198) }= 107.1, *p* < 0.0001 |

Treatment: *F*_{(3,22) }= 2.75, *p* = 0.07 |

Fig. 3*E* | Normal | One-way ANOVA | PI^{+}: *F*_{(3,22) }= 3.06, *p* = 0.049 |

Dunnett's multiple-comparisons test |

0–1 vs 1–2: *p* = 0.91, 0–1 vs 2–3: *p* = 0.99 |

vs 3–5: *p* = 0.033 |

GCaMP-filled: *F*_{(3,18) }= 1.7, *p* = 0.2 |

Dunnett's multiple-comparisons test |

0–1 vs 1–2: *p* = 0.97, 0–1 vs 2–3: |

*p* = 0.96 |

0–1 vs 3–5: *p* = 0.13 |

Fig. 5*C* | Nonparametric | Wilcoxon matched pairs test | *W* = 21, *p* = 0.031 |

Fig. 5*D* | Nonparametric | Wilcoxon matched pairs test | *W* = 21, *p* = 0.031 |

Fig. 5*F* | Nonparametric | Wilcoxon matched pairs test | *W* = 21, *p* = 0.031 |

Fig. 5*F* | Nonparametric | Wilcoxon matched pairs test | *W* = 8, *p* = 0.375 |

Fig. 6*B* | Normal | Two-way RM ANOVA | Interaction: *F*_{(9,369) }= 3.32, *p* = 0.0006 |

Depth: *F*_{(9,369) }= 48.1, *p* < 0.0001 |

Treatment: *F*_{(1,41) }= 8.51, *p* = 0.0057 |

Fig. 6*B* (inset) | Normal | Unpaired *t* test (two-tailed) | *t* = 3.19, df = 41, *p* = 0.002 |

Fig. 6*D* | Normal | Two-way ANOVA | Interaction: *F*_{(3,34) }= 0.97, *p* = 0.41 |

Depth: *F*_{(3,34) }= 4.57, *p* = 0.0091 |

Treatment: *F*_{(1,34) }= 15.4, *p* = 0.0004 |

Sidak's multiple-comparisons test |

<1 h: *p* = 0.67, 1–2 h: *p* = 0.21, 2–3 h: *p* = 0.023, 3–5 h: *p* = 0.011 |

Fig. 6*E* | Normal | Two-way ANOVA | Interaction: *F*_{(3,33) }= 1.4, *p* = 0.26 |

Depth: *F*_{(3,33) }= 0.71, *p* = 0.55 |

Treatment: *F*_{(1,33) }= 14.8, *p* = 0.0005 |

Sidak's multiple-comparisons test |

<1 h: *p* = 0.61, 1–2 h: *p* = 0.75, 2–3 h: *p* = 0.64, 3–5 h: *p* = 0.001 |

Fig. 6*F* | Normal | Two-way RM ANOVA | Interaction: *F*_{(9,369) }= 2.17, *p* = 0.023 |

Depth: *F*_{(9,369) }= 9.09, *p* < 0.0001 |

Treatment: *F*_{(1,41)} = 4.19, *p* = 0.047 |

Fig. 6*F* (inset) | Nonparametric | Mann–Whitney test | *U* = 86, *p* = 0.002 |

Fig. 6*G* | Normal | Two-way RM ANOVA | Interaction: *F*_{(9,369) }= 1.46, *p* = 0.038 |

Depth: *F*_{(9,369) }= 23.7, *p* < 0.0001 |

Treatment: *F*_{(1, 41) }= 2.8, *p* = 0.01 |

Fig. 6*G* (inset) | Normal | Unpaired *t* test (two-tailed) | *t* = 2.15, df = 41, *p* = 0.038 |

Extended Data Fig. 6-1*B* (right) | Lognormal | One-way ANOVA (Tukey's post hoc test) | ANOVA, *F*_{(2,30)} = 4.65. *p* = 0.017 |

Tukey's multiple-comparisons test |

aCSF vs MDL, *p* = 0.033 |

aCSF vs Cal 2-1, *p* = 0.046 |

MDL vs Cal 2-1, *p* = 0.910 |

Extended Data Fig. 6-1*B* (right) | Lognormal | One-way ANOVA (Tukey's post hoc test) | ANOVA, *F*_{(2,31)} = 5.79. *p* = 0.007 |

Tukey's multiple-comparisons test |

aCSF vs MDL, *p* = 0.005 |

aCSF vs Cal 2-1, *p* = 0.188 |

MDL vs Cal 2-1, *p* = 0.179 |