Fig. 3: Tracing layer-specific inputs to L2/3 pyramidal neurons across 1^st to 3^rd PNW.

a Schematic depicting experimental design. b Tracing inputs to L2/3 pyramidal neurons in the 1^st, 2^nd, and 3^rd PNWs. Ctip2 staining and neurite arborization of electroporated L2/3 neurons (GFP) are also shown to demonstrate layer delineation for 1^st PNW (left). Red: rabies virus (RbV) infected presynaptic cells; green: electroporated neurons in L2/3; yellow: starter cells. Blue: Ctip2 staining for L5 identification in 1^st PNW. c Confocal image of example starter cells in the 2^nd PNW, indicated by arrows. d Example of clusters of presynaptic cells labeled in the 1^st PNW, indicated by arrows. e Relationship between number of starters versus presynaptic inputs. Data fitted with linear regression, and R² is indicated on the plots. f Number of input cells per starter cell across development (One-way ANOVA with Bonferroni’s test; 1^st vs. 2^nd, 88.05 ± 14.10 vs. 44.00 ± 9.69, p = 0.018; 1^st vs. 3^rd, 25.76 ± 1.04, p = 0.0010; 2^nd vs. 3^rd, p = 0.54). Box plot whiskers indicate the minimum and maximum values. Bounds of the box are the 1^st and 3^rd quartile and the center line indicates the median. g–i Input distributions across cortical layers for 1^st (g), 2^nd (h), and 3^rd (i) PNW (One-way ANOVA with Bonferroni’s test for L4 vs. L5: 1^st, 19.38 ± 2.03 vs. 29.26 ± 2.26, p = 0.048; 2^nd, 30.98 ± 1.25 vs. 31.93 ± 1.07, p = 0.54; 3^rd, 34.23 ± 2.01 vs. 24.67 ± 1.84, p = 0.0011). Same 2^nd PNW dataset for age comparison here was used as a control dataset in a previous publication³¹. n = 5 brains for 1^st PNW, 5 brains for 2^nd PNW, and 6 brains for 3^rd PNW. Scale bar = 100 μm. *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant, p > 0.05. Bars represent the means ± SEM. Source data are provided as a Source Data file.