Fig. 3: Calcium imaging of thalamocortical axons during passive whisker stimulation.

a Schematic of viral vector injection and microprism implantation for in vivo calcium imaging of thalamocortical axons expressing GCaMP6s. b Intrinsic optical signal in wS1 during whisker stimulation. This experiment was repeated in 14 mice with similar results. c Two-photon image of VPM axons in wS1. Similar results were observed in three mice. d Left: Example of a region of interest for axonal segments with high calcium signal correlation in wS1 (red). Right: Corresponding calcium responses to C2 or B2 whisker stimulation in z-score across trials. The average calcium responses are shown below. e Normalized calcium responses averaged over all axons for each population (3 mice, n = 62 axons for VPM-FO; 3 mice, n = 101 axons for POm-FO; 8 mice, n = 86 axons for POm-HO). Inset: Response latency comparison (Kruskal–Wallis two-sided test with Bonferroni correction, **p = 0.008 for VPM-FO vs. POm-FO, **p = 0.004 for VPM-FO vs. POm-HO, p = 1 for POm-FO vs. POm-HO). Boxplot: central mark indicates the median and edges indicate 25th and 75th percentiles. Whiskers extend to the largest or smallest point comprised within 1.5× of the interquartile range from both edges. f Left: Two-photon image of the calcium response (ΔF/F) for C2 (green) and B2 (red) whisker stimulation overlaid on top of VPM axonal innervation (gray) in wS1. Middle: Distribution of whisker selectivity indices for the corresponding axonal population (3 mice, n = 62 axons). Right: Distribution for the absolute value of the whisker selectivity index. Red arrow: average value. g Same as f, but for POm-FO axons in wS2 (3 mice, n = 101 axons). Inset in the right panel: Response latency comparison between tuned (|WSI | ⩾0.75) and untuned (|WSI | < 0.75) axons (Kruskal–Wallis two-sided test, **p = 0.002). Boxplot statistics same as in (e) inset. h Same as f, but for putative POm-HO axons located in layer 1 (8 mice, n = 86 axons). Kruskal–Wallis test with Bonferroni correction comparing |WSI | : p = 0.003 for VPM-FO vs. POm-FO; p = 4 × 10⁻¹¹ for VPM-FO vs. POm-HO; and p = 4 × 10⁻¹¹ for POm-FO vs. POm-HO.