Fig. 2: Spatial registration of neuron subclasses in mouse primary somatosensory cortex (S1) by π-FISH rainbow multiplexed in situ detection.

a Diagram of spatial decoding of pyramidal neuron and interneuron subclasses in mouse S1 via two rounds of π-FISH rainbow hybridization and imaging. Nine excitatory neuron marker genes were detected in round 1, followed by signal wash-out and second-round hybridization of 12 interneuron marker genes. By combining two rounds of signals, spatial mapping of 13 interneuron subclasses in S1 were obtained. b Nine layer-specific excitatory neuron marker genes (Rasgrf2, Cux2, Plcxd2, Rorb, Kcnk2, Sulf2, Foxp2, Pcp4, and Ctgf) in S1 were simultaneously detected by π-FISH rainbow. White dashed lines indicated the boundaries of the cortex. Scale bar, 100 μm. c Decoding and mapping of signals from (b) showed the spatial distribution of nine neuron markers in the L1–L6 layers of the S1 cortex. The L1 and L6 of the cortex in S1 are indicated by the black dashed lines. d π-FISH rainbow accurately reproduced the spatial distribution of layer-specific marker genes, especially in the L5a and L6b sublayers. Scale bar, 100 μm. e The second-round hybridization for 12 interneuron marker genes (Gad1, Sst, Crh, Npy, Cck, Vip, Pvalb, Gda, Penk, Cxcl14, Cpne5, and Lphn2) in the same section (top). (i-viii) The higher magnifications images were shown at the bottom. The boundaries of S1 are marked with white dashed lines. Scale bars, 100 μm (top) and 5 μm (bottom). f The distribution of 13 Gad1⁺ interneuron subclusters in different layers based on two-round hybridization. Scale bar, 100 μm. g Bubble plot for layer distribution of 13 Gad1⁺ interneuron subclasses in S1. Int 9 and Int 10 subclasses were mainly distributed in the superficial layers (L2/L3), whereas Int 6 subclass was predominately located in the deep layers (L5 to L6) (light red frame). The number of interneuron subclasses is indicated by the spot size. n = 427 Gad1⁺ interneuron. Source data are provided as a Source Data file. Int1 to Int16 in (f) and (g) correspond to the interneuron subclasses defined by scRNA-seq from the publication by Zeisel et al.⁵.