Fig. 1: Population imaging in the ex vivo somatosensory preparation.

a Schematic of the ex vivo somatosensory preparation and neuronal labeling strategy. b Picture of the ex vivo somatosensory preparation with a typical recording region rostral of the L1-L2 dorsal root entry zone highlighted in red. c Low-magnification fluorescence image of the L2 dorsal root entry zone (scale bar, 100 µm) showing GCaMP6s expression in Vglut2-Cre positive excitatory neurons (green) and SPB neurons (red). N = 11 animals. Rostral (R), caudal (C), medial (M), and lateral (L) are indicated on the white cross. d Representative fluorescence in situ hybridization of Gcamp6s (green) and Vglut2 (magenta) with DAPI (blue) in the superficial dorsal horn (scale bar, 25 µm). e Percent of GCaMP6s SDH neurons which express Vglut2 (n = 4 mice, 60–76 GCaMP6s + nuclei quantified/mouse). Data are presented as mean values ± SEM. f Schematic of the multi-plane (XYZT) imaging protocol used for Ca²⁺ imaging. 3 separate optical planes with a spacing of 14 µm are imaged simultaneously allowing parallel access to lamina I and II neurons. Rostral (R), caudal (C), dorsal (D), ventral (V), medial (M), and lateral (L) are indicated for the labeled image planes. g Example maximum ΔF/F image with 5 brush responsive cells circled (scale bar, 50 µm). N = 11 animals. h ΔF/F Ca²⁺ traces from the 5 cells circled in g showing consistent responses to repeated brushing of the skin. i Top: stimulation type for mechanical and thermal stimuli. High threshold (HT) and low threshold (LT) stimuli were applied serially across an area of skin 15 × 15 mm, while all other stimuli covered the entire area with each trial. Shaded region represents the period during which responsiveness is considered. Bottom: representative ΔF/F Ca²⁺ traces from both interneurons and SPB neurons in response to the indicated cutaneous stimuli. Responsiveness to cooling (C, blue) mechanical stimulation (M, green) or heating (H, red) is indicated on the right.