Supplementary Figure 8: Contacts from boutons that contain somatostatin and VGLUT2 onto dynorphin cells that express the Sst_2a receptor.

A-C confocal image of part of a sagittal section from a Pdyn^Cre mouse that had received an intraspinal injection of AAV.flex.eGFP. eGFP is shown in green and sst_2a receptor in magenta. Nine eGFP⁺ cells are present in this field, and 8 of these (marked with asterisks) are sst_2a -immunoreactive, while 1 (arrowhead) lacks the receptor. Between 70 and 79 (mean 74.3) eGFP⁺ cells were identified in sections from the L3 segment of 3 mice, and 78.2% (68.9-78.5%) of these were sst _2a-immunoreactive. This is consistent with our finding that 90% of lamina I-II preprodynorphin neurons in hairy skin territory are inhibitory, and that 91% of inhibitory PPD cells are sst _2a-immunoreactive⁴. The box indicates the cell body of the neuron that is illustrated in D-I. D-I Contacts from boutons that co-express somatostatin (SST, red) and VGLUT2 (blue) onto the cell body (D-F) and dendrite (G-I) of a eGFP⁺ cell that was immunoreactive for sst _2a (magenta in D). Contacts are indicated with arrows. J. Plot of the contacts from SST/VGLUT2 boutons onto the cell bodies (yellow circles) or dendrites (blue circles) of 5 of the cells that were analysed. The top left cell is the one in illustrated in A (surrounded by box) and D-I. Confocal images in A-C are projected from 4 optical sections at 1 μm z-spacing. Those in D-F and G-I are projected from 3 and 5 (respectively) optical sections at 0.5 μm z-spacing. Scale bars: A-C = 50 μm, D-I = 5 μm, J = 50 μm. 4. Boyle, K.A. et al. A quantitative study of neurochemically defined populations of inhibitory interneurons in the superficial dorsal horn of the mouse spinal cord. Neuroscience 363, 120-133 (2017).