Anatomical evidence for primary afferent input to PrP-GFP and nNOS cells. a, Confocal image through a PrP-GFP cell in a parasagittal section from perfusion-fixed tissue. The scan shows GFP (green). Boxes represent the location of parts c and d. b–d, Parts of the field shown in a, scanned to reveal nNOS, IB4, and CGRP, respectively (all shown in magenta). The soma shows weak nNOS immunoreactivity, surrounding the nucleus (*). c, Two nearby dendritic spines belonging to the cell are in contact with IB4+ boutons, which lack CGRP (data not shown). d, A small CGRP+ bouton contacts the dendritic shaft of the cell. e, Projection of a short series of confocal images through a PrP-GFP cell that was processed for combined confocal and electron microscopy. The cell contains both GFP (green) and nNOS (blue) immunoreactivity, and a dendritic spine (marked with an arrow in the inset) receives a contact from a VGLUT1-immunoreactive (red) axonal bouton. f, g, Electron microscope images (the field shown in f corresponds to the box in e). The DAB reaction product labels both GFP+ and VGLUT1+ structures. f, Part of the dendritic shaft (d) can be seen together with the dendritic spine (arrow) and adjacent VGLUT1-immunoreactive axonal bouton. g, Higher-magnification EM image taken after tilting of the section shows an asymmetrical synapse between the axonal bouton (a) and the dendritic spine (s). h, Confocal images to show part of a lamina II nNOS-immunoreactive (green) cell in one of the RetCreER+;Ai34+ mice. The dendrite that emerges from the cell body has 2 spines (arrows). i, The same field scanned to reveal nNOS, tdTomato (red), and VGLUT1 (blue). The spines receive contacts from boutons that express tdTomato and are immunoreactive for VGLUT1, and therefore appear magenta. j, Part of the dendritic tree of the PrP-GFP neuron shown in a, with neurobiotin (NB) shown in red. This cell had an axon that entered lamina I and showed increased mEPSC frequency during bath application of capsaicin. k, l, Spines belonging to the cell (arrows and arrowhead) received contacts from VGLUT1-immunoreactive (blue) boutons. Confocal images are either single optical sections (d, k, l) or projections of 36 (a), 5 (b, c), 3 (e), 13 (h, i), or 7 (j) optical sections at 0.5 μm z-separation. Scale bars: a (also for parts b, j), 10 μm; c (also for parts d, h, i, k, l), 5 μm; e, 10 μm; f, 1 μm; g, 0.5 μm.