Electrophysiological properties of PrP-GFP neurons. a, Most of these exhibited tonic firing (99 of 138 cells, 72%) in response to suprathreshold square wave current injection (1 s), as shown in this figure. The remaining cells showed initial bursting (13%), single-spiking (7%), or unresponsive (9%) patterns. b, The relationship between firing frequency and injected current (f–I), with data pooled from tonic and initial bursting cells. Because individual cells fired across a slightly different range of injected currents, the numbers of cells that responded at different current steps varied, and these are shown in brackets. Error bars represent SEM. The f–I relationship demonstrates that most cells (101 of 117 cells, 86%) reached their maximal steady state with injections of <150 pA current.

Primary afferent input to PrP-GFP cells. a, b, Characterization of primary afferent input to PrP-GFP cells receiving monosynaptic Aδ with monosynaptic C fiber input, and monosynaptic C fiber input only, respectively. Left panels, Examples of eEPSCs resulting from low-frequency (0.05 Hz) dorsal root stimulation at Aβ (25 μA), Aδ (100 μA), and C fiber (500 μA) intensities; each trace is an average of three recordings. Right panels, Examples of eEPSCs resulting from high-frequency dorsal root stimulation (25 μA/20 Hz; 100 μA/2 Hz; 500 μA/1 Hz); each displays 20 superimposed traces. The cell shown in a receives polysynaptic C fiber input, in addition to the monosynaptic Aδ and monosynaptic C fiber input. The polysynaptic C component is seen as the shorter latency response revealed at 500 μA stimulation (arrow), which displays failures during high-frequency stimulation. There are larger amplitude responses superimposed upon the monosynaptic Aδ and monosynaptic C fiber eEPSCs, which display failures during high-frequency stimulation and indicate action potential firing. In addition to monosynaptic C-fiber input, the cell shown in b features a small polysynaptic Aδ fiber response, which is evoked during low-frequency stimulation at 100 μA and fails during high-frequency stimulation.

Responses of PrP-GFP neurons to TRP channel agonists and antagonists. a, Effects of capsaicin (2 μm) on PrP-GFP cells. ai, Representative raw traces show an increase in mEPSC events upon capsaicin application (2 μm). aii, A cumulative probability plot shows a significant decrease in the duration of interevent intervals of mEPSCs (Kolgomorov–Smirnov test, p < 0.05, 13 of 16 cells). aiii, Pooled data show a 16-fold increase in the rate of mEPSC events/min caused by capsaicin application (n = 13, paired t test, p < 0.01). b, The rate of baseline mEPSC events/min increased by fivefold when the temperature in the recording chamber was raised (n = 36 at room temperature and n = 24 at 32°C, unpaired t test, p < 0.01). c, Effects of icilin (20 μm) on PrP-GFP cells. ci, Representative traces show an increase in mEPSC events caused by icilin application (20 μm). cii, A cumulative probability plot shows a significant decrease in the duration of mEPSC interevent intervals (Kolgomorov–Smirnov test, p < 0.05, 14 of 24 cells). ciii, Pooled data show a twofold increase in mEPSC events/min by icilin application (n = 14, paired t test, p < 0.01). d, Pooled mEPSC events/min data showing the response of cells to icilin in the presence of A967079 (n = 5, paired t test, p < 0.05). This suggests that at least part of the action of icilin was mediated through TRPM8-expressing afferent terminals that are presynaptic to PrP-GFP cells. All recordings were made in the presence of TTX. Error bars represent SEM.

Neuronal morphology. a–e, Examples of Neurolucida reconstructions for five of the PrP-GFP neurons, which were recorded in parasagittal slices. In each case, soma and dendrites are shown in blue and axons in red. Top solid line in each drawing indicates the gray-white matter border. Dashed lines indicate the boundaries between laminae I, IIo IIi, and III. Note the variable dendritic morphology, and also the variation in the distribution of the axonal arborization. For the cells shown in a–c, part of the axon enters lamina I, where it gives rise to axonal boutons. f, Confocal optical section through the soma of the cell illustrated in e, showing GFP (green) and Neurobiotin (magenta). D, Dorsal; V, ventral. Scale bars: a–e, 100 μm; f, 20 μm.

Morphometric and electrophysiological data for the recorded neurons. a, Scatter plot of RC versus DV extent of the dendritic trees for the 81 neurons with well filled dendrites. The line indicates an RC/DV ratio of 3.5, which was used by ) to distinguish lamina II central cells in the rat. Very few of the cells have a ratio greater than this (i.e., fall below the line), and these therefore would not be classified as central cells based on this definition (see text). Blue and red symbols represent cells identified as nNOS or galanin immunoreactive. Black symbols represent cells that were not tested (n.t.) or showed neither type of immunoreactivity. This scheme also applies to parts c and f. b, Polar histogram of the dendritic tree for the cell illustrated in b (for further details, see text). The concentric circles represent 50, 100, and 150 μm. Bins corresponding to orientations between 45°–135° and 225°–315° were grouped (shown in dark blue) and represent predominantly RC orientation. Those for 315°–45° and 135°–225° (light blue) correspond to predominantly DV orientation. c, Scatter plots showing the values for RC and DV dendritic length obtained from the polar histograms of the 81 cells with well-filled dendrites. For nearly all cells RC > DV, the plots therefore fall below the line (which represents RC = DV). There is considerable variation in total dendritic length, which is the sum of RC and DV. d, A frequency histogram for the density of dendritic spines shows considerable variation in their density across the population. e, There is a highly significant positive correlation between spine number and mEPSC frequency, for those cells in which the frequency was measured in recordings made at room temperature. f, Scatter plot of axonal extent along the DV and RC axis. With the exception of one cell, which had an axon that traveled ∼700 μm in the ventral direction (arrow), all cells had rostrocaudally elongated axonal arbors, which were considerably longer than their dendritic trees (compare with part a). g, Electrophysiological properties that differed significantly between the nNOS⁺ and galanin⁺ cells. The nNOS⁺ cells (n = 10) had smaller input resistances than the galanin⁺ cells (n = 10), as well as greater action potential heights and shorter widths (n = 8 for the galanin cells, because 2 of these were unresponsive). Error bars represent SEM.

Neurochemistry of recorded PrP-GFP neurons. a, b, Part of the axon of a recorded cell, labeled with Neurobiotin (NB, magenta) shown in a projection of three optical sections (0.5 μm z-spacing) from a section immunostained with galanin antibody (green). Arrowheads indicate three boutons that show galanin immunoreactivity. c, Part of the axon of another cell labeled with Neurobiotin in a projection of 29 optical sections (0.5 μm z-spacing). Numbered boxes represent the regions shown in the insets, in which nNOS immunoreactivity (green) is also illustrated. Insets 1–3, Single optical sections; show nNOS in axonal boutons. Inset 4, Projection of three optical sections; shows nNOS in an intervaricose portion of the axon. Scale bar, 5 μm.

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.

Fos expression following intraplantar capsaicin injection in a transverse section from a PrP-GFP mouse. a–c, Immunoreactivity for GFP (green), nNOS (blue), and Fos (red) in the ipsilateral superficial dorsal horn in a confocal image stack (20 optical sections at 1 μm z-spacing). d, Merged image. Numerous Fos⁺ cells are present, and one of these (arrowhead) contains nNOS but not GFP. Three GFP⁺ cells that are nNOS immunoreactive, but lack Fos, are indicated with arrows. Scale bar, 20 μm.

Selective innervation of lamina I giant cells by GFP axons in the PrP-GFP mouse. a, Part of the soma (s) and dendrites of a giant cell seen in a horizontal section through lamina I of a perfusion-fixed mouse. The section was stained with antibodies against VGAT, VGLUT2, GFP, nNOS, and gephyrin, and this image is a projection of 24 optical sections at 0.3 μm z-spacing. The cell is outlined by numerous VGAT- and VGLUT2-expressing boutons. b–e, Higher-magnification view of the region indicated by the box in a in projections of 3 optical sections. b, The VGAT boutons in contact with the dendrite are associated with gephyrin puncta, indicating that these are sites of inhibitory synapses. c–e, The images reveal that many of these VGAT boutons contain both GFP and nNOS (some marked with arrows). f, Quantitative data for the inhibitory input to 25 lamina I giant cells. Each of these is shown in a separate column, and the vertical axis represents the percentage of the VGAT boutons apposed to gephyrin puncta on the cell that were immunoreactive for nNOS and/or GFP. The cells are ranked in decreasing order of the percentage of labeled boutons. For the great majority (21 of 25 cells), well over half of the inhibitory input is derived from boutons that contained both GFP and nNOS. Scale bars: a, b–e, 10 μm.

Innervation of lamina I NK1r⁺ neurons by PrP-GFP axons. a–d, Projected confocal images (8 optical sections at 0.3 μm z-spacing) to show immunostaining for NK1r (red), GFP (green), and VGAT (blue) in a horizontal section through lamina I from a PrP-GFP mouse. A cell with strong NK1r immunoreactivity receives numerous contacts from VGAT⁺ boutons, many of which contain GFP. d, Inset, Enlarged view of the region in the box, with three immunofluorescent channels visible: GFP (green), VGAT (blue), and gephyrin (red). The two GFP⁺/VGAT⁺ boutons that are indicated with arrowheads are associated with gephyrin puncta at the point where they contact the NK1r cell. e, Frequency histogram showing GFP⁺/VGAT⁺ boutons as a percentage of all of the VGAT boutons that were apposed to gephyrin puncta on the 60 NK1r⁺ cells. Scale bar: a–d, 20 μm.

Schematic diagram summarizing the inputs to and outputs from the PrP-GFP cells. Results from the present study suggest that these cells are innervated by several types of unmyelinated primary afferent, including peptidergic C fiber nociceptors that express TRPV1, cooling-responsive (TRPM8⁺) fibers, and nonpeptidergic C nociceptors that express Mrgd. In addition, they appear to be innervated directly by Aβ/Aδ LTMR afferents, although this is shown with a question mark, as it is not clear whether these form functional synapses. Some of these cells have axons that enter lamina I, where they preferentially innervate giant cells and some NK1r⁺ neurons. However, all of the cells also have extensive axonal arborizations in lamina II, and these are thought to include inhibitory islet cells (with which they make reciprocal connections) and excitatory vertical cells (data from ). Vertical cells in lamina II can form synapses on NK1r⁺ neurons in lamina I (); however, it is not known whether these include those cells that are directly innervated by the PrP-GFP cells, and so this connection is shown with a question mark. Dashed lines indicate the borders of lamina II.