Genetic identification of C fibres that detect massage-like stroking of hairy skin in vivo

Abstract

Stroking of the skin produces pleasant sensations that can occur during social interactions with conspecifics, such as grooming. Despite numerous physiological studies (reviewed in ref. 2), molecularly defined sensory neurons that detect pleasant stroking of hairy skin in vivo have not been reported. Previously, we identified a rare population of unmyelinated sensory neurons in mice that express the G-protein-coupled receptor MRGPRB4 (refs 5, 6). These neurons exclusively innervate hairy skin with large terminal arborizations that resemble the receptive fields of C-tactile (CT) afferents in humans. Unlike other molecularly defined mechanosensory C-fibre subtypes, MRGPRB4(+) neurons could not be detectably activated by sensory stimulation of the skin ex vivo. Therefore, we developed a preparation for calcium imaging in the spinal projections of these neurons during stimulation of the periphery in intact mice. Here we show that MRGPRB4(+) neurons are activated by massage-like stroking of hairy skin, but not by noxious punctate mechanical stimulation. By contrast, a different population of C fibres expressing MRGPRD was activated by pinching but not by stroking, consistent with previous physiological and behavioural data. Pharmacogenetic activation of Mrgprb4-expressing neurons in freely behaving mice promoted conditioned place preference, indicating that such activation is positively reinforcing and/or anxiolytic. These data open the way to understanding the function of MRGPRB4 neurons during natural behaviours, and provide a general approach to the functional characterization of genetically identified subsets of somatosensory neurons in vivo.

Figure 1. In vivo calcium imaging in genetically defined subsets of primary sensory neurons

a, Schematic illustrating AAV infection. LSL, loxP-STOP-loxP cassette. b-e, mGCaMP3.0 expression in somata (b, c) and central afferent fibers (d, e) of MrgprD⁺ (b, d) or MrgprB4⁺ (c, e) neurons in adult mice. Dashed lines indicate lateral margin of spinal cord. Scale bars in (b, d) = 50 and 45 μm, respectively. f, schematic illustrating imaging preparation; components not to scale. g-n, Calcium transients in the central projections of MrgprD⁺ (g, i, k, m) or MrgprB4⁺ (h, j, l, n) neurons, evoked by direct application of KCl to the spinal cord (i, j) or (in a different animal) peripheral injection of α, β-methylene ATP (k, l). Colored rectangles in (g, h) indicate Regions-Of-Interest (ROIs) used in (i, j), respectively; yellow boxes are regions for background subtraction. Scale bar in (g, h) = 40 and 20 μm, respectively. Red arrows (i-l) indicate time of stimulus delivery. (m, n) Quantification of peak ΔF/F values before (open bars) vs. after (filled bars) stimulation. **, p<.01; ***, p<.001. All data in this and other figures are mean±SEM.

Figure 2. Activation of MrgprD fibers by pinching

a, b, Schematics illustrating pinching (a) and stroking (b) stimuli. c, ROIs used for imaging in (d-i). Green rectangle (upper right) is region for background subtraction. Scale bar, 9 μm. d, Superimposed traces from different color-coded ROIs (c) in a single trial consisting of 4 pinch stimuli (gray bars). e, Average response to pinching in a single animal (n=4 trials, 7 stimuli total). See also Supplementary Fig. 5a-e. f, Response to 4 brushing stimuli (gray bars) delivered to pinch-sensitive digit (d), in same ROI (c). See also Supplementary Fig. 9g-l. g, Average response to brushing (n=1 mouse/2 trials, 7 stimuli). h, i, MPI ΔF/F_peak (upper) or integrated area (lower) from curves in (e, g), respectively. Open and filled bars are 5 frames before and 40 frames after stimulus delivery, respectively (see Supplementary Note 9). j, MPI ΔF/F_peak in two different ROIs (red and turquoise bars) from each of three mice. See also Supplementary Table 2.

Figure 3. Activation of MRGPRB4 fibres by stroking

a, b, Schematics illustrating brushing (a) and pinching (b) stimuli. c, F image of GCaMP3.0 at one frame point during stimulation and ROIs used for imaging in d–i. The dark-green rectangle (lower right) is the region used for background subtraction. Scale bar, 8.5 μm. d, Superimposed traces from different colour coded ROIs (c) in a single trial of three brush stimuli (turquoise bars). e, Average response to brushing from a single mouse (n=5 trials, ,~3-6 stimuli per trial). See also Supplementary Fig. 5g–j. f, Response to five pinching stimuli (turquoise bars) in brush-sensitive region (d), in same ROI (c). See also Supplementary Fig. 9a–f. g, Average response to pinching from the same animal (n=2 trials, 10 stimuli total). h, i, MPI ΔF/F_peak (upper) or integrated area (lower) calculated from the curves in e, g, respectively. Open and filled bars are 5 frames before and 20 frames after stimulus delivery, respectively. NS, not significant. j, MPI ΔF/F_peak in two different ROIs (red and blue graphs) from each of three independent mice. See also Supplementary Table 3. **P<0.01;***P<0.001. All data are mean +/− s.e.m.

Figure 4. Activation of MRGPRB4 neurons promotes conditioned place preference

a, b, Schematic of experiment (a) and CPP apparatus (b). I.N.P. and I.P. indicate initially non-preferred and preferred chambers, respectively (c, schematic, ‘pre-test’). c, Top: absolute time (s) in each chamber before (open bars; ‘pre’) versus after (filled bars; ‘post’) conditioning for the experimental group. Train drug indicates CNO or saline paired with the indicated chamber. Bottom: schematic of experimental design. Cham., chamber. d, Time in I.N.P. chamber for experimental (replotted from panel c for direct comparison) and control groups. **P< 0.01; ***P< 0.001; NS, not significant. See Supplementary Note 10 for F values and Supplementary Fig. 13. e–i, Difference scores ((time in indicated chamber after training) – (time in chamber before training)) for experimental (e, n=15) and control (f, g, h, i, n=9, 6, 8, 10, respectively) groups. j, Comparison of mean difference scores for the I.N.P. chamber for the experimental (e) and the pooled control (f, g, h, i) groups. There was no significant difference between control groups. *<P,0.05, ** P< 0.01; *** P< 0.001.