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[Preprint]. 2023 Dec 2:2023.12.01.569650. [Version 1] doi: 10.1101/2023.12.01.569650

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Figure 4: — A. Diagram of the trigeminal in vivo calcium imaging experiment with single hair-pull stimuli and genetic model for labelling Calca nociceptors. Calca^Cre mice were crossed with Ai95 Cre dependent GCamp6f strain. To postnatally limit the labelling to persistent Calca nociceptors offspring was injected at P1–P3 with AAV/PHP.S-CAG-Fl-tdTomato virus. B. Representative calcium transients recorded in Calca⁺ heat-insensitive hair follicle nociceptors (green) and heatresponsive polymodal nociceptors (magenta) neurons evoked by single hair-pull and heat stimuli. C. Forces activating HFNs (green) were markedly smaller (median threshold: 1 mN, n=20) than for polymodal nociceptors (magenta, median threshold: 3 mN, p=0.0037, n=21 for polymodal nociceptors, unpaired Student’s t-test). Data is shown as individual replicates with median and interquartile range overlay. D. Cumulative distribution of activation thresholds for HFNs (green) and polymodal nociceptors (magenta) neurons evoked by single hair-pull shows a markedly higher sensitivity to hair-pull in HFNs. Statistical differences were assessed by two-way ANOVA (F _{(1, 10)}=6.521, p=0.0287). E. Mouse HFNs express low levels of Trpm8. Sample images of fluorescence in situ hybridization co-localization of HFN’s markers Bmrp1b, Calca and Trpm8. F. Bmpr1b⁺ neurons express Calca and majority of them expresses low levels of Trpm8 transcripts. G. Genetic model of Piezo2 loss of function in HFNs. GCamp6f expressing conditional Piezo2 KO was generated by crossing the Trpm8^iCre mouse strain with double transgenic Ai95-Piezo2^fl/fl line to obtain homozygous Piezo2 cKO. To limit the labelling to persistent Calca nociceptors offspring was injected at P1–P3 with AAV/PHP.S-CAG-LSL-mNeptune. H. Representative calcium transients of functionally isolated HFNs recorded in control mice. Demonstrating high selectivity of HFNs. I. Hair follicle nociceptors (green) sensitivity to hair pull force (median threshold: 1 mN, n=29) was markedly diminished in the absence of Piezo2 (magenta, median threshold: 3.5 mN, n=21, p=0.0158, upaired Student’s t-test). Data is shown as individual replicates with median and interquartile range overlay. J. Percentage of cells responding to hair-pull in Piezo2cKO was diminished by almost half (3.8% in Piezo2 cKO vs 6.8% in control, p=0.0418, Fisher’s exact test). K. Additive effect of Piezo2 on hair follicle nociceptor responses during hair-pull. Cumulative distribution of hair follicle nociceptors activation scaled to total recruitment during hair-pull trial, (two-way ANOVA (F_{(1, 19)}=3556, p<0.0001, n=29 neurons from 7 mice for control and n=22 neurons from 9 mice for Piezo2KO).