Barry, Devin M.

Chen, Zhou-Feng

Gao, Fang

Liu, Benlong

Liu, Juan

Liu, Xian-Yu

Liu, Xue-Ting

Tao, Ailin

Wilhelm, Steven

Yang, Qianyi

Yin, Jun

Zeng, Xiansi

English

Digital Commons@Becker

  1  Supplementary Information    Exploration of sensory and spinal neurons expressing gastrin-releasing peptide in itch and pain related behaviors   Barry et al.      2   Supplementary Figure 1. Detection of Grp transcript in DRG. a IHC with eGFP antibody in GrpWT DRG.  b and c RNAScope method of Grp ISH in WT DRG (b) and Grp KO DRG (c). d Grp ISH using conventional DIG method in WT DRG. e The levels of Grp transcript in DRG are comparable at different age. P1, postnatal day 1. Data represent mean ± s.e.m., n = 3-4 mice per age group. Scale bars, 50 μm. Source data are provided as a Source Data file.      3   Supplementary Figure 2. Validation of GrptdTom neuorns in DRG, spinal cord and skin. a Epifluorescent image from GrpWT; Ai14 DRG. b IHC images of tdTom, CGRP and DAPI in GrpWT; Ai14 nape skin. Scale bars, 50 μm. c Epifluorescent images of tdTomato neurons in cervical spinal cord from GrptdTom mice. Scale bar, 100 μm. d ISH of Grp with tdTomato neurons. Scale bar, 20 µm. e Percentage of tdTom- or Grp-expressing neurons that express Grp or tdTom, respectively. n = 3 mice and 9 sections. f-h IHC images of tdTom, CGRP and NF-H in GrpWT; Ai14 glabrous skin. Image in g shows GrptdTom fiber with no NF-H (blue) expression and h shows NF-H fibers that do not express tdTom. Scale bar in f 50 μm and in g, 20 μm. i-m IHC images of tdTom, CGRP and DAPI in GrptdTom tongue (i), cornea (j), bladder (k) and skeletal muscle (l, m). Arrow indicates neuromuscular junction in m. Scale bars in l– m, 50 μm.       4    Supplementary Figure 3. Sensory GrptdTom neurons are mostly small sized neurons. a IHC of tdTomato and NF-H in GrptdTom DRG. Scale bar, 50 μm. b Diagram of DRG neurons with tdTomato and NF-H expression. c Frequency Distribution (%) of Perikarya Area (μm2) for GrptdTom, CGRP or IB4 neuron populations in DRG. Neurons from 6 DRG sections were measured.    5    Supplementary Figure 4. Sensory GrptdTom neurons co-express CGRP and TRPV1. a IHC image of tdTomato, CGRP and IB4 from GrptdTom DRG cultures. Arrows indicate GrptdTom neurons co-expressing CGRP and arrowheads indicate IB4-binding. b IHC image of tdTomato, TRPV1 and NF-H from GrptdTom DRG cultures. Arrows indicate GrptdTom neurons co-expressing TRPV1. Scale bar, 100 μm.      6   Supplementary Figure 5. Characterization of GrpChR2 neurons in DRG and fibers in skin. a IHC images of eYFP, CGRP and IB4 in GrpChR2 DRG. Scale bar, 50 μm. b eYFP image from GrpWT; Ai32 DRG. Scale bar, 50 μm. c IHC image of eYFP, βIII-Tubulin and DAPI in GrpWT; Ai32 nape skin. Scale bar, 100 μm.   d IHC image of eYFP and βIII-Tubulin in GrpChR2 cheek skin. Scale bar, 100 μm.   e IHC images of eYFP, CGRP and DAPI merge in GrpChR2 glabrous skin. Scale bar, 100 μm.   f and g IHC image of eYFP and DiI in GrpChR2 DRG (f) or TG (g) 10 days after i.d. nape or cheek injection of DiI tracer. Scale bar, 50 μm.     7   Supplementary Figure 6. Opto-stimulation of Grp fibers induces itch-specific behavior. a Number of scratches in 5 min induced by 3s – 1, 5, 10 or 20 Hz light stimulation of nape skin in GrpChR2 and GrpWT mice (b) Latency to scratch during 3s – 20 Hz light stimulation of skin in GrpChR2 mice. c Percentage of 20 Hz light stimulations eliciting scratching behavior in GrpWT, GrpChR2, GrpChR2 morphine-treated and GrpChR2 BB-sap-treated mice. d Number of spontaneous scratches in 30 min before and after the 5-min light stimulation experiment in GrpWT, GrpChR2, GrpChR2 morphine-treated and GrpChR2 BB-sap treated mice.  e and f Number of scratches (e) and wipes (f) in 30 min induced by CNO (1 mM) injections into the cheek skin in GrpGq and GrpWT mice. Data are presented as mean ± s.e.m.,  n = 8 – 10 mice in a-d, n = 3 mice in e and f, one-way ANOVA with Tukey post hoc in a and c, two-way RM ANOVA with Tukey post hoc in c, unpaired t test in e and f, **p < 0.01, ***p < 0.001, ns – not significant. Source data are provided as a Source Data file.      8   Supplementary Figure 7. Conditional deletion of sensory Grp is specific and does not affect pain responses. a ISH images of Grp expression in spinal cord sections from GrpF/F and GrpF/F; Nav1.8Cre mice. Right panels are high power images of the boxed areas in left panels. Scale bar, 100 μm for left panels, 20 μm for right panels.  b IHC of GRP in the suprachiasmatic nucleus from GrpF/F and GrpF/F; Nav1.8Cre mice. Scale bar, 50 µm. c- e Mean biting/licking time induced by i.d. calf injection of (c) CQ (100 μg), (d) SLIGRL-NH2 (100 μg), and (e) BAM8-22 (100 μg) in GrpF/F and GrpF/F; Nav1.8Cre littermates.  f Mean biting/licking time induced by i.d. calf injection of saline (10 µl) and CQ (100 μg). g Mean biting/licking time induced by i.d. calf injection of CQ (100 μg) following i.t. injection of saline (10 µl) or morphine (0.3 nmol). h Mean biting/licking time induced by i.d. calf injection of CQ (100 μg) on control mice and BB-sap treated mice. Data are presented as mean ± s.e.m., n = 6-7 mice. *p < 0.05, ***p < 0.001, unpaired t test. Source data are provided as a Source Data file.    9   Supplementary Figure 8. Ablation of GrptdTom neurons is restricted to the spinal cord.  a Epifluorescent images of tdTomato neurons in DRG, cingulate cortex and hippocampus from GrptdTom control and GrptdTom ablated mice. Scale bars, 50 μm for DRG, 100 μm for cingulate cortex and 100 μm for hippocampus, respectively. b IHC images of tdTom with PKC or Pax2 and ISH images of Grpr or Npr1 expression in cervical spinal cord sections from GrptdTom control and ablated mice. Scale bars, 20 μm for tdTom and 100 μm for ISH. c Mean number of PKC, Pax2, Grpr or Npr1 neurons from control and ablated mice. Data are presented as mean ± s.e.m., n = 3 mice and 10 sections, unpaired t test in c. Source data are provided as a Source Data file.    10     Supplementary Figure 9. Spinal GRP neurons are heterogenous. a ISH images of Grp and Nmbr in the superficial dorsal horn. b Diagram of overlapping expression of Grp and Nmbr in the superficial dorsal horn. c ISH images of Grp and Tacr3 in the superficial dorsal horn. d Diagram of overlapping expression of Grp and Tacr3 in the superficial dorsal horn. e Single-cell qRT-PCR results from GrptdTom dorsal horn neurons.  n = 10-15 neurons.   11  Supplementary Table 1. Fiber projections in GrptdTom mice.    * GrptdTom fibers were observed only in nerve bundles going through muscle but did not innervate any muscle fibers.  Tissue Fibers Tissue Fibers Skin (Hairy, Glabrous, Mystacial)  Retina +(rare) Unmyelinated:  Cornea +(rare) Epidermal free nerve endings + Tongue +(rare) Circular/penetrating follicle neck endings + Bladder +(rare) Bush/cluster endings +     Skeletal muscle -* Myelinated:  Esophagus - Circumferential nerve endings - Heart - Lanceolate/club endings - Intestine - Meissner corpuscles - Kidney - Merkel cells and endings - Liver Lung - Sebaceous gland endings - Testis - Vibrissa follicle-sinus complex (Vib) - Smooth Muscle -     

Exploration of sensory and spinal neurons expressing gastrin-releasing peptide in itch and pain related behaviors

https://digitalcommons.wustl.edu/cgi/viewcontent.cgi?filename=0&article=10027&context=open_access_pubs&type=additional

Exploration of sensory and spinal neurons expressing gastrin-releasing peptide in itch and pain related behaviors

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