Fig 2: Increased Grp+, but not Tac1+, neurons and upregulation of Grp mRNA levels in Dry skin-induced chronic itch DRGs. (a)–(d) Cervical DRG images of Grp ISH. Grp ISH is absent in Grp KO (a). Comparison of control (b) and dry skin (c) DRG indicates the percentage of Grp+ neurons is nearly doubled in dry skin mice (d). (e)–(h) Cervical DRG images of Tac1 ISH. Tac1 ISH is unaffected in Grp KO (e). Comparison of Tac1 ISH from control (f) and dry skin (g) shows no significant difference in percentage of Tac1+ neurons in DRG (h). (i) Gel electrophoresis images of RT-PCR products also shows an increase of Grp mRNA in dry skin DRG samples compared to control and, whereas Tac1 mRNA increase in dry skin DRG is less apparent and Actb levels appear similar between the two groups. No products were detected in H2O and no-RT (?RT) samples, whereas RT samples had a single band detected for each mRNA. DL, DNA ladder. (j, k) Quantitative RT-PCR results from control and dry skin DRG show a approximately 7 fold increase in Grp mRNA levels (j) and a approximately 1.2 fold increase in Tac1 mRNA levels (k) in dry skin. Scale bar = 100 µM. Data are presented as mean ± SEM. n = 3 mice per group and 10–12 DRG sections per group in. n = 4–6 mice per group and 20–24 DRGs per animal in i-k. Unpaired t-test in d, h, j, and k. * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 3: Grp expression is enriched in a subset of sensory neurons with both peptidergic and non-peptidergic markers.a, c, e IHC image of tdTomato with CGRP (a), IB4 (c), and TRPV1 (e) in GrptdTom DRG. Arrowheads indicate double stained neurons. Scale bar, 50 µm. b, d, f Venn Diagram of DRG neurons with tdTomato/CGRP expression, tdTomato/IB4-binding, and tdTomato/TRPV1 expression from GrptdTom mice. g ISH of Grp and Mrgpra3 in WT DRG. Scale bar, 20 µm. h Diagram of DRG neurons with Grp and Mrgpra3 expression. i ISH of Grp and Hrh1 in WT DRG. j Diagram of DRG neurons with Grp and Hrh1 expression. n = 3 mice and 9 sections.

Fig 4: Dorsal root rhizotomy causes a dramatic loss of Pap and Cgrp but significantly smaller changes of bombesin and Pkc? in dSC. L4, L5, and L6 central roots of two month old wild type mice were transected, and mice were examined two weeks following surgery. Sections of the lumbar enlargement were immunostained to examine the loss of proteins and peptides caused by dorsal root axonal degeneration and the contralateral side of the same spinal cord section was used as the control. (A-F) Sections were immunostained with antibodies against bombesin (red) and Pkc? (green), a protein highly synthesized in dSC neurons (control side (A-C) and transected side (D-F)). Comparable and modest loss of both bombesin and Pkc? immunoreactivity are found after the dorsal root rhizotomy. Enhanced green fluorescence and abnormal morphology of the transected dorsal root were also noted, which may be caused by axonal inflammation after injury. (G-L) Sections immunostained with antibodies against bombesin (red) and Pap (green). As noted, dSC Pap is dramatically decreased after the dorsal root rhizotomy. (M-R) Sections immunostained with antibodies against bombesin (red) and Cgrp (green), another marker derived from DRG neurons. Similar to Pap, dSC Cgrp is also greatly reduced following the dorsal root rhizotomy. Scale bar = 100 µm. (S) Quantification and statistical analysis of dynamic changes of dSC Grp, Pkc?, Pap, and Cgrp following the dorsal root rhizotomy. The loss of dSC Grp after dorsal root rhizotomy is significantly different from those of Pap and Cgrp (P < 0.01), and the same is true for dSC Pkc?. In contrast, no significant differences are found between Grp and Pkc? (P = 0.44) or Pap and Cgrp (P = 0.25).

Fig 5: Schematic representations of the connections between peptidergic neuronal types. The numbers and the arrow thickness indicate the proportion of cells receiving more than or equal to three appositions (statistics in Tables 2, 3). A, In WT mice, there was a preferential unilateral direction of communication between AVP-VIP and GRP-CALR (blue). While the other connections indicated are all reciprocal, there are few AVP to VIP contacts and more GRP to CALR contacts than the converse (***p < 0.001, **p = 0.008). B, In contrast to WT mice, in VIP-KO mice, there was no evidence of preferential direction of communication between peptidergic cell types.