Fig 2: In vitro calcium responses to histamine, but not algogenic agonists, are enhanced in Dgki-/- mouse DRG neurons.Calcium activity of DRG neurons dissected and cultured from WT and Dgki-/- mice in response to 100 µM histamine (A,B), 1 µM capsaicin (C), 10 µM lysophosphatidic acid (LPA; D), or 100 µM uridine triphosphate (UTP; E). Each experiment ended with exposure to 100 mM KCl to ensure neuron health. Average calcium responses over time in WT and Dgki-/- DRG neurons are shown for histamine experiments in (A). The area under the curve representing the 90 s of agonist exposure was calculated for each responding neuron, plotted in (B-E). F) The proportion of all healthy neurons (neurons respond to KCl) responding to the indicated agonists. For (A-E) the number of responding neurons is indicated in parentheses, and graphs show mean ± SEM. For (F) on each bar the top number indicates the number of neurons responding to the agonist and the bottom number indicates the total number of healthy neurons.

Fig 3: Loss of Dgki enhances histamine-induced scratching in male and female mice.An acute scratching assay was used to test itch responses in Dgki-/- mice following an injection of 500 µg histamine (A-B), 200 µg chloroquine (C-D), or 500 µg ß-alanine (E-F) in WT and Dgki-/- male (A,C,E) and female (B,D,F) mice in 5 minute intervals. The number of mice of each sex tested for all pruritogens is indicated in parentheses in (A) and (B). Graphs show mean ± SEM. p< *0.05, **0.01, ***0.001, ****0.0001.

Fig 4: Dgki deletion does not alter sensitization or recovery in chronic inflammatory or neuropathic pain models.Withdrawal latencies to heat in the Hargreaves assay for 7 days following injection of complete Freund’s adjuvant (CFA) into the plantar surface of the ipsilateral and contralateral hindpaws in male (A) and female (B) Dgki-/- and WT mice. BL = baseline. Response rates to von Frey filaments of the indicated forces in male (C,E) and female (D,F) Dgki-/- and WT mice at baseline (C-D) and 14 days after spared nerve injury (SNI) surgery (E-F) in the ipsilateral and contralateral hindpaws. The number of mice tested is indicated in parentheses. Graphs show mean ± SEM. *p<0.05.

Fig 5: DGKI is enriched in peripheral nociceptors and is absent in neuronal tissues of Dgki-knockout mice.(A) Protein immunohistochemical staining shows that DGKI protein is cytoplasmic in small-diameter (arrowheads) WT mouse DRG neurons and is absent in Dgki-/- mouse DRG neurons. Scale bars = 50 μm. (B-D) Immunoblotting with lysates from female and male WT and Dgki-/- mouse DRG (B), spinal cord (C), and cortex (D) showing that genetic deletion of Dgki eliminates the DGKI protein. DGKI and β-actin bands paired together in each panel are from the same blot. (E-F) Analysis of immunohistochemical staining in WT mouse DRG neurons reveals the percent of neurons expressing different markers that also express DGKI (E) and the percent of DGKI-expressing neurons that also express different markers (F). (G) Histogram showing the soma diameter distribution of DRG neurons expressing DGKI. Number of sections plotted in (E) and (F) labeled on graph in (E). Graphs in (E) and (F) show mean ± SEM.