Calcium imaging

Similar to previous work [10, 33], on the day of imaging cells were washed 2 times with assay buffer: Hanks’ balanced salt solution (14025–126, Thermo Fisher; 140 mg/L CaCl₂, 100 mg/L MgCl₂-6H₂O, 100 mg/L MgSO₄-7H₂O, 400 mg/L KCl, 60 mg/L KH₂PO₄, 350 mg/L NaHCO₃, 8 g/L NaCl, 48 mg/L Na₂HPO₄, 1 g/L D-glucose) supplemented with 2.4 g/L HEPES, 2 g/L D-glucose, and 0.1% w/v fatty acid–free BSA (A6003, Sigma-Aldrich), at pH 7.3. Cell were then incubated with 2 μM Fura-2, AM (F1221, Invitrogen) in 0.02% Pluronic F-127 (P-3000MP, Invitrogen) in assay buffer for 1 h at room temperature. Cells were washed 3 times with assay buffer, then maintained at room temperature for 30 min before imaging. Assays were performed on an Eclipse Ti microscope (Nikon, Tokyo, Japan) with a CFI Plan Fluor 20x objective (Nikon) and a DG-4 light source (Sutter, Novato, California). Cells were alternately excited for 500 ms at 340 nm and for 250 ms at 380 nm. Emission was measured at 510 nm and recorded using a Clara DR-328G-C01-SIL CCD camera (Andor, Belfast, United Kingdom) and NIS Elements imaging software (Nikon). During imaging, coverslips were placed in a chamber (Model RC-26GLP, Warner Instruments, Hamden, Connecticut) mounted on microscope stage adapter and were perfused with 30°C buffers (heated with SH-27B inline heater, Warner Instruments) at a flow rate of 4.2 mL/min (using a Minipuls 3 Peristaltic Pump, Gilson, Middleton, Wisconsin). After collecting 90 s of baseline Fura-2 ratios while perfusing with assay buffer, cells were perfused with agonist for 90 s, followed by assay buffer for 180 s, then 100 mM KCl for 30 s to test for neuron health. Agonists and 100 mM KCl solutions were made with assay buffer.

The agonists used were 100 μM histamine, 1 μM capsaicin, 10 μM lysophosphatidic acid (LPA), or 100 μM uridine triphosphate (UTP). Histamine activates the H1 receptor on sensory neurons to induce itch responses [19]. Capsaicin is the chemical ligand for the ion channel TRPV1 (transient receptor potential vanilloid 1), an important receptor for itch and pain signaling [19, 34]. Lysophosphatidic acid acts on LPA₁, LPA₂, and LPA₃ receptors (Gα_q-GPCRs) and induces nociception in peripheral neurons [35, 36]. Uridine triphosphate binds to P2Y receptors and activates and sensitizes small-diameter DRG neurons [37, 38]. We used histamine because of the in vivo scratching phenotypes we observed in response to histamine injection. We used capsaicin, LPA, and UTP for algogens because their receptors are expressed in a relatively high percentage of rodent DRG neurons [37, 39, 40].

For analysis, we calculated the Fura-2 ratio (ratio of the emission following excitation at 340 nm/380 nm). We excluded cells that had high baseline Fura-2 ratios (>1.0) or failed to respond to KCl (did not reach ratio of at least 1.0 during KCl exposure). To normalize to baseline, the 60 s of Fura-2 ratios measured immediately preceding agonist exposure were averaged for a given cell, and that average was subtracted from each data point for that cell throughout the whole assay period. Only cells that responded to agonist (had a minimum increase in Fura-2 ratio of 0.1 over baseline average for 2 consecutive time points during period of agonist exposure) were included in our analyses. Area-under-the-curve (AUC) values were calculated on a cell-by-cell basis, using the baseline-normalized values, for the 90-s period of agonist exposure.

DRG cultures from one WT and one Dgki^-/- mouse (cultured concurrently) were tested each day, alternating coverslips in a random order. For histamine, we used 4 male and 3 female mice each for WT and Dgki^-/- (14 mice total); here, we present responses from the 114 WT neurons and 124 Dgki^-/- neurons that were activated by histamine. For capsaicin, we used 2 males of each genotype, and we present the 110 WT and 86 Dgki^-/- neurons that responded to capsaicin. For LPA, we used 1 male and 1 female of each genotype, and we present the 116 WT neurons and 93 Dgki^-/- neurons that responded to LPA. For UTP, we used 4 males of each genotype, and we present the 116 WT neurons and 165 Dgki^-/- neurons that responded to UTP.