Depolarization activates PKA-II in nociceptive neurons. (A) HCS microscopy images of rat DRG neurons stimulated with solvent (Ctrl) or KCl (40 mM) for 1 min. Cultures were immunolabeled with UCHL1 to identify the neurons and pRII to quantify PKA-II signaling activity. Red frames mark enlarged image sections; green or red encircled neurons in the left panel indicate the mask to selected or rejected objects, respectively (see Materials and methods). Scale bars, 100 µm. (B) Time course of pRII intensity in DRG neurons stimulated with KCl (40 mM). (C) Dose–response curve of pRII intensity in DRG neurons exposed to KCl (0–80 mM, EC50 = 10 mM) for 3 min. (D) Time course of pRII intensity in DRG neurons treated with veratridine (VT; 100 µM) to open VGSCs in comparison to the KCl (40 mM) response. (E) Size and RIIβ intensity distributions of control and KCl-stimulated DRG neurons used to determine the gating thresholds for the following subgroup analysis. PDE, phosphodiesterase. (F) Cell density plots showing single-cell data of pRII intensities versus the neuronal areas of KCl (40 mM)–stimulated sensory neurons. Dashed lines indicate gating thresholds used to calculate the percentage of cells in the respective quadrant. Combined data of n = 4 experiments with a total of >3,000 neurons per condition. (G) Quantification of responding smaller (<1,400 µm2) and larger (>1,400 µm2) neurons in n = 4 replicate experiments with a total of >3,000 neurons per condition. (H) Cell density plots showing single-cell data of pRII versus RIIβ intensities in the same neurons shown in F. (I) Quantification of responding RIIβ− and RIIβ+ neurons. HCS data in B–D are means ± SEM; n = 3–4 independent experiments; >2,000 neurons/condition; two-way ANOVA with Bonferroni’s test; **, P < 0.01; ***, P < 0.001 indicate significance levels between baseline and stimulated conditions at the respective time point.
Figure 1.

Depolarization activates PKA-II in nociceptive neurons. (A) HCS microscopy images of rat DRG neurons stimulated with solvent (Ctrl) or KCl (40 mM) for 1 min. Cultures were immunolabeled with UCHL1 to identify the neurons and pRII to quantify PKA-II signaling activity. Red frames mark enlarged image sections; green or red encircled neurons in the left panel indicate the mask to selected or rejected objects, respectively (see Materials and methods). Scale bars, 100 µm. (B) Time course of pRII intensity in DRG neurons stimulated with KCl (40 mM). (C) Dose–response curve of pRII intensity in DRG neurons exposed to KCl (0–80 mM, EC50 = 10 mM) for 3 min. (D) Time course of pRII intensity in DRG neurons treated with veratridine (VT; 100 µM) to open VGSCs in comparison to the KCl (40 mM) response. (E) Size and RIIβ intensity distributions of control and KCl-stimulated DRG neurons used to determine the gating thresholds for the following subgroup analysis. PDE, phosphodiesterase. (F) Cell density plots showing single-cell data of pRII intensities versus the neuronal areas of KCl (40 mM)–stimulated sensory neurons. Dashed lines indicate gating thresholds used to calculate the percentage of cells in the respective quadrant. Combined data of n = 4 experiments with a total of >3,000 neurons per condition. (G) Quantification of responding smaller (<1,400 µm2) and larger (>1,400 µm2) neurons in n = 4 replicate experiments with a total of >3,000 neurons per condition. (H) Cell density plots showing single-cell data of pRII versus RIIβ intensities in the same neurons shown in F. (I) Quantification of responding RIIβ− and RIIβ+ neurons. HCS data in B–D are means ± SEM; n = 3–4 independent experiments; >2,000 neurons/condition; two-way ANOVA with Bonferroni’s test; **, P < 0.01; ***, P < 0.001 indicate significance levels between baseline and stimulated conditions at the respective time point.

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