Figure 4.

Receptor-induced decrease of PI(4,5)P₂ in DRG neurons and HEK 293 cells. A, Confocal images of WT mouse DRG neurons transduced with green PI(4,5)P₂ sensor BacMam and human muscarinic cholinergic receptor 1 (M1). Images are representatives of reporter distribution before and after application of either the inflammatory cocktail (same as in Fig. 1) or control solution (2 mm Ca²⁺ NCF). Measurements were conducted in 2 mm Ca²⁺ NCF solution. B, Graphs correspond to fluorescence intensities plotted along the lines indicated in each image in A before (red) and after (black) application of inflammatory cocktail or control. C, Representative traces of time course changes for green PI(4,5)P₂ sensor BacMam in response to cocktail followed by 100 μm carbachol in DRG neurons. D, Averaged time course of fluorescence density changes for green PI(4,5)P₂ sensor BacMam in response to cocktail followed by 100 μm carbachol in DRG neurons. Four GFP⁺ neurons showed no changes in red fluorescence in response to cocktail application and only one of those four responded to carbachol; these cells were excluded from the summary. E, Statistical analysis of fluorescence density at baseline, 40 s after application of cocktail just before application of carbachol and 40 s after application of carbachol in neurons (n = 31) from two different DRG neuron preparations from two mice. Bars represent mean ± SEM; statistical significance was calculated with one-way ANOVA (F_(3,124) = 21.07, p = 4.5 × 10⁻¹¹). Fluorescence density values were normalized to baseline. F, Time course of fluorescence density changes for green PI(4,5)P₂ sensor BacMam in response to two applications of control solution (2 mm Ca²⁺ NCF) in DRG neurons (n = 12). G, Confocal images of HEK 293 cells transduced with green PI(4,5)P₂ sensor BacMam as reporters of PI(4,5)P₂ and human muscarinic receptor 1 (M1) receptor. Images are representatives of reporter distribution before and after application of either 100 μm carbachol or control solution (2 mm Ca²⁺ NCF). Measurements were conducted in 2 mm Ca²⁺ NCF solution. H, Graphs correspond to fluorescence intensities plotted along the lines indicated in each image in G before (red) and after (black) application of 100 μm carbachol or control. I, Representative traces of time course changes for green PI(4,5)P₂ sensor BacMam in response to carbachol. J, K, Average time course of fluorescence density changes for green PI(4,5)P₂ sensor BacMam in response to carbachol or control (2 mm Ca²⁺ NCF) in HEK 293 cells. L, Statistical analysis of fluorescence density at baseline and after application of carbachol (n = 18) or control (n = 11). Bars represent mean ± SEM; statistical significance was calculated with one-way ANOVA (F_(2,44) = 60.03, p = 2.7 × 10 −¹³). Fluorescence density values were normalized to baseline.