Fig. 2

The impact of the HCN channel on the membrane potential and intracellular calcium concentration [Ca²⁺]_i.*p < 0.05, **p < 0.01, ***p < 0.001 compared to control; #p < 0.05, ##p < 0.01, ###p < 0.001 compared to the untreated experimental group measured at the same time interval. a HCN channel characterized by a fluorescence resonance energy transfer (FRET)-derived measurement of the membrane potential. The schematic illustration was modified from the description by Thermo Fisher Scientific. The FRET pair was composed of a highly fluorescent, mobile, voltage-sensitive acceptor oxonol (DiSBAC2) and a fluorescent, membrane-bound coumarin-phospholipid FRET donor (CC2-DMPE). In resting cells, both members of the FRET pair bind to the outer surface of the cell membrane, resulting in efficient FRET. When the cells are depolarized, the oxonol dye translocates to the inner surface of the cell membrane, resulting in diminished FRET. The emission rate (the ratio of the donor emission to acceptor emission) reports changes in the membrane potential and is low in polarized cells and increases in depolarized cells. Representative images of primary microglia supplied with VSPs. Fluorescence microscopy revealed the emission of 460 nm (blue) and 580 nm (red; scale bars = 100 μm). b Microglia exposed to the FRET pair CC2-DMPE and DISBAC2 were untreated or pre-treated with ZD7288 (10 and 30 μM) 10 min before the emission at 580 nm, and 460 nm was measured with a BMG FLUOstar Omega reader. Data reveal the calculation of the emission rate (460/580). Potassium served as a positive control. Data were normalized to the untreated control (H(3) = 17.188, p = 0.001). c Response rate was calculated as the fraction of the acute emission rate after treating cells with high potassium (165 mM KCl), and the emission rate before potassium was added. Data reveal the response rate of untreated microglia compared to microglia that were pretreated with 10 μM and 30 μM ZD7288 10 min before measurement started (H(2) = 9.379; p = 0.009). d To asses changes in the intracellular Ca²⁺ concentration, primary microglia were loaded with fluorescent Fluo-4 (Fluo-4 AM), and images were captured every second during the experiment. The schematic illustration gives an overview of the experimental setup. Thirty micromolar of ZD7288 and 200 μM ATP were added 150 s and 550 s after the start of the experiment, respectively. The time-resolved fluorescence intensity at baseline (F0), as well as the change of fluorescence (ΔF), was analyzed for each time point during measurement. Representative images depict primary microglia in the brightfield technique, as well as with fluorescence microscopy in untreated status and after treatment with 200 μM ATP. e Ca²⁺ response of one representative primary microglia cell was examined in the presence of 30 μM ZD7288 (after 150 s of baseline measurement) and subsequent ATP-application (200 μM, 400 s after ZD7288 application). Data are presented as ΔF/F0 for each time point. Rectangle depict 100-s intervals that were used to assess the f area under curve (AUC; ΔF/F0 × s). Data are shown as mean values of 38 controls and 38 ZD7288-treated cells. Only cells that responded to 200 μM ATP were used for data acquisition (MWU was performed for each time interval: 250–350 s, p = 0.004; 350–450 s as well as 450–550 s p < 0.0001)