Figure 5. Stimulation of YSpH-labelled vesicles by ATP and NH₄Cl reveals the number of YSpH molecules present on a single astrocytic vesicle.

(a) Time series frames of a representative YSpH-positive vesicle (circle) recorded by CLSM before and after successive stimulations with ATP and NH₄Cl. Bar, 1 mm. (b) Examples of time-dependent changes in the fluorescence intensity of individual vesicles: left panel, ATP failed to evoke an increase in fluorescence intensity, however, NH₄Cl elicited an increase; middle panel, application of ATP elicited a sustained increase in fluorescence intensity that was augmented by NH₄Cl; and right panel, ATP elicited a transient increase in fluorescence intensity, which was followed with NH₄Cl-elicited increase. Horizontal grey bars indicate the timing of ATP/NH₄Cl applications. (c) Amplitude distributions of the change in YSpH fluorescence intensity (ΔF) recorded in nine astrocytes before and after stimulations (bin width =1.5 a.u.). Left panel, the change in YSpH intensity plot represents a background, which was obtained by monitoring changes in YSpH intensity before and after addition (at 30 s for 10 s) of ATP (bottom horizontal line) in recordings where ATP failed to elicit a significant increase in YSpH. Middle panel, after ATP, and, right panel, subsequent NH₄Cl applications, the intensity distribution incrementally shifts to the right as vesicles alkalinize due to the pore formation (ATP) and subsequent chemical alkalinization (NH₄Cl), respectively. (d) Combined plot of the change in YSpH intensity distributions for all vesicles (n = 1,045) from nine astrocytes: background (white) and after stimulation with ATP (dark grey) and NH₄Cl (light grey). The Gaussian curves are fitted by keeping the s.d. as determined in the background shown in left panel and using Equation 1 (where x = ΔF (a.u.), for fitting parameters see Table 1). (e) The peaks of the Gaussian fits of different modes of intensities (obtained in d) are plotted as a function of modes, that is, number of YSpH packets; the relationship can be described using a liner regression in the form: ΔF (a.u.) = (4.95 ± 0.04) × apparent # of YSpH+(−0.02 ± 0.05), where # is the number or multiples of YSpH molecules (R² = 0.997; significantly different from zero, P< 0.001; ANOVA).