Figure 1.

The diameter of the UV light pulse and the rate of the Ca²⁺ wave are estimated. a, Astrocytes with large processes oriented parallel to the line scan were patch clamped with pipettes containing 200 μm Ca²⁺ indicator dye OGB-1 and 400 μm caged IP₃. The blue line is the line scan position. The red circle is the estimated diameter of the UV light pulse (see below). b, IP₃ uncaged during a 10 sec line scan revealed a Ca²⁺ increase that did not occur simultaneously in all parts of the cell. The portion of the soma in which Ca²⁺ increased simultaneously had a diameter of ∼2.5 μm. This provided an estimate of the UV beam diameter. In the remaining parts of the cell, Ca²⁺ increased after a delay that was longer with distance from the uncaging point, indicating a Ca²⁺ wave. c, The line scan image in b was fitted with a threshold function to see the Ca²⁺ increase in the astrocyte more easily. The red box represents the beam diameter estimate and the yellow lines represent distance, time, and slope. The estimate in this cell was restricted to the proximal process, as more distally the process dipped out of focus. d, A zoomed-in image of c showing that the Ca²⁺ signal traveled 7.0 μm in 286 msec, for an estimate of 24.6 μm/sec. e-g, UV light alone did not produce Ca²⁺ elevations in astrocytes. f, 40 sec line scan showing no effect of a 10 msec UV light pulse on fluorescence of an astrocyte patched with OGB-1 only. The trace below the line scan indicates the relative change in fluorescence in the astrocyte soma over the duration of the scan. The 10 msec light artifact (arrow) is produced when neutral density and UV wavelength filters are removed from the laser. This results in a beam of high intensity that indicates when the UV pulse was applied. g, To make sure that the astrocyte was capable of producing a Ca²⁺ increase, 50 μm tACPD was added to the bathing solution. Scale bars, 10 μm.