Fig. 3. An activation of mechanosensitive Ca2+ channels and subsequent Ca2+ entry induced by direct mechanical poking. (a) Schematic illustration of astrocyte Ca2+ imaging in inverted microscope using ratiometric Ca2+ dye, Fura-2 AM (left). The inset showing representative bright-field image (top right) and Fura-2 AM image (bottom right) of an RGC. (b) The representative bright field image of an RGC and 340/380 ratio images of Ca2+ imaging taken at 10 s, 26 s, and 354 s (top). Representative 340/380 ratio trace of poked RGC (bottom). (c) Pie chart showing that the RGCs responsive to poking account for 56% (n=27 out of 48 single cells) and non-responsive RGCs account for 44% (n=21 out of 48 single cells). (d) Representative 340/380 traces in which two sequential peaks were induced by two sequential poking with 20 min interval (top left) and the 2nd peak was significantly decreased under extracellular Ca2+-free condition (bottom left). Summary bar graph showing 2nd/1st peak ratio of 340/380 trace of Ca2+ response with and without Ca2+ in external solution (right). Unpaired two-tailed t-test (**p<0.01). Diamond indicates the timing of mechanical stimulation.
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