Figure 7.

Calcium measurements with yellow cameleon YCX2.60. (A) Responses in individual HeLa cells to ionomycin applied to saturate yellow cameleons (YCs) with calcium (n = 5), and to BAPTA-AM applied to deplete YCs of calcium (n = 4). The dynamic range of YCX2.60 is twice as large as that of YC2.60 (Rmax/Rmin = 6.20 vs 3.17). The apparent dissociation constants of YC2.60 and YCX2.60 for calcium were calculated as 80 nM and 220 nM, respectively (Y.N. and A.M., unpublished results). (B-D) In vivo two-photon imaging of calcium signals in Rasgrf2-2A-dCre;Camk2a-tTA;Ai92(TITL-YCX2.60) mice. (B) Visualization of the uniform expression in layer 2/3 at 190-μm depth within the cranial window one week after TMP induction (field of view size 1.7 × 1.7 mm). Note the shadows from surface blood vessels. The image on the right is a magnified view of the boxed area in the left image, showing neuronal somata labeled with YCX2.60. (C) Representative 60-s example of spontaneous activity in the neuron marked with an arrow. Raw, unfiltered calcium transients are expressed once as YFP:CFP ratio R, which in principle can be calibrated in terms of absolute calcium concentration, or as relative percentage change of the ratio R (ΔR/R). The expanded view of the trace segment in the box highlights fast calcium transients presumably evoked by few or single action potentials. (D) Evoked activity in two example neurons following whisker stimulation. The principal whisker was repeatedly stimulated at 10 Hz for 2 seconds (onsets indicated with dashed lines). YCX2.60 AR/R traces are shown for two example layer 2/3 neuron for three trials, each comprising five stimulation periods. Note responses at stimulus onset, during stimulation, and at stimulus offset as well as spontaneous activity in between. Large calcium transients likely correspond to bursts of action potentials whereas small-amplitude transients may reflect occurrence of only few or single action potentials.