2pMAPG induction of action potentials in a layer 2/3 cell and detection of Ca2+ transients in a dendritic spine of a layer 5 cell. (A) A Z-stacked image of a representative layer 2/3 pyramidal cell filled with Alexa Fluor 594. The depth of the soma was 82 μm from the slice surface. The red region was divided into 16 × 8 pixels in a single plane and 2pMAPG was performed at each pixel at depths of 30, 80 and 130 μm from the top of the slice. (B) Each trace represents the membrane potential derived from 2pMAPG performed at each pixel in the orange-boxed region in (A) at a depth of 80 μm. Red bars indicate the time of 2pMAPG. (C) Green, yellow, and red pixels indicate AP-evoking pixels detected at any one of the three depths (30, 80 or 130 μm), at any two of these three depths, or at all of the three depths, respectively. (D) A single electrical stimulation near the dendrite induced a Ca2+ transient in one of the dendritic spines in the imaging region (arrow). Red (Alexa Fluor 594) fluorescence and green (Fluo-5F) fluorescence are overlaid. The overlapping signals appear yellow. The time interval between images was 194 ms. The time from the onset of sequential imaging is indicated to the right of each figure. (E) The G intensity/R intensity (G/R) trace in the spine is indicated with an arrow in (D) for 10 electrical stimuli at 0.2 Hz. (F) Expanded G/R trace is boxed in (E). Four dots indicate the acquisition times of the four imaging frames shown in (D). (G) The mean amplitudes of Ca2+ transients (black circle) and the mean 5 coefficients of variance of G/Rbase (blue rectangle) are plotted against spine size. The regression line for Ca2+ transients is shown. Bars, standard error of mean.