Induction of Ca2+ transients in the dendritic spines of a layer 5 cell by 2pMAPG mapping in layer 2/3. (A) A Z-stacked image of a representative layer 5 pyramidal cell filled with Alexa Fluor 594. White broken lines indicate laminar borders. The depth of the soma was 53 μm from the slice surface. The red boxes indicate the 2pMAPG mapping area. The sky-blue rectangle indicates the Ca2+ imaging region. The purple box indicates the field of view under the 25× objective lens. (B) Ca2+ imaging region corresponding to the sky-blue rectangle in (A). Red (Alexa Fluor 594) fluorescence and green (Fluo-5F) fluorescence are overlaid. The time interval between images was 236 ms. Dendritic spines indicated by yellow (a) and green (b) arrows exhibited Ca2+ transients after 2pMAPG at the yellow and green pixels in (A), respectively. (C) Five sequential images from the yellow-boxed region in (B). Numbers at the top of the image indicate the onset time of each imaging frame relative to that of the first imaging frame. The yellow horizontal bar in the second frame indicates the time of 2pMAPG at solid yellow pixel 1 in (A). (D) G/R traces in spines shown by the arrows marked a and b in (B) are shown at the top and bottom, respectively. Traces were obtained during 2pMAPG mapping at a depth of 130 μm. Arrows indicate Ca2+ transients immediately after 2pMAPG at the yellow and green pixels in (A). The numbered arrows correspond to the numbered pixels in (A) and the spines shown in (B). Asterisks indicate Ca2+ transients observed throughout the dendrite, possibly due to bursting activity. (E) The image at the time corresponding to the asterisk in (D). (F) The G/R trace in the spine indicated by the green arrow in (B) was obtained by fast imaging of the green-boxed region in (B). The time interval between images was 48 ms. During imaging, 2pMAPG was performed at the green closed pixels in (A) at 0.2 Hz (red bars). (G) Expanded G/R trace boxed in (F). (H) Seven images before, during, and after 2pMAPG in (G).