Fig. 7.

The site of Ca²⁺ wave initiation moves to the region where subthreshold [InsP₃] was rapidly and locally applied. (A–D) Carbachol (CCh, 6 s; Ci) applied via a puffer pipette evoked an increase in [Ca²⁺]_c in the form of a Ca²⁺ wave (Ai,Bi). The wave initiated at a central site (region ii, Ai, panel 1) and propagated from there in either direction (Ai). Caged InsP₃ (Dii), at an empirically determined concentration that was just subthreshold did not produce an increase in [Ca²⁺]_c when focally photoreleased (Bii) ∼90 seconds later in a 20 µm region at a site distant from the carbachol-evoked Ca²⁺ wave initiation site (region iv in Ai panel 1; note the flash artefact shown in Bii). In the next part of the experiment, carbachol was again applied but this time the subthreshold caged InsP₃ was locally photoreleased (Diii) 3 seconds after the start of the carbachol application (Ciii) and before the Ca²⁺ wave initiated. In this case, the site of initiation was altered and the Ca²⁺ wave began (Aii,Biii) at the site where [InsP₃] rapidly increased (region iv in Ai, panel 1). The [Ca²⁺]_c images (Ai and Aii) are derived from the time points indicated by the corresponding numerals in Bi and Biii. [Ca²⁺]_c changes in Ai and Aii are represented by colour; blue is low and red is high [Ca²⁺]_c. Changes in the fluorescence ratio with time (Bi,Bii,Biii) are derived from 3×3 pixel boxes in regions i to iv in A, panel 1. The regions are drawn as larger yellow circles to facilitate visualisation. (E) Brightfield image of the cell; the whole-cell electrode can be seen (left side). The carbachol-containing puffer pipette is located to the right of the cell outside the field of view. (F) Direction of Ca²⁺ wave from the initiation site either before (left panel) or after (right panel) photolysis of subthreshold InsP₃ (n = 4).