(A) AP-evoked Ca2+ signals measured in a spine (green) and nearby dendrite (blue) during tonic firing. Signals are averages of 3 individual AP-evoked Ca2+ transients. (B) Ca2+ signals as in A, for glutamate uncaging near the spine head while holding at −63 mV. Signals are averages of 6 uncaging trials. (C) Spine and dendrite Ca2+ signals evoked by uncaging glutamate during tonic firing. Dotted lines represent the ‘linear sum’, defined as ‘AP-alone’ plus ‘uncage- alone’ signals from panels A and B. (D) Comparison of spine Ca2+ signals and corresponding linear sums for trials in which glutamate uncaging occurred at either an early phase (left) or intermediate phase (right) of the firing cycle. Data are from the same spine as panels A–C. Dashed lines indicate baseline signals before the uncaging pulse. In the early phase example, the Ca2+ signal displayed an AP-evoked increase before the uncaging pulse that was not measured as part of the glutamate-evoked response (arrow). Inverted triangles in the mid-phase example indicate the peaks of the glutamate-evoked Ca2+ signal measured before (open symbol) and after the subsequent AP (closed symbol). (E) Top: Normalized spine Ca2+ amplitude plotted against the phase at which glutamate uncaging occurred for measured data (green) and linear sums (black). Bottom: Plot of membrane potential immediately prior to glutamate uncaging versus phase. The black trace shows a typical interspike interval. (F) As in E, except glutamate-evoked Ca2+ signals were only measured before the onset of the subsequent AP.