(a) Left, Color-coded maximal ∆F/F of OGB-1 imaged in a L5 pyramidal neuron axon in response to an a-EPSP (five subthreshold current injections at 100 Hz) overlaid with a z-projection of biocytin-streptavidin (grey) of the same neuron. White arrowheads indicate regions of interest from which example traces are shown right. Scale bar, 20 µm. Right, ∆F/F traces from locations specified left. For illustrative purposes, ∆F/F traces represent averages of ~400 trials. Top to bottom scale bars, 10 mV, 1 nA, 5% ∆F/F, 100 ms. (b) Left, higher magnification of the regions of interest shown in a. Right, maximal z-projection of biocytin-streptavidin (blue) and ßIV spectrin (red). White arrows indicate nodes or Ranvier. Sites with a-EPSP-evoked [Ca2+]i transients were all positive to ßIV spectrin (n = 15 AISs and 23 nodes from n = 15 axons). Scale bar, 5 µm. (c) Same axon and locations as in a with color-coded maximal ∆F/F in response to an AP. Scale bars, 20 mV, 0.5 nA, 10% ∆F/F, 100 ms and 20 µm. (d) Top, Population data for peak ∆F/F in response to a-EPSPs in the AIS (n = 13), first internode (IN, n = 9), the first Node (n = 9) and basal dendrite (Dend, n = 4), one-way ANOVA with Tukey’s multiple comparisons test, p<0.0001, Bottom, peak ∆F/F in response to a single AP in the AIS (n = 10), first internode (IN, n = 6), the first Node (n = 6) and basal dendrite (Dend, n = 4), one-way ANOVA with Tukey’s multiple comparisons test, p<0.0001. Open circles represent individual cells and bars show the population mean ± s.e.m. Data available in Figure 1—source data 1. See also Figure 1—figure supplement 1.