Figure 4: Somatic depolarization flattens the profile of Na⁺ entry along the axon.

(a) Image: granule cell filled with 1 mM SBFI (for example, collage of averaged 20–30 μm z-stacks, λ_x^2P=800 nm); dotted segments 1–3, recording sites; inset, example linescan at site 1 (dotted arrows, scan position). (b) Characteristic linescan traces in response to five APs at 20 Hz as shown in a. (c) Axonal distribution of relative ΔF/F amplitude (evoked by five APs at 20 Hz). To improve S/N ratio, two largest ΔF/F (out of 3–10 traces) within each bin were averaged (n=60 recorded sites in total). (d) Average SBFI ΔF/F signal (magenta, top scale) near the spike-initiation segment (as in a,b), and the best-fit model representation (black, bottom scale) of Na⁺ entry; see details in Methods and Supplementary Fig. 4. (e) Traces: SBFI fluorescence (example) in proximal axon evoked by five spikes at 20 Hz (as in a) under baseline conditions (black, −70 mV) and during somatic pre-depolarization (to −40 mV; red). Plot: summary, the SBFI ΔF/F amplitude versus distance from the soma (data at >20 μm shown to reduce bias due to somatic Na⁺ diffusion sink); 25 °C. (f) Relative changes in ΔF/F from experiments in e; line, negative linear regression (n=15, Pearson’s R=−0.54, P<0.04). (g) Left traces: somatic voltage during electrical stimulation of presynaptic perforant path fibres (as in Fig. 1) with stronger (green) and weaker (orange) stimuli; dotted lines, higher spiking threshold upon slower depolarization. Right traces: SBFI ΔF/F recorded simultaneously in the proximal axon (colour-coded accordingly); 33 °C. (h) Summary of experiments shown in g: colour-coded accordingly; ***P<0.002, n=16 cells. (i) Summary of experiments shown in g: increases in AP-evoked Na⁺ entry SBFI ΔF/F amplitude) due to reduced spike latency (faster depolarization) are weaker with greater distances from soma; data average at 20 μm are significantly above zero (*P<0.02, n=6, t-test); dotted line, best-fit linear slope.