Fig. 1. ATP induces intracellular Ca²⁺ transients in distal axon regions and exerts a negative effect on axon growth. (A) Fluorescence image of hippocampal neurons loaded with Fura-2 dye. Two different areas along the axon (regions 2 and 3) and the soma (region 1) were analyzed. The graphs represent the time course of Fura-2 emission as the 340 (F₃₄₀) and 380 (F₃₈₀) wavelength ratio. Solid bars represent the period of ATP or KCl treatment. Neurons were stimulated with 1 mM ATP (b) and then with 60 mM KCl (d) to test their viability. The right panel shows representative images of changes in Fura-2 fluorescence recorded at four different times during the experiment (a, b, c and d). Scale bar: 50 µm. (B) Hippocampal neurons cultured for 3 days in the presence or absence of ATP (1 mM). Neurons were stained for tyrosinated -tubulin to identify the neuronal morphology and with phalloidin–Alexa-Fluor-594 to identify the growth cones. Scale bar: 50 µm. (C) Graphs represent the mean ± s.e.m. of the axon length, axon ramifications and the ratio between ramifications and total axon length in three different experiments (n=100). Statistical differences were analyzed using a paired t-test (*P<0.05, ****P<0.0001 versus control).