Fig. 4. Functional P2X7 receptors are restricted to the distal region of the axon and growth cones. (A) Hippocampal neurons cultured for 3 DIV were stained with antibodies against tyrosinated -tubulin and P2X7. Higher-magnification views of the boxed areas show the distal region of the axon stained for tyrosinated -tubulin or P2X7 receptor. Scale bar: 50 µm. (B) Distal region of an axon stained with anti--tubulin and anti-P2X7 antibodies. Note that -tubulin staining, unlike that of tyrosinated -tubulin, does not display an increasing distal gradient. (C,D) Graphs represent the fluorescence intensity of tubulin (red) and P2X7 (green) along the axon in the neurons shown in A (C) and B (D), quantified using the ImageJ program. (E) Images of the most distal region of the axon and the growth cone of hippocampal neurons stained with anti-tyrosinated--tubulin and anti-P2X7 antibodies. Note the absence of P2X7 staining in axons running parallel to a P2X7-positive distal region of an axon, where P2X7 is located in the microtubule domain of the axon and in the actin-rich domain (inset). (F,G) Images show hippocampal neurons loaded with Fura-2 dye. Insert in G shows a fluorescence image of a whole hippocampal neuron loaded with Fura-2. Different areas along the axon were analyzed (numbers in G and F). (H,I) Time course of the changes in Fura-2 fluorescence recorded in the axonal areas selected in F and G. The graph represents the ratio of the Fura-2 intensity at the 340 (F₃₄₀) and 380 (F₃₈₀) wavelengths. (H) Increase in intracellular Ca²⁺ induced by 1 mM ATP in the presence or absence of extracellular Mg²⁺ ions. Note that Ca²⁺ influx induced by ATP is higher in the absence of extracellular Mg²⁺. (I) Intracellular Ca²⁺ influx induced by 1 mM ATP was abolished when neurons were pre-incubated with BBG (1 µM) in the absence of extracellular Mg²⁺. The solid bars indicate the periods of stimulation; a KCl pulse (60 mM) was also applied to test the viability of the neurons.