Fig. 3: Visualization of inter-neuronal communication of nucleotides mediated by gap junctions.

a Wildtype or SARM1-knockout DRG neurons were seeded in two corners of a well of an 8-well Chambered Coverglass as two small islands (represented by orange dots in the illustration) and cultured until the outgrowing axons contacted each other. The culture was treated with 25 μM PC11, with or without 200 μM CZ-48, for 16 h. Fluorescence signals in the axons within the red-dotted areas were captured using a confocal microscope. “L” and “R” represent the axons projected from the left and right islands, respectively. Scale bar: 10 μm. b Quantification of fluorescence intensity in the images from a. c Similar experiments to the third-row co-culture in a were conducted, except that the images were captured before or after the axons contacted. Scale bar: 10 μm. d, e Wildtype DRG neurons were seeded as two islands. When the axons projected from these islands made contact, the cultures were pretreated with 5 μM mefloquine (right charts) or vehicle as a control (left charts) for 2 h, followed by the addition of 200 μM CZ-48 (d) or axotomy (e), together with 25 μM PC11. Fluorescence of PAD11 within the squared areas was monitored at the indicated time points, and fluorescence intensity was quantified. f Wildtype and SARM1-knockout DRG neurons were seeded as a third-row co-culture in a and infected with AAV-PHP.eB encoding Cx36-targeting shRNA or a scramble shRNA, indicated by mScarlet expression. The culture was treated with 25 μM PC11 and 200 μM CZ-48, for 6 h, and fluorescence of PAD11 and mScarlet was captured under a confocal microscope. Scale bar: 10 μm. g Endogenous expression level of Cx36 in the DRG neurons in f was measured by Western blot. Quantification of the PAD11 fluorescence intensity in f. Signals from the right side were normalized to those from the co-cultured left axons. All experiments were repeated at least three times, and data are presented as means ± SDs (n = 3). Statistical significance was determined by Student’s t test in b, following two-way ANOVA test; Student’s t test in d, e, and g (ns no significance; **P < 0.01; ***P < 0.001; ****P < 0.0001).