Figure 1.

The P2X₃ receptor is retrogradely transported in the axons of DRG neurons. (A, B) Sciatic nerves were ligated at the middle-thigh level and processed for immunostaining and immunoblotting. Similar to TrkA, P2X₃ receptors accumulated at the distal (d) and proximal (p) segments of the ligated nerves. NGF and synaptophysin (Syn) accumulated at the distal and proximal segments, respectively. Actin served as a loading control. Scale bar, 100 μm. (C) PNGase F treatment shifted the P2X₃ receptors in the DRG lysate from 53 kD to predominantly 45 kD, which is consistent with the predicted molecular weight of this receptor. (D) The freshly dissected and cultured nerves were cut into four segments. P2X₃ receptors accumulated at both the proximal and distal segments after culture. (E) The distal end of the sciatic nerve connected with DRG was incubated with a P2X₃ receptor antibody or a control IgG for the indicated time. The signal for retrogradely transported P2X₃ receptors in DRG neurons became visible in the receptor-positive neurons at 6 h and lasted up to 12 h after culture. Scale bar, 30 μm. ^* P < 0.05 versus the signal in P2X₃ receptor-negative neurons at 6, 9 or 12 h (n = 3). (F) Live cell imaging showed the retrograde transport of P2X₃ receptors (arrowheads) in cultured DRG neuron axons. The mean and active retrograde velocity profiles were assembled with 97 puncta. Scale bar, 5 μm.