Holt had already shown that, at the beginning of the pathway traversed by frog retinal axons, netrin leads the axons out of the eye. To study the rest of the pathway, Shewan achieved the finicky feat of culturing the entire pathway. He confirmed that netrin could later act as a repellent that probably helps to prevent overshoot of the axons' final target.
That was a nice result. But the surprise was yet to come. When the team cultured a chunk of retina, which lacked the rest of the optic pathway and had not yet initiated axon outgrowth, they saw that after two days there was the same switch from netrin attraction to netrin repulsion. Thus, the switch appears to be intrinsic.
“It was when we did the control experiment that we realized [that the switch to repulsion] was happening even without the pathway experience,” says Holt. “It was one of those strange twists.”
The initial, acute switch during outgrowth may still be influenced by pathway cues—possibly a combination of laminin, which Holt's group has shown can flip the netrin switch, and another guidance molecule called Robo, whose receptor in the spinal cord can silence the netrin receptor. But the more long-lasting switch in netrin responsiveness may be a result of dropping levels of cyclic AMP (cAMP), which Holt's group shows is correlated with the aging of the retinal neurons. Boosting cAMP can restore the youthful attraction to netrin, but the ultimate cause of the age-dependent dip in cAMP is not yet known. ▪