Figure 3. Horizontal axons exhibit pause behavior in response to high-frequency photostimulation directed at the soma of labeled neurons.

A, A horizontal axon from a cell cotransfected with DsRed and ChR2-EYFP was observed. Photostimulation (4 Hz, 1 min) with 475-nm excitation was applied to the cell body after a 60-min observation of axon growth at 560-nm excitation. After the photostimulation at 475-nm excitation, the axon was further observed at 560-nm excitation. Scale bar, 5 µm. B, Growth of the horizontal axon shown in A. The arrow indicates the time of photostimulation. C, Growth of individual axons before and after photostimulation. Despite differences in growth rates, most axons display pause behavior after high-frequency photostimulation (arrow), n = 11. D, Axonal growth rates before and after high-frequency stimulation. After high-frequency photostimulation, axonal growth rate decreased significantly and this decline persisted for 20 min (Student’s t-test, *p<0.005). E, Axonal tip morphology before and after photostimulation. Scale bar, 2 µm.