Figure 1.
Horizontal axon growth in cortical slice culture.
A, DsRed and ChR2-EYFP plasmids were introduced into several layer 2/3 cells at 1 DIV, and labeled axons were observed by confocal microscopy (560 nm excitation) at 4 DIV. This excitation does not activate ChR2. Note that a horizontally elongating axon is clearly labeled. The interrupted lines indicate the pial surface and the presumed layer 2/3 borders. Scale bar, 100 µm. B, Laminar localizations of the cells projecting horizontal axons which were used for the time-lapse study. Red dots indicate the locations of the cell bodies. Arrows indicate the presumed borders of layer 1, 2/3, 4, 5 and 6. C, Growth of the horizontal axon revealed by time-lapse study. The axon was followed every 10 min. Scale bar, 5 µm. D, Growth curve of the horizontal axon shown in C. E, Average growth of horizontal axons observed in cortical slice cultures from 3 to 10 DIV (n = 32).
Figure 2.
Electrophysiological responses of ChR2-expressing cortical neurons to photostimulation.
A and B, ChR2-expressing cells among layer 2/3 neurons were subjected to patch-clamp recording at 4 DIV. Interrupted lines in A indicate the presumed layer 2/3 borders. The white line in B indicates the pial surface. Bar, 200 µm. C, Each flash with 475-nm wavelength light (50-msec duration in the upper trace and 200-msec duration in the lower trace) elicited depolarization and a single action potential.
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.
Figure 4.
Horizontal axon growth before and after high-frequency stimulation to the axon.
A, High-frequency photostimulation (4 Hz) elicited retraction of a representative growth cone. 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 (arrow). D, Horizontal axon growth in the presence of TTX after high-frequency photostimulation. TTX (1 µM) was added to the culture medium 1 h prior to the observation. Photostimulation was applied at 4 Hz for 1 min to the cell body (n = 2).
Figure 5.
Axonal growth after low-frequency stimulation of the soma.
A, B, Photostimulation at 1(A) or 0.1 Hz (B) for 1 min was applied (arrows) during the time-lapse study. C, A total of 120 pulses was applied to a single axon at low frequency (0.1 Hz) and subsequently at high frequency (4 Hz). Pause behavior was induced by high-frequency stimulation but not by low-frequency stimulation, even though the number of pulses was the same in both cases.
Figure 6.
Occurrence of pause behavior after photostimulation with different frequencies.
Histograms show the percentage of axons displaying pause behavior after photostimulation with different frequencies. The numbers in parentheses are the numbers of samples examined for each frequency.