Figure 1.

The Temporal Sequence of Afferent Activity Is Transformed into the Spatial Order of Axon Projections

(A) Experimental design to assay the transformation of the temporal sequence of stimulation into a spatial representation of sequential axon activity. One tectal lobe is ablated so that the RGC axons from both eyes project to the remaining tectum. By stimulating the eyes sequentially with LEDs, the two groups of the convergent RGC axons are activated in a sequence. RGCs in the left eye are sparsely labeled with tdTomato (red) for in vivo time-lapse imaging of dynamic changes in axon arbor morphology and analysis of changes in branch tip positions. The sequence of activity is schematized under each panel. Left panel: The left eye with the labeled axons is stimulated 15 ms earlier (dt = −15ms) than the right eye. Right panel: The left eye is stimulated 15 ms later (dt = +15ms) than the right eye. The animals are raised in dark until the stimulation protocol begins. The eyes are stimulated for 10 h per day starting after the images were collected on Day 0.

(B and C) Examples of the directional axon morphology branch tip shift with opposite temporal sequences of retinal activity. The left eye was stimulated 15 ms earlier or later than the right eye, as schematized in (A), for earlier or later stimulation of the eye with the labeled axons, respectively. Left: Time-lapse confocal images of z series through axons imaged before and 2 days after the stimulus protocol, superimposed on differential interference contrast images of the tectum. Right: Axon reconstructions from images collected on Day 0 (blue), Day 1 (green), and Day 2 (red) are superimposed. The colored arrow shows the overall direction of branch shift over the 3 days of imaging. The rostrocaudal (R<->C) and mediolateral (M<->L) orientation of the tectum is shown in the inset. (B and C) show 2 and 1 axons, respectively.

(D) Quantification of branch tip movement. The relative distance from each axon branch tip (A) to the rostral and the caudal poles (B) was measured for each time point. Changes in the relative positions between time points were determined and expressed as a shift toward the rostral or caudal pole.

(E) Histogram of the proportion of total branch tips that shift toward the rostral or caudal poles for axons stimulated earlier (red) or later (blue), where 0 indicates no shift. Bin = 1.5 μm. Bootstrap (N = 10,000) was used to determine the significance (p = 0.0013) of the difference in the mean values.

Scale bar, 100 μm in (B, C: left) and 67 μm in (B, C: right). N = 144 and 219 branches in 7 and 7 animals for earlier and later conditions, respectively.