Figure 3.

Two-photon in vivo imaging reveals migration of cells with multipolar morphology. a, Schematic of the experimental setup for in vivo time-lapse imaging. New neurons were labeled by injecting an oncoretroviral vector carrying GFP into the VZ adjacent to HVC. Mature HVC_X neurons (red dots) were retrogradely labeled with DiI injections into area X (red circle). We imaged HVC, from 4 to 22 dpi, with a two-photon microscope (2PM). Field of view was ∼700 × 1000 × 200 μm (depth). b, Example of a maximum intensity projection of a multipolar GFP⁺ cell (green) within HVC imaged in vivo at 7 dpi using two-photon microscopy. HVC_X somata are labeled with DiI (red). Scale bar, 50 μm. c, Maximum intensity projection of two GFP⁺ multipolar cells (labeled in green and marked by a blue asterisk and a blue arrowhead) and DiI⁺ somata of HVC_X neurons (red) at 8 dpi. Arrowhead indicates the same multipolar cell shown in b. Top shows the relative position of the two GFP⁺ cells. Scale bar, 100 μm. Bottom shows a reconstruction of the migratory trajectory of the multipolar cell in b (marked with a blue arrowhead) over 120 h (white line); white circles indicate the position of the cell body of the migrating cell recorded at 12 h intervals. Note that the cell moved a short distance over the first 12 h of its recorded trajectory; thus, the white circles for the first and second time points are partially overlapping. d, Reconstructed morphology for the multipolar cell shown in b and c at 12 h intervals. Time in hours is indicated in the top left corner. Red line indicates approximate location of the border of HVC. Grid spacing is 80 μm. A, Anterior; L, lateral; V, ventral.