Short-Term Dendritic Dynamics of Neonatal Cortical Neurons Revealed by In Vivo Imaging with Improved Spatiotemporal Resolution

One-hour interval in vivo time-lapse imaging of L4 neurons in the neonatal barrel cortex. A, Experimental design of time-lapse in vivo imaging. TCA-GFP Tg mice were used for visualizing the barrel map. L4 neurons in the TCA-GFP mouse barrel cortex were sparsely labeled with Supernova-mRFP via IUE at E14. The cranial window was made in the morning on P4, and the 1 h interval in vivo imaging started 1 h after the surgery. In every imaging session, the pup for in vivo imaging was head fixed to the imaging stage under a two-photon microscope with light anesthesia. The brain of the in vivo imaged mouse was collected after 9 (h0 to h8) imaging sessions, and then fixed. Tangential sections were obtained for post hoc confocal analyses. B, An example of 1 h interval in vivo imaging of Supernova-mRFP-labeled L4 neurons at P4; z-stacked images from the top view. Scale bar, 100 μm. C, The post hoc confocal image (z stacked) of the tangential section of the same area in B. The neuron pointed with a yellow arrow in C is the same neuron pointed in the h8 session of B. The same neurons were identified by their relative positions with each other and dendritic patterns. The in vivo imaged neurons were located on C1−C4 barrels in this example mouse. The sample orientations in B and C were similar. A, Anterior; L, lateral. Scale bar, 100 μm. D, An example of time-lapse images of dendritic morphologies of an L4 neuron in 8 h of imaging sessions. Top views of an L4 neuron (z-stacked), which was located at the edge of the C1 barrel. Snapshots were taken from the 3D reconstruction software Imaris. The neuron was rotated to the same angle and at the same magnification from 9 imaging sessions. The green line in the h8 imaging session showed the C1 barrel edge. Scale bar, 20 μm. E, The corresponding confocal image (z-stacked) of the neuron (yellow arrow) shown in D at a similar angle and magnification. An example neuron is located at the C1 barrel edge. Scale bar, 20 μm.

Detection of rapid dendritic dynamics with improved spatiotemporal resolution in vivo imaging. A, A basal dendritic tree (arrowhead) that newly emerged between h7 (left) and h8 (right) sessions. Snapshot images taken in the Imaris software. Scale bar, 5 μm. B, A basal dendritic tree that disappeared between h0 (left) and h1 (right) sessions. Snapshot images taken in the Imaris software. Scale bar, 5 μm. C, A transient basal dendritic branch (arrowheads) emerged between h0 and h1 sessions and disappeared between h2 and h3 sessions. Scale bar, 10 μm. D, D′. After a branch (red arrowheads) was eliminated between h1 and h2 sessions, another branch (yellow arrowheads) emerged at a similar position between h2 and h3 sessions (D); it is highly likely that these two branches (red and yellow arrowheads) are recognized as an identical dendrite if only h0 and h5 images are available (D′). Snapshot images taken in the Imaris software. Scale bar, 5 μm. Examples in A–C, D, and D′ are from Neuron 1 and Neuron 3 (Table 1), respectively.

Reconstruction of dendritic patterns imaged in vivo. A, A two-dimensional view of 3D reconstructed dendritic morphology from the neuron shown in Figure 2D. Representative basal dendritic trees (tr 2 and tr 7) that originated from the barrel-side half (IN) of the cell body and the other half (OUT), respectively, are labeled in red. Scale bars, 20 μm. B, A schematic that enables simple tracking of dendritic pattern changes during imaging sessions. The representative schematic for the example neuron in A is shown. Imaging session numbers (h0 to h8) are shown on the top. The same basal dendritic trees (tr 1−8) were arranged in the same rows in schematics. The same dendritic segments from different imaging sessions were placed at the same angles. Individual dendritic segments were classified into barrel-inner (IN; cyan) and barrel-outer (OUT; magenta) according to their location. The newly emerged inner and outer branches (and trees) were marked with cyan and magenta arrowheads, respectively. The lengths of individual dendritic trees and segments are approximately proportional. Gray lines, Apical dendrite. Scale bar, 20 μm.

General properties of the dendritic dynamics of L4 neurons at P4. A, Changes in basal dendritic tree numbers from each neuron during 8 h of imaging. The gray and black lines represent data of individual neurons and the average, respectively. n = 19 neurons, 3 mice. B, Numbers of emerged, eliminated, and transient trees from each neuron in 8 h of imaging. +, The mean value; horizontal line in the boxplot, the median. Each dot represents one neuron. n = 19 neurons, 3 mice. C, Survival ratio of newly emerged trees. In this analysis, only dendritic trees that were first detected at h1, h2, h3, h4, or h5 imaging sessions were used. The imaging session at which the tree was first detected was defined as the time point 0 (n = 33 trees); 27 (81.8%), 19 (57.6%), and 16 (48.5%) of the 33 trees were still present at time points 1, 2, and 3, respectively. n = 16 neurons, 2 mice. Three of 19 in vivo imaged neurons have no trees that emerged between h1 and h5 sessions. D, Changes in dendritic tip numbers from each neuron during 8 h of imaging. The gray lines and black lines represent data of individual neurons and the average, respectively. n = 19 neurons, 3 mice. E, Numbers of emerged, eliminated, and transient tips (per mm) from each neuron in 8 h of imaging. +, The mean value, and the line in the boxplot represents the median value. Each dot represents one neuron. n = 19 neurons, 3 mice. F, Survival ratio of the newly emerged dendritic tips. Only dendritic tips that emerged between h1 to h5 of the in vivo imaging sessions were used. At the time point 0, n = 214 tips; 105 (49.1%), 74 (34.6%), and 61 (28.5%) of 214 dendritic tips were still present at time points 1, 2, and 3, respectively. n = 19 neurons, 3 mice. G, Histogram of dendritic tip length changes in 1 h. The x-axis shows the length change (μm), and the y-axis shows the event number of corresponding length changes. Length changes >3 μm, smaller than −3 μm, and between −3 and +3 μm were classified as E (28.1%), R (28.5%), and stable (S; 43.4%), respectively. n = 2089 dendritic tips (from 19 neurons, 3 mice). H, Frequencies of tip behavior in which individual tips continued to elongate or retract in 2 consecutive imaging sessions (EE and RR) and those of tip behavior in which dendritic tips changed the motility direction from E to R or R to E (ER and RE) were compared. Other behaviors (ES, RS, SE, SR, and SS) were excluded from comparison. +, The mean value and the horizontal line in the boxplot represents the median. Each dot represents one neuron. p = 0.022, Mann–Whitney test. n = 19 neurons, 3 mice.

Comparison of dynamic properties between the inner and outer dendrites. A, B, Changes in the numbers of inner (IN) and outer (OUT) trees (A) and tips (B) from each neuron across 9 imaging sessions. Gray lines, Data for individual neurons; black lines, Average of all neurons. n = 19 neurons, 3 mice. C, There were no significant differences between IN and OUT trees in the numbers of emerged, eliminated, and transient trees. p = 0.796, 0.556, and 0.234, respectively; Wilcoxon matched-pairs signed-rank tests; n = 19 neurons, 3 mice. Red lines, Average. D, There were no significant differences between IN and OUT tips in the numbers of emerged, eliminated, and transient tips (per mm basal dendritic length; p = 0.738, 0.568, and 0.441, respectively; Wilcoxon matched-pairs signed-rank tests; n = 19 neurons, 3 mice). The dendritic tip numbers were normalized with the IN or OUT basal dendritic length of the neuron (average length from 9 imaging sessions). Red lines, Average. E, There were no significant differences between IN and OUT dendritic tips in 1 h length changes (μm). p = 0.304; Mann–Whitney test; IN tips, n = 945; OUT tips, n = 852 (from 19 neurons, 3 mice). F, There were no significant differences between IN and OUT basal dendritic trees of high-OBI neurons in the numbers of emerged, eliminated, and transient trees. p = 0.648, 0.750, and >0.999, respectively; Wilcoxon matched-pairs signed-rank tests. n = 9 high-OBI neurons, 3 mice. Red lines, Average. G, There were no significant differences between IN and OUT dendritic tips of high-OBI neurons in the numbers of emerged, eliminated, and transient tips (per mm IN or OUT basal dendritic length; p = 0.820, 0.570, and 0.250, respectively; Wilcoxon matched-pairs signed-rank tests). n = 9 high-OBI neurons, 3 mice. Red lines, Average. H, There were no significant differences between IN and OUT tips of high-OBI neurons in 1 h length changes (μm). p = 0.260; Mann–Whitney test; high-IN tips, n = 568; high-OUT tips, n = 340 (from 9 high-OBI neurons, 3 mice).

Comparison of dynamic properties of dendrites between high-OBI and low-OBI neurons. A, Numbers of basal dendritic trees that emerged (p = 0.024), eliminated (p = 0.106), and were transient (p = 0.030) were compared between high-OBI and low-OBI neurons. +, The mean value; horizontal line in the boxplot, the median value. Each dot represents one neuron. Mann–Whitney tests; high-OBI neurons, n = 9; low-OBI neurons, n = 8. B, Numbers of basal dendritic tips that emerged (p = 0.047), eliminated (p = 0.059), and were transient (p = 0.036) were compared between high-OBI and low-OBI neurons. The dendritic tip number was normalized with the total basal dendritic length of the neuron (average length from 9 imaging sessions). +, The mean value; horizontal line in the boxplot, the median value. Each dot represents one neuron. Mann–Whitney tests. high-OBI neurons, n = 9; low-OBI neurons, n = 8. C, Reconstructed dendritic pattern for an example dendritic tree (yellow) from a high-OBI neuron, which is located near the barrel edge (green). An outward basal dendritic tip did not change its direction or start to retract at the barrel edge and elongated outward further (arrow at h8). Two inward tips were generated near the barrel-edge, but both were eliminated immediately (arrowheads at h1 and h6 sessions). Scale bar, 20 μm.

Dendrite tip features represent their behavior. A, An example image of a dendritic branch (tip-segment) whose tip appeared thick at Time 0 (red arrow) was becoming longer and longer between the Time −1 and Time +1 sessions. Red and yellow arrows indicate the distal end of the branch. Snapshot images taken in the Imaris software. Scale bar, 10 μm. B, An example image of a dendritic branch whose tip appeared thin at Time 0 (red arrow) was becoming shorter and shorter between the Time −1 and Time +1 sessions. Red and yellow arrows indicate the distal end of the branch. Snapshot images taken in the Imaris software. Scale bar, 10 μm. C, Example diagrams of quantitative analysis for TTI. Left, An example for a thin tip, TTI = 0.26. Right, An example for a thick tip, TTI = 1.68. The distal part of the tip-segment was divided into 3 units: T, middle, and S. The length of each unit is 5 μm. ROIs (magenta areas) of T and S units were determined according to their morphologies. Snapshot images taken in the Imaris software. Scale bar, 10 μm. D, Quantification of correlations of TTIs and length changes of tip-segments in 2 h (between Time −1 and Time +1). The x-axis is in log₂ units. y = 3.689 * x – 4.826. The black curve represents the fitted curve, and the dashed line represents the 95% confidence interval. Each inverted triangle represents individual dendritic tips. n = 79 tips, from 14 neurons, 3 mice. E, TTIs of tip-segments that continued to extend (EE) were compared with TTIs of tip-segments that continued to retract (RR) in 2 consecutive hours. In this analysis, TTIs were calculated from the middle time points of continuous elongation or retraction. +, The mean value; horizontal line in the boxplot, the median. Each dot represents one neuron. p = 0.003, Mann–Whitney test. n = 5 EE and 7 RR tip-segments, from 5 neurons, 2 mice.