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The retinotectal projection in Xenopus laevis is topographically organized. During the early development of the Xenopus visual system, the optic tectum increases considerably in volume, and retinotectal axons and dendrites undergo extensive activity-dependent remodeling. We have previously observed marked changes in the three-dimensional layout of the tectal retinotopic functional map over the course of a few days. This raised the question of whether such functional reorganization might be attributable to the migration and structural remodeling of tectal neurons as the brain grows. To examine changes in map topography in the context of individual tectal neuron morphology and location, we performed calcium imaging in the optic tecta of GCaMP6s-expressing tadpoles in parallel with structural imaging of tectal cells that were sparsely labeled with Alexa 594-dextran dye. We performed functional and structural imaging of the optic tectum at two developmental time points, recording the morphology of the dextran-labeled cells and quantifying the changes in their positions and the spanning volume of their dendritic fields. Comparing anatomical growth to changes in the functional retinotopic map at these early stages, we found that dendritic arbor growth kept pace with the overall growth of the optic tectum and that individual neurons continued to receive widespread visual field input, even as the tectal retinotopic map evolved markedly over time. This suggests a period of initial growth during which inputs to individual tectal neurons maintain diffuse connectivity and broad topographic integration.
FIGURE 1. Morphometric changes in the tadpole tectum between stages 45 and 48. (a) Illustration of the tadpole and tadpole tectum at Stages 45 and 48. The neuropil of the tectum is represented by darker colors. (b) Volume of the tectum and tectal neuropil increased significantly from Stages 45 to 48. (c) (Left) Schematic of a tadpole with hemimosaic GCaMP6s expression restricted to the left half of the animal. (Right) Two‐photon optical section showing GCaMP6s expression limited to tectal cells in the left and RGC axons in the right tectal hemisphere. A single postsynaptic cell in the left tectal hemisphere was labeled with Alexa Fluor 594 dextran fluorescent dye. (d) (Left) Maximum projection images of the left tectal hemisphere of the same tadpole at Stages 45 and 48, with a single postsynaptic tectal neuron labeled with Alexa 594 dextran. (Right) Reconstructions of the dendritic tree of the dextran‐labeled cell, with colored patches showing the boundaries of its 3D spanning volume. (e) Dendritic total branch length increased from Stages 45 to 48. (f) Dendrite density, calculated as total branch length divided by neuropil volume, did not change significantly. (g) Dendrite spanning volume increased from Stages 45 to 48. (h) Dendrite coverage, calculated as dendrite spanning volume divided by neuropil volume, did not change significantly. (i) Position of the cell soma along the rostrocaudal (R–C) axis was calculated as the distance from the soma to the caudal limit of the tectum divided by the full length of the R–C axis. (j) Labeled neuronal somata shifted rostrally from Stages 45 to 48. (k) Position of the cell soma along the dorsoventral (D–V) axis. From Stages 45 to 48, cell somata shifted deeper below the dorsal surface of the tectum. (l) β‐Tubulin and CldU labeling in the tectum of an animal treated with 10 mM CldU for 2 h at Stage 45 and sacrificed at Stage 48. CldU labeling can be seen distributed in the cell body layer in a laminar fashion, consistent with new tectal cells being added in layers from the caudal end of the tectum. CldU puncta can also be seen sparsely distributed in the tectal neuropil. All paired comparisons were done using Wilcoxon matched pairs test, n = 10 animals, *p < 0.05, **p < 0.01.
FIGURE 2. Visual receptive fields represented in the tectal neuropil at different developmental stages. (a) Azimuth and elevation receptive field maps from the same animal at Stages 45 and 48. Receptive field (RF) positions were calculated as the phase of the response to a repeated drifting bar stimulus in the corresponding axis. Pixel intensities indicate signal‐to‐noise ratio (SNR). Images were taken in stacks of 10 optical sections starting at approximately 40 µm depth from the surface of the tectum, with 7.5 µm between sections. Scale bar is 40 µm. (b) 3D renderings of the phase maps from (a), showing only voxels in the neuropil with SNR > 2. (c) Neuropil receptive field positions from (b) (all optical sections) mapped onto the stimulus display field. (d) Mean neuropil phase did not differ between early (Stages 45 and 46) and late (Stage 48) stages for both azimuth and elevation axes (n = 9 animals, Wilcoxon matched pairs test). (e) Cumulative probability distribution of neuropil receptive field phase values. Thin lines show data from individual animals (n = 9), down‐sampled to 2000 random data points for each animal. Thick lines show pooled data from all animals. Pooled data show a small but significant shift in the RF distributions between early and late stages for both azimuth and elevation (Kolmogorov–Smirnov test, ****p < 0.0001).
FIGURE 3. Receptive fields measured at the dendrites of single tectal neurons at different developmental stages. (a) Example optical section from the same animal imaged at Stages 46 and 48: (Left) Morphology of the tectum with a single‐cell electroporated dextran‐labeled neuron. (Middle) Elevation receptive field maps (pixel intensities indicate SNR). (Right) Elevation receptive field maps overlaid with mask of dextran labeling. (b) Cumulative distribution of receptive field phase values recorded from areas in the neuropil with dextran labeling (n = 9 animals, 10 optical sections for each animal). Thin lines show data from individual animals, downsampled to 200 data points for each animal. Thick lines show pooled data from all animals. Phase values were corrected by mean centering (see Section 4). Pooled data from all nine animals show significant difference between early and late stages for both azimuth and elevation (Kolmogorov–Smirnov test, ****p < 0.0001). (c) RF phase values from the animal in (a) (data from 10 optical sections) mapped onto the stimulus display field. Colored scatter points represent data points from the neuropil; black scatter points represent data points from areas in the neuropil with dextran labeling. (d) Heat maps showing the data from (c) binned into 2D histograms (50 × 50 bins. Bin counts were normalized so that the sum of all bin counts in each histogram equals 1). (e) The match value (see Section 4) between RF histograms for neuropil and dextran‐labeled cells was high (median 0.58 for early stage and 0.62 for late stage) and didn't change significantly between early and late stages (Wilcoxon matched pairs test, n = 9 animals).
FIGURE 4. Receptive fields of tectal neuron cell bodies across developmental stages. (a) Azimuth and elevation cell body receptive field maps from the same animal and showing the same optical sections as in Figure 2a. RF phase values were calculated from the mean ΔF/F trace for each cell body ROI. Only cells with SNR > 2 are shown. Scale bar is 40 µm. (b) Mean cell body RF phase (n = 6 animals). There was no significant difference between early and late stages (Wilcoxon matched pairs test). (c) Cumulative probability distributions of cell body RF phase values. Thin lines show data from individual animals (n = 6), downsampled to 100 cells for each animal. Thick lines show pooled data from all animals. Pooled data reveal a small difference between early and late stages for both azimuth and elevation (Kolmogorov–Smirnov test, ****p < 0.0001). For the analyses in (b) and (c), only cells with SNR > 2 were included, corresponding to 42.8% of total segmented cells for Azimuth/early, 55.9% for Azimuth/late, 45.4% for Elevation/early, and 62.3% for Azimuth/late.
FIGURE 5. Stage‐dependent change in retinotectal input wiring mechanisms during development. The initial development of the retinotectal projection is characterized by broad integration of multiple RGC inputs representing a large portion of the visual field, as demonstrated by results in the current study. In this phase, the dendritic arbors of tectal neurons grow at a pace proportionate to the overall growth of the optic tectum. In later development, there is a refinement of visual receptive fields, presumably as a result of activity‐dependent input selection. Throughout development, the retina grows radially by adding RGCs at the ciliary margin, whereas the tectum adds cells in a caudomedial proliferative zone, displacing older cells toward the anterior tectum. To maintain an orderly topographic representation, RGCs and tectal neurons must shift their connections. RGC, retinal ganglion cell.
