Refinement of the retinogeniculate synapse by bouton clustering

Abstract

Mammalian sensory circuits become refined over development in an activity-dependent manner. Retinal ganglion cell (RGC) axons from each eye first map to their target in the geniculate and then segregate into eye-specific layers by the removal and addition of axon branches. Once segregation is complete, robust functional remodeling continues as the number of afferent inputs to each geniculate neuron decreases from many to a few. It is widely assumed that large-scale axon retraction underlies this later phase of circuit refinement. On the contrary, RGC axons remain stable during functional pruning. Instead, presynaptic boutons grow in size and cluster during this process. Moreover, they exhibit dynamic spatial reorganization in response to sensory experience. Surprisingly, axon complexity decreases only after the completion of the thalamic critical period. Therefore, dynamic bouton redistribution along a broad axon backbone represents an unappreciated form of plasticity underlying developmental wiring and rewiring in the CNS.

Figure 1. Development of BD-RGC axon terminals in the LGN

(A) Schematic of functional pruning at the retinogeniculate synapse. LGN neurons (yellow ovals) and RGC inputs (black lines) of varying strength (black circles). Adapted from Hong and Chen, 2011. (B) Confocal images of BD-RGC labeled in the retina of BD-YFP double transgenic mice. Arrowhead: primary axon. (C) Dense labeling of BD-RGC axons in the dorso-lateral region of LGN (sagittal view). (D) Single axon labeling by dose of tamoxifen. Dashed lines indicate LGN boundary. (E) Examples of reconstructed BD-RGC axon arbors. Quantification of (F) total axon length, (G) average segment length, number of (H) segments, (I) branch points, (J) terminal endings, and (K) maximum branch order. B-D: Image gray values inverted for clarity. Scale bars: 100um. See also Figure S1.

Figure 2. Boutons cluster over development

(A-C) Single YFP-labeled BD-RGC axons at different ages. Confocal images at P8 and P20 coimmunolabeled against YFP (D,G), VGLUT2 (E,H) and merged images (F, I; VGLUT2, red; YFP, green). (J, K) Example outcomes of mean-shift clustering analysis for boutons in x, y, z space for axons shown in A and C. Colored dots represent boutons within clusters; grey dots lie outside clusters. (L-P) Quantification of bouton diameter, NN distances, cluster size (boutons/cluster) determined by mean-shift clustering analysis (>2 boutons), % boutons within clusters, and # boutons per axon. Maximum projection images of confocal stacks of P22 LGN labeling BD-RGC (green) SMI-32 (magenta) (Q,Q’); VGLUT2 (red) and SMI-32 (cyan) (R, R’). Q’,R’: magnified images of single optical section of inset shown in (Q, R), respectively. Scale bars: A-C: 50μm, D-I: 5 μm, Q, R: 10μm, Q’,R’; 2μm . See also Figure S2, S3.

Figure 3. Sensory-dependent Axonal Remodeling

Maximum Projection images of (A) LR and (B) LDR BD-RGC axons. Quantification of average axon (C-E) and bouton (F-I) parameters between P20 and P31 in LR (black) and LDR (red) mice. P20 data set is the same as in Figure 1-2, shown for comparison. Scale bars: 50μm.

Figure 4. Structural axonal pruning occurs between P31-60

Axon backbone (A-D) and bouton (E-H) parameters at P31 (grey) and P60 (red). P31 data set is the same as Figure 3. (I) Representative retinogeniculate slice recordings at P30 and P67. (J) Cumulative probability distribution of single fiber AMPAR strength shows no significant difference. (K) The fiber fraction increases with age. J-K, grey includes P27-34, and red, P60-75. (L) Summary schematic of structural plasticity of RGC axons in LGN. (1) During retinotopic refinement and eye-specific segregation, the axon arbor elaborates and becomes more complex in the monocular layer. (2) During the period of functional synapse elimination and strengthening, the overall structure of the BD-RGC axon backbone is maintained, but the synaptic boutons are redistributed to form clusters and grow in size. (3) During the vision-sensitive period, LDR results in declustering of boutons and decrease in bouton size when compared to LR mice. (4) Finally, between P30-P60, further axonal and synaptic pruning takes place, with a reduction in axon size and complexity. See also Figure S4.