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Comparative Study
. 2010 Dec 1;30(48):16376-82.
doi: 10.1523/JNEUROSCI.3455-10.2010.

Emergence of Lamina-Specific Retinal Ganglion Cell Connectivity by Axon Arbor Retraction and Synapse Elimination

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Free PMC article
Comparative Study

Emergence of Lamina-Specific Retinal Ganglion Cell Connectivity by Axon Arbor Retraction and Synapse Elimination

Ting-Wen Cheng et al. J Neurosci. .
Free PMC article

Abstract

Throughout the nervous system, neurons restrict their connections to specific depths or "layers" of their targets to constrain the type and number of synapses they make. Despite the importance of lamina-specific synaptic connectivity, the mechanisms that give rise to this feature in mammals remain poorly understood. Here we examined the cellular events underlying the formation of lamina-specific retinal ganglion cell (RGC) axonal projections to the superior colliculus (SC) of the mouse. By combining a genetically encoded marker of a defined RGC subtype (OFF-αRGCs) with serial immunoelectron microscopy, we resolved the ultrastructure of axon terminals fated for laminar stabilization versus those fated for removal. We found that OFF-αRGCs form synapses across the full depth of the retinorecipient SC before undergoing lamina-specific arbor retraction and synapse elimination to arrive at their mature, restricted pattern of connectivity. Interestingly, we did not observe evidence of axon degeneration or glia-induced synapse engulfment during this process. These findings indicate that lamina-specific visual connections are generated through the selective stabilization of correctly targeted axon arbors and suggest that the decision to maintain or eliminate an axonal projection reflects the molecular compatibility of presynaptic and postsynaptic neurons at a given laminar depth.

Figures

Figure 1.
Figure 1.
Lamina-specific axonal refinement of genetically identified OFF-αRGCs. A, Schematic of retinotopic refinement, SC lamina development, and lamina-specific axon targeting for OFF-αRGCs in the mouse. Retinotopy and SC laminae develop by ∼P4/5, whereas lamina-specific refinement occurs from P4/5 to P12 (Edwards et al., 1986; McLaughlin and O'Leary, 2005; Huberman et al., 2008a). B–E, Lamina-specific refinement of GFP+ OFF-αRGC axons in the SC. Arrows, GFP+ axons in the uSGS that are destined for removal. Asterisk, Non-neural cells that transiently express GFP at the pia. SO, Stratum opticum. Scale bar (in E): B–D, 100 μm; E, 150 μm.
Figure 2.
Figure 2.
Ultrastructure of boutons formed by developing OFF-αRGC axons in the SC. A, Electron micrographs of GFP+ boutons (electron-dense immunoperoxidase reaction products) in the developing SC. GFP+ boutons in the uSGS or lSGS of the SC were examined at P4, P6, P8, and P11 (scale bars, 0.5 μm). Insets, High-magnification images of synapses [arrowheads; scale bars (in main panels), 0.2 μm]. Two serial ultrathin sections of the same bouton were presented for P6 lSGS. P11 3D, Serial EM reconstructions of P11 boutons (5–6 serial ultrathin sections per bouton, 0.35–0.42 μm in thickness) showing the synaptic organization (yellow, PSD) of GFP+ boutons (green) and postsynaptic profiles (purple). B, GFP+ bouton perimeter (mean ± SEM) as a function of age and laminar position. Boutons in the uSGS are significantly smaller than boutons in the lSGS during laminar refinement (p = 0.01, two-way ANOVA; n = 56–62). In the uSGS, bouton size increased from P4 to P6, followed by a reduction from P6 to P11 (*p < 0.007, one-way ANOVA Hochberg tests; n = 12–17). C, Percentage of synapses containing boutons from P4 through P11. Synapses were first observed at P6, and similar percentages were detected in both uSGS and lSGS from P4 to P11 (n = 12–18).
Figure 3.
Figure 3.
Characteristics of developmental versus lesion-induced RGC axon pruning. A–H, Confocal images of the SC of CB2-GFP mice, after unilateral enucleation at P8. GFP+ RGC axons (green) and DAPI+ cell bodies (blue), in the SC ipsilateral (control) and contralateral (enu.) to the enucleated eye. High-magnification images of the uSGS (outlined boxes in A–D) are shown in E–H, respectively. Fragmented axons were observed in the enu. SC 4–6 h after enucleation. Degeneration of RGC axons progressed from the distal toward the proximal (arrowheads) aspect of GFP+ axons. I, Iba1+ (red) microglia. J, Number of Iba1+ glial cells (mean ± SEM). More Iba1+ cells were present in the enu. SC versus the control SC 4–6 h after enucleation (p = 0.03, two-way ANOVA; n = 3 per time point). K, L, GFP+ axons in the SC of two P11 C1q−/−::CB2-GFP mice. L, CTB-Alexa594 (red) labeling of all RGC axons. GFP+ axons were restricted to the lSGS as in wild-type CB2-GFP mice. M–R, Electron micrographs of P8 GFP+ boutons located in the uSGS at 5 h after enucleation. In the control SC hemisphere (M–O), intact boutons with synapses (arrowhead, inset) were observed (M, N). O, 3D reconstruction of serial sections (17 serial sections including M and N; 1.2 μm thick) demonstrating the synaptic contact (green, bouton; purple, postsynaptic profile; yellow, PSD; gray square, section plane). In the enu. SC (P–R), GFP+ axon fragments were surrounded by glial processes (blue). R, 3D reconstructions (14 serial sections including P and Q; 0.98 μm thick) showing GFP+ axon fragments (green) separated from mitochondria (pink), both of which are contained within the glial cell (blue). Scale bars: A–D, K, L, 100 μm; E–I, 25 μm; M–Q, 0.5 μm; inset in M, 0.2 μm (in main panels). Also see supplemental movie (available at www.jneurosci.org as supplemental material).
Figure 4.
Figure 4.
Quantification of OFF-αRGC axonal boutons in the developing SC. A, PSD length as a function of age and laminar position. PSD length was not significantly different in the uSGS versus lSGS (p = 0.33, two-way ANOVA; n = 5–9). B, lSGS bouton perimeter as a function of age and synaptic contact. In the lSGS, the size of boutons stayed the same from P6 through P11 regardless of the presence or absence of synapses (p = 0.72, two-way ANOVA; n = 5–9). C, uSGS bouton perimeter as a function of age and synaptic contact. In the uSGS, bouton size decreased, regardless of the presence or absence of synapse (age p = 0.02, synapse p = 0.36, two-way ANOVA; n = 5–9). Values are mean ± SEM.

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