Characteristic patterns of dendritic remodeling in early-stage glaucoma: evidence from genetically identified retinal ganglion cell types

Abstract

Retinal ganglion cell (RGC) loss is a hallmark of glaucoma and the second leading cause of blindness worldwide. The type and timing of cellular changes leading to RGC loss in glaucoma remain incompletely understood, including whether specific RGC subtypes are preferentially impacted at early stages of this disease. Here we applied the microbead occlusion model of glaucoma to different transgenic mouse lines, each expressing green fluorescent protein in 1-2 specific RGC subtypes. Targeted filling, reconstruction, and subsequent comparison of the genetically identified RGCs in control and bead-injected eyes revealed that some subtypes undergo significant dendritic rearrangements as early as 7 d following induction of elevated intraocular pressure (IOP). By comparing specific On-type, On-Off-type and Off-type RGCs, we found that RGCs that target the majority of their dendritic arbors to the scleral half or "Off" sublamina of the inner plexiform layer (IPL) undergo the greatest changes, whereas RGCs with the majority of their dendrites in the On sublamina did not alter their structure at this time point. Moreover, M1 intrinsically photosensitive RGCs, which functionally are On RGCs but structurally stratify their dendrites in the Off sublamina of the IPL, also underwent significant changes in dendritic structure 1 week after elevated IOP. Thus, our findings reveal that certain RGC subtypes manifest significant changes in dendritic structure after very brief exposure to elevated IOP. The observation that RGCs stratifying most of their dendrites in the Off sublamina are first to alter their structure may inform the development of new strategies to detect, monitor, and treat glaucoma in humans.

Keywords: dendrites; glaucoma; retinal ganglion cells.

Figure 1.

Elevation of IOP with bead occlusion. A, B, Microbeads (10 μm) injected in the anterior chamber of one eye and confirmed by in vivo fluorescent microscopy. Beads prevent the aqueous outflow through the trabecular meshwork, thus inducing elevation of IOP. C, Quantification of maximum IOP (at any point before or within the week after bead injection) in control and bead-injected eyes, before (baseline; black) and after bead injection (red). Bead injection causes a significant increase in IOP relative to baseline and to controls. D, Plot showing the daily average IOPs in the eyes of the control group (empty circles) and the eyes of the bead-injected group (filled circles). Arrow, IOP after bead injection. N = 17 mice. Error bars: ±SEM; *p < 0.05; **p < 0.01 (Student's t test).

Figure 2.

Transgenic mouse lines for examination of the effects of elevated IOP on specific, distinct subtypes of RGCs. A, Whole-mount retina, with GFP-expressing RGCs. Scale bar, 100 μm. B–D, Individual GFP-expressing RGCs (B) targeted with a glass pipette (C) and filled with fluorescent dye to reveal its complete dendritic structure (D). Scale bar: (in C) B–D, 50 μm. E–H, Maximum intensity projection images showing the morphology of an individual RGC that was GFP+ (E) and filled with Alexa Fluor 555 (F). G, Merged image. Green, GFP; white, targeted fill. H, Neurolucida three-dimensional reconstruction of cell body, proximal axon, dendritic branching, and stratification depth. I, Schematic of laminar organization of the retina. The vitreal or “On sublamina” half of the inner plexiform layer (ipl) is shown in red. The scleral “Off sublamina” half of the ipl is in black. prl, photoreceptor layer; opl, outer plexiform layer; inl, inner nuclear layer; GCL, ganglion cell layer. J–M, Lucida reconstructions of representative examples of each of the RGC subtypes examined in this study: On DSGCs (J) and aOn-Off DSGC (K) labeled in Hoxd10-GFP transgenic mice; tOff-α RGCs (L) labeled in CB2-GFP transgenic mouse mice; and type-M1 ipRGCs (M) visualized through immunostaining with anti-melanopsin antibodies. Dendritic stratification patterns are shown below as side-view images (red: soma, axon, and dendrites localized to On sublamina; black: dendrites localized to the Off sublamina). Arrowheads in H and J–M indicate the axon (red). Scale bar, 50 μm.

Figure 3.

tOff-α RGCs rapidly alter their dendritic structure in response to elevated IOP. A–F, Maximum intensity projection confocal images of tOff-α RCCs from control (A–C) and IOP-elevated retinas (D–F). CB2-GFP somas (A, D, inset image) were filled with Alexa Fluor 555 hydrazide dye (A, D) and reconstructed (C, F). Arrowhead, Axon. B, E, Side views showing the stratification depth of the dendritic arbor within the Off sublamina (green, cell fill; purple, ChAT and VAChT immunostaining; bottom panels show area in dotted rectangle). G–L, Quantification of various morphological parameters examined for RGCs in control (white bars) and IOP-elevated/bead-injected (black bars) eyes. No significant differences (n.s.) were found in soma diameter (G) and total number of dendritic branches (H). Dendritic length (I; t test *p < 0.05) and dendritic field area of tOff-α RGCs were significantly reduced in IOP-elevated retinas (J; t test **p < 0.01), which was corroborated by Sholl ring analysis (K; t test statistical significance: 20–90 μm, *p < 0.001; 100–110 μm, *p < 0.01; 210–220 μm, *p < 0.05), and DOi analysis (L, **p < 0.01). M, N, representative example of the Sholl ring analysis used to generate the dendritic orientations values in L for control (M) and IOP-elevated neurons (N). For G–L, error bars represent ± SEM; O, P, Examples of tOff-α RGCs obtained from control retinas (O) and IOP-elevated retinas (P). Side views shown at the bottom of each neuron (red: soma and axon; black: Off sublamina dendrites). Scale bars: A, P, 50 μm.

Figure 4.

Change in symmetry of tOff-α RGCs is not due to changes in GFP expression patterns. A, Neurolucida reconstructions of tOff-α RGCs in CB2-GFP mice showing asymmetric morphologies. For most cells, the dendrites are pointed away from the temporal axis. D, dorsal; N, nasal; T, temporal; V, ventral. Scale bar, 50 μm. B–E, Axonal projections patterns of tOff-α RGCs are maintained to and within central visual targets after IOP elevation, indicating that there is no change in the GFP expression patterns caused by the bead injections. dLGN, dorsal LGN; vLGN, ventral LGN; SO, stratum optimum; lSGS, lower stratum griseum superficialis; uSGS, upper stratum griseum superficialis; SZ, stratum zonale. D, E, dashed line delineates the border of the dLGN. Asterisks indicate the shell of the dLGN, which is devoid of CB2-GFP axonal projections (Huberman et al., 2008, 2009). Scale bar, 100 μm.

Figure 5.

On DSGCs are structurally resistant to the early phase of elevated IOP. A–F, Same format as for Figure 3 but GFP cells are On DSGCs from Hoxd10-GFP mice. G–K, Quantification of various morphological parameters examined for RGCs in control (white bars) and IOP-elevated (black bars) eyes. No significant differences (n.s.) were found for any of the morphological parameters studied, including soma diameter (G), total number of branches (H), dendritic field area (I), dendritic length (J), as well as the percentage dendritic length in the On versus Off sublaminae (K). L, Moreover, no changes were noted when examining the overall dendritic architecture using Sholl ring analysis. For G–L, error bars represent ±SEM. M, N, Representative examples of On DSGCs in control (M) and IOP-elevated retinas (N). For C, F, M, and N, red represents the soma, axon (arrowhead), and dendrites found in the On sublamina, while dendrites present in the Off sublamina are depicted in black. Scale bars: A, N, 50 μm.

Figure 6.

aOn-Off DSGCs show laminar-specific alterations in their dendritic structure. A–F, Same as in Figure 5 but for aOn-Off DSGCs labeled in Hoxd10-GFP mice. G–I, No significant changes were noted for soma size (G), dendritic length (H), and dendritic field area (I) of aOn-Off DSGCs in control (white bars) and IOP-elevated retinas (black bars). J–M, RGCs had a larger number of total dendritic branches (J) in IOP-elevated retina, caused by an increase in the length of the dendrites present in the On sublamina (K, L) and a reduction of Off-sublamina dendrites (K, M). N–P, Sholl analysis revealing the increased branching of On-sublamina dendrites (O, asterisk) and reduction of Off-sublamina ones (P, asterisk). For G–P, t test *p < 0.05 and **p < 0.01; error bars represent ±SEM. For C–F, the soma, axon (arrow), and On dendrites are shown in red, and Off-sublamina dendrites are shown in black. Scale bar: A, 50 μm.

Figure 7.

Additional examples of aOn-Off DSGCs in Hoxd10-GFP retinas. A, B, RGCs from control retinas (A) and IOP-elevated retinas (B). The soma, axon, and dendrites found in the On sublamina are shown in red, while dendrites present in the Off sublamina are depicted in black. Note the increase in On-sublamina dendrites (red arrows) and the reduction in Off-sublamina dendrites (black arrows; Fig. 6). Scale bar, 50 μm.

Figure 8.

M1 ipRGCs have reduced dendritic branching in response to elevated IOP. A–E, Maximum-intensity projection images showing the morphologies of M1 ipRGCs obtained by immunostaining for melanopsin in control (A–C) and IOP-elevated retinas (D–F). Reconstructed M1 cells (B, E; arrow, axon). G–K, Quantitative morphological comparison of control (white bars) and IOP-elevated (black bars) RGCs showed no significant differences (n.s.) for soma diameter (G) and dendritic field area (H). Reduction was significant for total number of branches (I) and dendritic length (J). K, Sholl analysis showing reduction of branches along distinct locations of the dendritic arbor denoted by asterisks. For G–K, error bars represent ±SEM; t test *p < 0.05. L, M, Representative examples of M1 ipRGCs are shown from control (L) and IOP-elevated (M) retinas. Scale bars: A, M, 50 μm.

Figure 9.

Subtype-specific RGC loss in response to IOP elevation. A, Bar graph showing the normalized fraction of RGCs in the different transgenic mouse lines between control (empty bars) and IOP-elevated retinas after 1 (gray bars) or 2 (dark gray bars) weeks after IOP elevation. Total counts of GFP+ somata revealed a subtype-specific cell loss in bead-injected retinas, in which the highest degree of cell loss was observed for tOff-α RGCs compared with other RGC subtypes. B–D, Bar graphs showing the normalized fraction of RGCs between the two eyes of normal, untreated animals in the different transgenic mouse lines, CB2-GFP (B) and Hoxd10-GFP (C), as well as for melanopsin-positive RGCs (D). Note that in normal conditions, there are no significant differences (n.s.) in the number of RGCs between the two eyes. For A–D, error bars represent ±SEM. t test *p < 0.01.

Figure 10.

RGC dendritic susceptibility is not correlated to soma size. Scatter plot showing lack of correlation between soma size and the DCI for RGC subtype that showed changes in IOP-elevated retinas. Different colors represent the distinct subtypes of RGCs. Line represents a linear regression fit, with the r² value of 0.004.