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. 2013 Oct 30;33(44):17444-57.
doi: 10.1523/JNEUROSCI.5461-12.2013.

Differential Progression of Structural and Functional Alterations in Distinct Retinal Ganglion Cell Types in a Mouse Model of Glaucoma

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Free PMC article

Differential Progression of Structural and Functional Alterations in Distinct Retinal Ganglion Cell Types in a Mouse Model of Glaucoma

Luca Della Santina et al. J Neurosci. .
Free PMC article

Abstract

Intraocular pressure (IOP) elevation is a principal risk factor for glaucoma. Using a microbead injection technique to chronically raise IOP for 15 or 30 d in mice, we identified the early changes in visual response properties of different types of retinal ganglion cells (RGCs) and correlated these changes with neuronal morphology before cell death. Microbead-injected eyes showed reduced optokinetic tracking as well as cell death. In such eyes, multielectrode array recordings revealed that four RGC types show diverse alterations in their light responses upon IOP elevation. OFF-transient RGCs exhibited a more rapid decline in both structural and functional organizations compared with other RGCs. In contrast, although the light-evoked responses of OFF-sustained RGCs were perturbed, the dendritic arbor of this cell type remained intact. ON-transient and ON-sustained RGCs had normal functional receptive field sizes but their spontaneous and light-evoked firing rates were reduced. ON- and OFF-sustained RGCs lost excitatory synapses across an otherwise structurally normal dendritic arbor. Together, our observations indicate that there are changes in spontaneous activity and light-evoked responses in RGCs before detectable dendritic loss. However, when dendrites retract, we found corresponding changes in receptive field center size. Importantly, the effects of IOP elevation are not uniformly manifested in the structure and function of diverse RGC populations, nor are distinct RGC types perturbed within the same time-frame by such a challenge.

Figures

Figure 1.
Figure 1.
Correlations between IOP elevation, RGC death, and optokinetic responses of microbead-injected and control eyes. A, Plots of IOP of bead- and saline-injected control eyes. Shown here are the IOP measurements for 6 animals that survived for 30 d after injection. Black lines and symbols are saline-injected and gray lines and symbols are bead-injected. Within each group, each animal is represented by a different symbol. The solid lines represent the averaged data for each group. B, Plots of SFT for the same animals as in A. C, Nuclear staining (TO-PRO3, gray) and Brn3a immunostaining (red) in the GCL of control and microbead-injected eyes. D, Average density of cells (gray histograms) and Brn3a+ cells (red histograms) in the GCL of the retinas from A and B. Each plot quantifies cell loss in a specific retinal quadrant, as shown in the inset (T, temporal; V, ventral; N, nasal; D, dorsal). Statistics: Wilcoxon–Mann–Whitney rank sum test. E, Correlation between IOP elevation and SFT for each eye of the animals in A and B. F, Correlation between IOP elevation and cell loss for each animal. For all plots (A–F), each symbol represents the saline- and bead-injected eyes from an individual animal.
Figure 2.
Figure 2.
Spontaneous activity of RGCs is reduced following IOP elevation. A, Spike raster plots of representative RGCs showing spontaneous activity and their responses to square-wave full-field light stimulation (4 s ON, 5 s OFF). Shown here are RGCs recorded from saline-injected (black lines) or microbead-injected eyes (gray lines), 30 d after injection. B, Five major types of RGCs were identified based on their response profiles to light ON and OFF, shown here for one recording. RON and ROFF are the average spike rate in response to light ON or OFF, respectively. y-axis: Values closer to 1 indicate ON RGCs; values closer to −1 indicate OFF RGCs, values close to zero indicate ON–OFF RGCs. x-axis: Values approaching 1.0 indicate relatively sustained responses. See Materials and Methods for definition of these parameters. C, Plots of IOP and SFT for mice recorded 15 or 30 d after injection (n = 5 animals each). D, Quantification of the average spontaneous spike rates of each major RGC type for the animals whose IOPs and SFTs are shown in C. Numbers inside histograms represent number of recorded cells. Statistics: Wilcoxon–Mann–Whitney rank sum test.
Figure 3.
Figure 3.
OFF transient RGCs rapidly demonstrate altered receptive field center size after IOP elevation. A, Spatial representations of the preferred stimuli for representative RGCs in saline- or microbead-injected eyes. Each image is the average stimulus taken at the maximum (for ON RGCs) or minimum (for OFF RGCs) of the STA temporal profile, during checkerboard Gaussian white noise stimulation. B, Quantification of the average receptive field center size of the cells in Figure 2 revealed a significant reduction only for OFF-transient RGCs after microbead injection. Numbers in histograms indicate number of cells recorded; 5 mice per group. Statistics: Wilcoxon–Mann–Whitney rank sum test.
Figure 4.
Figure 4.
Nonlinearities in the light responses are differentially affected among RGC types. A, Average spike rates as a function of the generator signal. Cumulative distribution functions were fitted (lines) to the experimental data points. B–D, Average values of threshold (B), gain (C), and maximal spike rate (D) are differentially affected among the RGC types. See Materials and Methods for the definition of these parameters. Cell numbers are noted in the histograms; 5 animals per group. Statistics: by Wilcoxon–Mann–Whitney rank sum test.
Figure 5.
Figure 5.
Dendritic arbor of three RGC types in control and microbead-injected eyes. Maximum intensity projections of confocal image stacks of RGCs in Thy1-YFP retinas from recorded saline-injected and microbead-injected mice. Orthogonal rotations of the arbors are provided below the x–y views. Blue labeling represents staining of cell nuclei in the ganglion cell layer and inner nuclear layer with TO-PRO3. Scale bars, 20 μm. IPL, Inner plexiform layer.
Figure 6.
Figure 6.
Reduction in receptive field size of OFF-transient RGCs occur before pronounced dendritic changes. A–F, Measurements of dendritic territory, total dendritic length, and number of dendrites (A) and Sholl analysis for OFF-transient RGCs (B), OFF-sustained RGCs (C, D), and for ON-sustained RGCs (E, F) in control saline and bead conditions. These measurements were performed on RGCs labeled in retinas from Thy1-YFP mice after multielectrode array recording. See Materials and Methods for definitions of the morphological measurements. Cell numbers are noted in the histograms; 5 animals per group. Statistics: Wilcoxon–Mann–Whitney rank sum test.
Figure 7.
Figure 7.
OFF transient RGCs show decreased synaptic density in addition to reduced dendritic arborization. A, Example of biolistically labeled OFF transient-RGCs in control and microbead-injected eyes. Cells coexpressed PSD95-CFP and tdTomato. B, Magnified views of the dendrites within the boxed area in A. C, Distribution of identified PSD95-CFP puncta of the cells in A, represented by yellow dots. Insets show “heat-maps” of the linear density of PSD95 of the cell. Hotter colors indicate higher density of PSD95 puncta (Morgan et al., 2008). D, Average linear density of PSD95 puncta across conditions, from retinas of animals in Figure 1. Cell numbers are provided in the histograms (n = 5 mice each condition). Statistics: Wilcoxon–Mann–Whitney rank sum test. E, Average linear density of PSD95 puncta as a function of distance from the soma position. Solid line: average value of sampled cells; colored band: SEM.
Figure 8.
Figure 8.
OFF-sustained RGCs show decreased synaptic density despite a normal dendritic morphology. A, Example of biolistically labeled OFF sustained RGCs in microbead-injected and control eyes showing coexpression of PSD95-CFP and tdTomato. B, Bottom are magnified views of the dendrites in the boxed regions. C, Distribution of identified PSD95-CFP puncta, represented by yellow dots. Insets show “heat-maps” of the linear density of PSD95 of the cell. Hotter colors indicate higher density of PSD95 puncta. D, Average linear density of PSD95-CFP puncta of RGCs labeled in retinas obtained from animals in Figure 1. Cell numbers are noted on the histograms (n = 5 mice for each condition). Statistics: Wilcoxon–Mann–Whitney rank sum test. E, Average linear density of PSD95-CFP puncta of the cells in C, as a function of distance from the soma. Solid line: average value of sampled cells; colored band: SEM.
Figure 9.
Figure 9.
ON-sustained RGCs show decreased synaptic density despite normal dendritic morphology. A, Immunolabeled PSD95 on the dendrites of ON-sustained RGCs across conditions. B, Higher magnifications of the area within white rectangles in A. Shown are as follows: (top) raw images of PSD95 labeling (yellow) and dendrites (red), (middle) PSD95 within the dendritic mask, and (bottom) PSD95 channel only (for masked dendrite). C, Distribution of PSD95 puncta. Each yellow dot is a punctum. Insets are heat map representations of the linear puncta density of the arbor. D, Comparison of average linear density of cells from saline- and bead-injected eyes. Cell numbers are provided on the histograms (n = 5 mice each condition). Statistics: Wilcoxon–Mann–Whitney rank sum test. E, Average linear density of PSD95 puncta for the cells in D, plotted as a function of distance from the soma position.
Figure 10.
Figure 10.
Summary of physiological and morphological alterations of various RGC types due to IOP elevation upon microbead injection.

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