Transduction of retinal ganglion cells (RGCs) in <i>rd1</i> mice following intravitreal administration of an adeno-associated virus (AAV) vector carrying the gene for enhanced green fluorescent protein (EGFP).

<p>Representative retinal flat mount shows extensive GFP expression throughout the whole retina. Some GFP expressing cells have the size and dendritic morphology of RGCs and possess an axon leading from the cells to the optic disc (arrows), indicative of RGCs. The entire dendritic tree structure of some RGCs is clearly visualized by GFP expression (inset). Scale bars, 1 mm (100 µm in inset).</p

Cell populations in the ganglion cell layer of <i>rd1</i> mouse retinas.

<p><b>A–B,</b> Images of DAPI labeled cells in the ganglion cell layer were taken from the retinal regions located 1 mm superior to the center of the optic nerve head at two different ages. Scale bar, 10 µm. <b>C,</b> Quantification of DAPI labeled cells in the ganglion cell layer of <i>rd1</i> mice at three different ages. Values are mean ± SD, with n indicating the number of retinas counted.</p

Determining the dendritic stratification level of RGCs in the IPL.

<p><b>A</b>, One representative image of AAV-GFP transduced RGCs in a cross section of <i>rd1</i> retinas. In this illustration, the RGC has a big soma (arrow) and a broad dendritic tree (arrowheads). <b>B</b>, To define the stratification level of RGC dendrites in the IPL, we stained cell nuclei with diamidinophenylindole (DAPI). The INL and GCL delineate the proximal (0%) and distal (100%) boundaries of the IPL, respectively. <b>C</b>, Merged image shows that the dendrite of the RGC are centered at 25% of the IPL. <b>D</b>–<b>F</b>, One representative image of AAV-GFP transduced RGCs in a cross section of WT mouse retinas was shown as comparison. The transfection in WT was similar to what we observed in rd1 mice. <b>G</b>–<b>I</b>, To distinguish RGCs from diaplaced amacrine cells, AAV-GFP transduced retinas were co-labeled with an antiserum against Thy-1, a marker for all RGCs. Single image section was taken from the ganglion cell layer with focus on cell bodies, and the axon of one AAV-GFP transduced cell was out of focus (arrow). The transduced cell with an axon colocalized with Thy-1 (solid arrowhead), while another transduced cell without an axon did not colocalize with Thy-1 (open arrowhead). INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars, 20 µm in A–F, and 10 µm in G–I.</p

Summary of RGC classification encountered in this study.

<p>Summary of RGC classification encountered in this study.</p

Somatic and dendritic-field sizes of SMI-32 positive α RGCs and their density distribution in the <i>rd1</i> retina.

<p><b>A</b>: Histogram of mean dendritic field diameters of α ganglion cells stained by SMI-32. N indicates the number of α RGCs studied. <b>B</b>: Histogram of mean soma diameters of α ganglion cells stained by SMI-32. There was no significant difference in either dendritic-field sizes or soma sizes of α ganglion cells between WT and <i>rd1</i> retinas (p>0.05, one-way ANOVA analysis). <b>C</b>, The diagram illustrates six sampling areas, regularly spaced along the dorsal-ventral axis of retinal whole-mounts, were surveyed for all cell counting. ONH, optic nerve head. <b>D,</b> The graphs show the density distribution of SMI-32 positive RGCs from both WT and <i>rd1</i> mouse retinas at two different ages.</p

Measurements of morphological parameters.

<p>Measurements of morphological parameters.</p

Representative A2 RGCs classified using nomenclature of Sun et al. (2002), from both WT and <i>rd1</i> mice of different ages.

<p><b>A–F,</b> A2 RGCs were injected with neurobiotin, stained with fluorochrome-conjugated streptavidin, and reconstructed from mosaics of confocal Z-series. Arrows indicate axonal processes emerging from the somata of the A2 RGCs. Arrowhead in <b>B</b> indicates an axon of another injected RGC passing through this A2 RGC. <b>G</b>: Histogram of mean dendritic field diameters of A2 RGCs. There was no significant difference in dendritic-field sizes of A2 RGCs between WT and <i>rd1</i> retinas (p>0.05, one-way ANOVA analysis). <b>H</b>: Histogram of mean soma diameters of A2 RGCs. There was no significant difference in soma sizes between WT and <i>rd1</i> retinas (p>0.05, one-way ANOVA analysis). Scale bars, 50 µm.</p

A large-scale survey of RGCs in <b><i>rd1</i></b><b> retinas of 18 month-old.</b>

<p>Individual RGCs are visualized by GFP expression. Representations of nine types of RGCs are shown together with their dendritic diameters and stratification in the IPL. Numbers in parentheses correspond to the cell types distinguished by Sun et al. (2002). Insets show the dendritic stratification of RGCs in the IPL. In the inset, green circle shows the location of soma, and green line indicates dendrites. The dendrites of Off and On RGCs stratified in the proximal (0–40%) and distal (40–100%) parts of the IPL, respectively. Note that this representation shows the depth of the processes only and does not attempt to show their spread. Arrows indicate axons of individual representative RGCs. INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars, 50 µm.</p

SMI-32 immunostaining in the <i>rd1</i> retina and the cell types it reveals.

<p><b>A–C,</b><b> </b> Antibody to SMI32 was reacted against flat mounted retinas of <i>rd1</i> mice of different ages, developed using DAB-immunohistochemistry. Arrows point to α ganglion cells. <b>A’–C’,</b> Highly magnified image from the boxed regions in <b>A–C</b>. Scale bars, 50 µm in A–C, and 25 µm in A’–C’.</p

RGC Populations in <i>rd1</i> mouse retinas.

<p><b>A–B,</b> RGCs were revealed by using an antiserum against Thy-1, a marker for all RGCs in the mouse retina. Two representative images were taken from <i>rd1</i> retinas at the age of 3 months (<b>A</b>) and 18 months (<b>B</b>). Thy-1 is a surface glycoprotein uniquely expressed in RGC’s membrane (Arrows). Scale bar, 10 µm. <b>C,</b> Quantification of Thy-1 positive cells in the ganglion cell layer of both WT and <i>rd1</i> retinas at two different ages. Values are mean ± SD, with n indicating the number of retinas counted.</p