Ectopic photoreceptors and cone bipolar cells in the developing and mature retina

Abstract

An antibody against recoverin, the calcium-binding protein, labels photoreceptors, cone bipolar cells, and a subpopulation of cells in the ganglion cell layer. In the present study, we sought to establish the origin and identity of the cells expressing recoverin in the ganglion cell layer of the rat retina. By double labeling with rhodopsin, we demonstrate that early in development some of the recoverin-positive cells in the ganglion cell layer are photoreceptors. During the first postnatal week, these rhodopsin-positive cells are eliminated from the ganglion cell layer, but such neurons remain in the inner nuclear layer well into the first postnatal month. Another contingent of recoverin-positive cells, with morphological features equivalent to those of bipolar cells, is present in the postnatal retina, and approximately 50% of these neurons survive to maturity. The incidence of such cells in the ganglion cell layer was not affected by early transection of the optic nerve, a manipulation that causes rapid loss of retinal ganglion cells. These recoverin-positive cells were not double-labeled by cell-specific markers expressed by photoreceptors, rod bipolar cells, or horizontal and amacrine cells. Based on their staining with recoverin and salient morphological features, these ectopic profiles in the ganglion cell layer are most likely cone bipolar cells. Collectively, the results provide evidence for photoreceptors in the ganglion cell and inner nuclear layers of the developing retina, and a more permanent subpopulation of cone bipolar cells displaced to the ganglion cell layer.

Fig. 1.

Confocal images of recoverin expression patterns in the rat retina during prenatal and postnatal development. Ages are indicated in each photomicrograph. Gestation in the rat is 21 d. Recoverin is expressed by developing cells in the neuroblast layer of the rat retina as early as E16. The number of cells that express recoverin increases markedly during the last days of gestation and the first week of postnatal life. Note that the migration of these cells into the ganglion cell layer (GCL,arrows) precedes normal migration into the inner nuclear layer (INL). In this and all of the other figures, the ganglion cell layer is oriented down. NBL, Neuroblast layer; ONL, outer nuclear layer;OPL, outer plexiform layer; IPL, inner plexiform layer. Scale bars, 20 μm.

Fig. 2.

Top, Distribution of recoverin-positive cells in the ganglion cell layer of P6 and P8 whole-mounted retinas. Shaded regions had incomplete staining. Scale bar, 1 mm. Note that the P8 retina is sized to be comparable with the P6 retina. N, Nasal;T, temporal. Bottom, Selected low- and high-power images from P6 (top row) and P8 (bottom row) whole-mounted retinas. Scale bars, 50 μm in low-power images and 20 μm in high-power images.

Fig. 3.

Confocal images of rhodopsin-labeled cells (left) and recoverin-labeled cells (middle) in the rat retina during postnatal development (P0–P7). As indicated by arrows in the right panels, some recoverin-positive cells are rods that have migrated beyond the photoreceptor layer. GCL, Ganglion cell layer; NBL, neuroblast layer; OPL, outer plexiform layer; IPL, inner plexiform layer;INL, inner nuclear layer. Scale bars, 20 μm.

Fig. 4.

Bright-field images of rhodopsin labeling in the P20 and adult retinas. Note that in the adult retina there are no rhodopsin-positive cells in the inner retina. Inset, High-power image of P20. Scale bars, 10 μm in low-power images and 5 μm in high-power images.

Fig. 5.

Confocal images of recoverin-expressing cells (arrows) in the ganglion cell layer of adult retinas (Normal) and in retinas of comparable age in which all ganglion cells were depleted by an optic nerve transection (ONT) at P0 or P1. The presence of such recoverin-positive neurons after early ONT suggests that these were not ganglion cells with centrally projecting axons. In some cases, the axon of a recoverin-expressing cell was found to project upward toward the inner plexiform layer, as shown in the left middle panel. Scale bar, 20 μm.

Fig. 6.

Confocal images of Thy1.1-, vimentin-, parvalbumin-, and PKC-labeled cells (left) and recoverin-labeled cells (middle) in the rat retina. Overlapping images are shown at the left. Note that none of the recoverin-positive cells in the ganglion cell layer are double-labeled. Note also that the axonal process of recoverin-positive cells in the ganglion cell layer is directed toward the inner plexiform layer in the vimentin–recoverin panel. Scale bar, 20 μm.