Laminar restriction of retinal ganglion cell dendrites and axons: subtype-specific developmental patterns revealed with transgenic markers

Abstract

Retinal ganglion cells (RGCs), which transfer information from the eye to the brain, are heterogeneous in structure and function, but developmental studies have generally treated them as a single group. Here, we investigate the development of RGC axonal and dendritic arbors using four mouse transgenic lines in which nonoverlapping subsets of RGCs are indelibly labeled with a fluorescent protein. Each subset has a distinct functional signature, size, and morphology. Dendrites of each subset are restricted to specific sublaminae within the inner plexiform layer in adulthood, but acquire their restriction in different ways: one subset has lamina-restricted dendrites from an early postnatal stage, a second remodels an initially diffuse pattern, and two others develop stepwise. Axons of each subset arborize in discrete laminar zones within the lateral geniculate nucleus or superior colliculus, demonstrating previously unrecognized subdivisions of retinorecipient layers. As is the case for dendrites, lamina-restricted axonal projections of RGC subsets develop in different ways. For example, while axons of two RGC subsets arborize in definite zones of the superior colliculus from an early postnatal stage, axons of another subset initially occupy a deep layer, then translocate to a narrow subpial zone. Together, these results show that RGC subsets use a variety of strategies to construct lamina-restricted dendritic and axonal arbors. Taking account of these subtype-specific features will facilitate identification of the molecules and cells that regulate arbor formation.

Figure 1.

Transgenic lines that mark RGC subsets. a, Transgenic lines used in this study. b, c, e, f, Portions of retinas showing YFP-marked J-RGCs in a JxTSY mouse (b), BD-RGCs in a BDxTSY mouse (c), W3-RGCs in a TYW3 mouse (e), and W7-RGCs in a TYW7 mouse (f). d, g, Retinas from a BDxTSY mouse (d) and a TYW7 mouse (g) with reduced density of labeling resulting from manipulation of Cre as shown in a and described in results. Scale bar: (in g) b–g, 100 μm.

Figure 2.

Morphology of J-, BD-, W3-, and W7-RGCs. a–g, Confocal stacks of J-, BD-, W3-, and W7-RGCs. W7a and W7b indicate RGCs with dendrites in SL1–SL2 and SL4 or only in SL4, respectively. a′–g′, Z rotations of cells shown in a–g. h, i, Dendritic field and soma areas of labeled RGCs, measured from images such as those in a–g (n = 13–27 cells per type). Error bar indicates SEM. Scale bar: (in g) a–g, 50 μm.

Figure 3.

Lamina-restricted dendrites of J-, BD-, W3-, and W7-RGCs. RGCs were labeled with anti-GFP (green), starburst amacrines with anti-ChAT or VAChT (red), and somata with Neurotrace or TO-PRO (blue). INL, Inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar: (in d) a–d, 20 μm.

Figure 4.

Visual response properties of J-, BD-, W3-, and W7- RGCs. a–d, Sample responses to a spot flashing on (white) and off (gray) over the receptive field center. Raster plot of spikes on six repeats. e–h, Responses to flashing spots as a function of the spot radius. Plotted is the number of spikes during the light off (closed circles) or light on (open circles) period normalized to the maximum. i–l, Responses to small spots moving across the receptive field center in different directions. The polar plot shows the relative number of spikes fired for each of the eight directions. The corresponding direction selectivity index (DS index) values are indicated on the right. m, The optimal spot radius, a measure of the receptive field center size, for each of the RGC types. Error bars denote SEM. Sample size is 17, 6, 23, and 15 for J, BD, W3, and W7, respectively. n, The radius of the dendritic field plotted against the radius of the optimal spot for each of the four types. Ordinate shows the radius of a circle with the same area as the dendritic field. o, Direction selectivity (DS) index for each of the RGC types, calculated as described in Materials and Methods. Sample size is 16, 10, 11, and 4 for J, BD, W3, and W7, respectively. All error bars indicate SEM.

Figure 5.

Subtype-specific patterns of dendritic development in the inner plexiform layer. Retinal sections were from P5, P8, and P12–P13 mice. Staining and abbreviation are as in Figure 3. All subtypes have lamina-restricted dendrites by P12–P13 (c, f, i, l; compare with Fig. 3), but the extent and nature of postnatal remodeling varies among subtypes. Scale bar: (in l) a–l, 20 μm.

Figure 6.

Lamina-restricted arbors of RGC axons within the retinorecipient zone of the superior colliculus. a–j, Vibratome sections were immunostained with anti-GFP (green) and counterstained with Neurotrace or TO-PRO (blue). a, c, e, g, i, Coronal sections from mice that had been monocularly enucleated to ablate retina axons from the side marked “ipsi.” b, d, f, h, j, Sagittal sections from other mice that had not been enucleated. Brackets indicate the span of axonal arbors of RGC subtypes. Transgenic lines are indicated at the left; mice were 40 d of age or older. k, The entire retinorecipient zone is marked by injection of cholera toxin b subunit (CTB, red) into one eye, and corresponds to that marked in Chx10-Cre × TSY mice in a. Arrows in a and k show sparse ipsilateral projections, which terminate in a deep region. l, Schematic showing distinct axonal restriction of each type of RGCs and ipsi projection. Scale bar: (in k) a–k, 300 μm.

Figure 7.

Lamina-restricted arbors of RGC axons in the lateral geniculate nucleus. Coronal sections were stained with anti-GFP (green) and Neurotrace (blue) after enucleation and cholera toxin administration (CTB, red). a, b, J-RGCs. c, d, BD-RGCs. Cholera toxin-positive regions in b and d contain projections from the ipsilateral retina. D, Dorsal, V: ventral. Scale bar: (in d) a–d, 150 μm.

Figure 8.

Subtype-specific patterns of axonal development in the superior colliculus. Sagittal sections from P5–P6, P8, P11–P13, and P17–P18 mice are shown. Axonal arborization from each RGC subtype was visualized by immunostaining to anti-GFP (green). i′–p′, High magnification of boxes marked in i–p to emphasize distinct patterns of axonal arborization in W3- and W7-RGCs. The pia surface is indicated by a red line. Blue, Cell bodies. Scale bars: (in p) a–p, 200 μm; (in p′) i′–p′, 100 μm.

Figure 9.

Subtype-specific patterns of axonal development in the lateral geniculate nucleus. Sections were immunostained by anti-GFP (green) at P5–P6, P7–P8, and P11. The dotted lines mark the boundary of LGN. Scale bar: (in f) a–f, 100 μm.

Figure 10.

Summary of subtype-specific patterns of dendritic and axonal development. See Results for details.