Identification of retinal neurons in a regressive rodent eye (the naked mole-rat)

Abstract

The retina consists of many parallel circuits designed to maximize the gathering of important information from the environment. Each of these circuits is comprised of a number of different cell types combined in modules that tile the retina. To a subterranean animal, vision is of relatively less importance. Knowledge of how circuits and their elements are altered in response to the subterranean environment is useful both in understanding processes of regressive evolution and in retinal processing itself. We examined common cell types in the retina of the naked mole-rat, Heterocephalus glaber with immunocytochemical markers and retrograde staining of ganglion cells from optic nerve injections. The stains used show that the naked mole-rat eye has retained multiple ganglion cell types, 1-2 types of horizontal cell, rod bipolar and multiple types of cone bipolar cells, and several types of common amacrine cells. However, no labeling was found with antibodies to the dopamine-synthesizing enzyme, tyrosine hydroxylase. Although most of the well-characterized mammalian cell types are present in the regressive mole-rat eye, their structural organization is considerably less regular than in more sighted mammals. We found less precision of depth of stratification in the inner plexiform layer and also less precision in their lateral coverage of the retina. The results suggest that image formation is not very important in these animals, but that circuits beyond those required for circadian entrainment remain in place.

Fig. 1

A captive naked mole-rat (Heterocephalus glaber) investigating an opening in its tunnel system. Although its visual system is greatly reduced, the naked mole-rat retains an eyelid allowing the small eye to be directly exposed to light. Note also the tiny ear (raised area, behind the eye).

Fig. 2

The general structure of the mole-rat (A) and rat (B) retinas are shown with differential interference contrast photography. The structures of the mole-rat retina are less well delineated, but the major differences are the thinner plexiform layers, fewer ganglion cells, and a shortening of the photoreceptor inner and outer segments. OPL: outer plexiform layer; IPL: inner plexiform layer; ONL: outer nuclear layer; INL: inner nuclear layer; and GCL: ganglion cell layer.

Fig. 3

A diversity of ganglion cell types is revealed by staining with DiI or with antibodies to calcium-binding proteins. (A) DiI applied to the optic nerve stains cells both in the GCL (red) and INL (green). Stacks of 0.5-μm sections were taken separately from the two layers and pseudocolored for contrast. (B) Retrograde DiI staining of ganglion cells is shown in a rat retina for comparison. (C) Combined staining with antibodies to parvalbumin (green) and calretinin (red) suggest at least three types of ganglion cells exist in the naked mole-rat retina.

Fig. 4

An antibody to calretinin stains a variety of cells in mole-rat (A) and rat (B) retina. (A) Anti-calretinin stains some types of ganglion, amacrine, horizontal, and bipolar cell in the naked mole-rat. Calretinin immunoreactivity is primarily found in non-AII amacrine cells in the rat retina, as well as some ganglion cells. The nerve fiber layer is also stained. (C) The primary type of amacrine cell stained in the mole-rat retina has the general morphology of AII amacrine cells, as a stout process descends from the soma, contains apparent lobular appendages (arrowheads) in sublamina a, and has processes descending further into sublamina b. (D) A horizontal cell (arrow) is stained by anti-calretinin in the mole-rat; likely bipolar cells are also stained (left). (E) A flatmount view of the calretinin immunoreactivity in the naked mole-rat further shows the lobular appendages near the somas of the putative AII amacrine cells (arrowheads). Each micrograph is a stack of 0.5-μm confocal sections.

Fig. 5

An antibody to the dopamine-synthesizing enzyme tyrosine hydroxylase stains no cells in the naked mole-rat retina (A) at 1:500 dilution. In rat retina (B), this dilution stains the dopaminergic amacrine cells (arrowhead) as well as some other types of amacrine cell (arrows) and probable bipolar cells (stars). Higher dilution (1:10,000) in rat retina stains only the dopaminergic amacrine cell (data not shown).

Fig. 6

An antibody to the acetycholine synthesizing enzyme, choline acetyltransferase (ChAT), stains amacrine cells in both the INL and GCL. (A, B) Radial sections of the mole-rat (A) and rat (B) retinas. Staining in the mole-rat retina lacks the obvious bands present in other mammalian species. (B) Staining in the rat retina has a more even distribution of cell bodies and produces the narrow, parallel bands typical of mammalian ChAT staining. Wholemount views of the mole-rat INL (C) at the level of the amacrine cells and of the GCL (D) show more numerous and intense staining of cells in the INL than in the GCL, consistent with the more distinct labeling in the distal portion of the IPL.

Fig. 7

An antibody to calbindin stains a horizontal cell in the mole-rat retina. Many other unidentified somas are also stained in the outer portion of the INL. The large caliber dendrites marked by arrows lead to a dim soma (long arrow). The brighter horizontal cell has thinner dendrites. No axon is readily detectable emanating from either cell. Inset: A z-rotation shows a radial view of the same horizontal cell prominent in the wholemount view, as well as some amacrine cells located in the proximal INL.

Fig. 8

A radial section of PKC staining (red) in the naked mole-rat. Rod bipolar cells are most brightly stained (arrowheads), which are also lightly stained with CaBP immunoreactivity (green). Some cone bipolar cells may also be stained by PKC, as there are red somas lacking the bushy dendritic arbor of rod bipolar cells and also stained with CaBP immunoreactivity (large stars). Some weakly CaBP-immunoreactive bipolar cells lacking PKC immunoreactivity (small stars) are also presumably cone bipolar cells. PKC immunoreactivity appears diffusely throughout the IPL. The PKC-positive bipolar cell processes extend further than the bottom of the IPL (short arrows), sometimes wrapping around ganglion cell somas and even branching into the nerve fiber layer. CaBP immunoreactivity also labels a putative horizontal cell (long arrow).

Fig. 9

A flatmount view of PKC immunoreactivity (green) and calretinin immunoreactivity (red) in the mole-rat retina. The inset shows comparable staining in the rabbit retina, where each PKC-immunoreactive rod bipolar cell endfoot is contacted by an calretinin-immunoreactive AII amacrine cell process. In the mole-rat, the calretinin-immunoreactive AII amacrine cell processes do not cover the entire surface of the retina; many rod bipolar cell endfeet are apparently missed. Some AII varicosities appear to contact (unlabeled) processes other than rod bipolar cell endfeet. The variation in the size of the rod bipolar terminals is large, compared to the rabbit. Scale bar: 12 μm in figure and 25 μm in inset.