Immunohistochemical Characterisation of the Whale Retina

Abstract

The eye of the largest adult mammal in the world, the whale, offers a unique opportunity to study the evolution of the visual system and its adaptation to aquatic environments. However, the difficulties in obtaining cetacean samples mean these animals have been poorly studied. Thus, the aim of this study was to characterise the different neurons and glial cells in the whale retina by immunohistochemistry using a range of molecular markers. The whale retinal neurons were analysed using different antibodies, labelling retinal ganglion cells (RGCs), photoreceptors, bipolar and amacrine cells. Finally, glial cells were also labelled, including astrocytes, Müller cells and microglia. Thioflavin S was also used to label oligomers and plaques of misfolded proteins. Molecular markers were used to label the specific structures in the whale retinas, as in terrestrial mammalian retinas. However, unlike the retina of most land mammals, whale cones do not express the cone markers used. It is important to highlight the large size of whale RGCs. All the neurofilament (NF) antibodies used labelled whale RGCs, but not all RGCs were labelled by all the NF antibodies used, as it occurs in the porcine and human retina. It is also noteworthy that intrinsically photosensitive RGCs, labelled with melanopsin, form an extraordinary network in the whale retina. The M1, M2, and M3 subtypes of melanopsin positive-cells were detected. Degenerative neurite beading was observed on RGC axons and dendrites when the retina was analysed 48 h post-mortem. In addition, there was a weak Thioflavin S labelling at the edges of some RGCs in a punctuate pattern that possibly reflects an early sign of neurodegeneration. In conclusion, the whale retina differs from that of terrestrial mammals. Their monochromatic rod vision due to the evolutionary loss of cone photoreceptors and the well-developed melanopsin-positive RGC network could, in part, explain the visual perception of these mammals in the deep sea.

Keywords: cetacean; evolutionary neuroscience; glia; neuron; retina; retinal ganglion cell (RGC); visual system; whale.

FIGURE 1

Scheme of the whale retinal areas. Scheme of a whale retina in which the four different areas of the retina are represented as concentric circles in red. The areas were classified based on their distance from the optic nerve: center, a 2-cm thick band near the optic nerve; middle-center, a 2.5-cm thick band outside the center band; middle-periphery, a 2.5-cm thick band adjacent to the middle-center band; and periphery, a 2-cm thick band at the edge of the retina and the furthest from the optic nerve.

FIGURE 2

Retinal ganglion cell (RGCs) and astrocytes in the pig and fin whale retina. The cytoskeleton of astrocytes (green) was labelled with an antibody against GFAP and the nuclei of the RGCs (red) with an antibody against Brn3a in both pig (A,C) and fin whale (B,D) retinas. Whole mount retinas (A,B) and sections (C,D) are shown. Whale astrocytes do not completely surround the vessels in the whale retina as they do in pigs (white arrowheads A,B). The nuclei in the retinal sections were labelled with DAPI (blue C,D): ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner nuclear layer; GCL, ganglion cell layer. Scale bar = 50 μm.

FIGURE 3

Retinal ganglion cell quantification in the fin whale retina. Image of a whole mount whale retina labelled with an antibody against RBPMS (red) to label RGCs (A). The densities of RGCs (RGCs/mm²) in different regions of the whale retina are shown in a histogram (B). Scale bar = 100 μm.

FIGURE 4

Neurofilament analysis of RGCs in the fin whale retina. Images of whole mount whale retinas (A,B), and retinal sections (C) labelled with antibodies against different types of neurofilaments: NF-H (red) and NH-HP (green) (A); NF-H (red) and NH-M (green) (B); and NF-L (red), NH-M (green), and DAPI (C). Note that each type of NF has a specific labelling pattern and not all the neuron structures were labelled with each NF (arrows). The percentage of RGCs labelled with each type of NF was quantified (D). Scale bar = 50 μm.

FIGURE 5

Melanopsin positive cell quantification and classification in the fin whale retina. Melanopsin positive RGCs (green) in the whole mount whale retina (A). Histogram of the total number of melanopsin positive cells/mm² in different regions of the whale retina (B). Melanopsin cells can be classified based on their soma location, typically in the ganglion cell layer (GCL), while their dendrites stratify in the inner plexiform layer (IPL) (C). The proportion of M1, M2, and M3 subtypes of melanopsin positive cells, and their distribution is represented in a histogram (D). Scale bar = 100 μm.

FIGURE 6

Other RGC markers expressed by Melanopsin positive cells in the whale retina. Images of whole mount whale retinas labelled with antibodies against melanopsin (green) and other specific RGC markers (red): βIII tubulin (A), Brn3a (B), NF-HP (C), and NH-M (D). Note that the melanopsin positive ipRGCs, were not stained with any of the other markers. Scale bar = 100 μm.

FIGURE 7

Photoreceptors the in rat, pig and fin whale retina. Sections of rat (A,D,G,J,M), pig (B,E,H,K,N), and whale (C,F,I,L,O) retinas. The retinas were labelled with antibodies against rhodopsin (red, A–C), M/L opsin (green, D–F), S opsin (green, G–I), rat cone arrestin (green, J–L), and human cone arrestin (red, M–O). The nuclei were labelled with DAPI (blue): OS, outer segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner nuclear layer; GCL, ganglion cell layer. Scale bar = 50μm.

FIGURE 8

Bipolar cells in the rat, pig, and fin whale retina. Sections from rat (A), pig (B), and whale (C) retinas labelled with an antibody against PKC-α (red) to identify bipolar cells. The nuclei were stained with DAPI (blue): OS, outer segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner nuclear layer; GCL, ganglion cell layer. Scale bar = 50 μm.

FIGURE 9

Amacrine cells in the fin whale retina. Sections of whale retinas labelled with antibodies against calbindin (red, A) and calretinin (green, C) to stain amacrine cells. The merged image of the two antibodies is also shown in panel (B). The nuclei were stained with DAPI (blue): OS, outer segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner nuclear layer; GCL, ganglion cell layer. Scale bar = 50 μm.

FIGURE 10

Müller cells in the fin whale and pig retina. Sections from whale retinas labelled with different antibodies to label Müller cells: CRALBP (A), glutamine synthetase (GS, B), p75NTR (C), S100 (D), and vimentin (E). A section of the pig retina was also labelled with vimentin (F). The nuclei were stained with DAPI (blue): ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner nuclear layer; GCL, ganglion cell layer. Scale bar = 50 μm.

FIGURE 11

Microglia in the rat and fin whale retina. Microglial cells were labelled with an antibody against Iba1 (red) in rat (A,C) and fin whale (B,D) whole mount retinas. Images of the microglia were taken close to the ganglion cell layer (A,B) and to the inner nuclear layer (C,D) in the whole mount retinas. Scale bar = 50 μm.

FIGURE 12

Differences in whale retinas due to tissue conservation. Images of whole mount retinas from fin whale (Balaenoptera physalus A,C,E) and sei whale (Balaenoptera borealis B,D,F) fixed 24 and 48 h post-mortem, respectively. The retinas were labelled with antibodies against RBPMS (red, A,B), NF-H (C,D), and melanopsin (E,F). Scale bar = 100 μm.

FIGURE 13

Thioflavin S (ThS) in the fin whale retina. Images of a whole mount whale retina from Balaenoptera physalus stained with ThS (red, A) and labelled with an antibody against βIII tubulin (green, B) to label RGCs. Scale bar = 50 μm.