Seasonally Changing Cryptochrome 1b Expression in the Retinal Ganglion Cells of a Migrating Passerine Bird - PubMed (original) (raw)

Seasonally Changing Cryptochrome 1b Expression in the Retinal Ganglion Cells of a Migrating Passerine Bird

Christine Nießner et al. PLoS One. 2016.

Abstract

Cryptochromes, blue-light absorbing proteins involved in the circadian clock, have been proposed to be the receptor molecules of the avian magnetic compass. In birds, several cryptochromes occur: Cryptochrome 2, Cryptochrome 4 and two splice products of Cryptochrome 1, Cry1a and Cry1b. With an antibody not distinguishing between the two splice products, Cryptochrome 1 had been detected in the retinal ganglion cells of garden warblers during migration. A recent study located Cry1a in the outer segments of UV/V-cones in the retina of domestic chickens and European robins, another migratory species. Here we report the presence of cryptochrome 1b (eCry1b) in retinal ganglion cells and displaced ganglion cells of European Robins, Erithacus rubecula. Immuno-histochemistry at the light microscopic and electron microscopic level showed eCry1b in the cell plasma, free in the cytosol as well as bound to membranes. This is supported by immuno-blotting. However, this applies only to robins in the migratory state. After the end of the migratory phase, the amount of eCry1b was markedly reduced and hardly detectable. In robins, the amount of eCry1b in the retinal ganglion cells varies with season: it appears to be strongly expressed only during the migratory period when the birds show nocturnal migratory restlessness. Since the avian magnetic compass does not seem to be restricted to the migratory phase, this seasonal variation makes a role of eCry1b in magnetoreception rather unlikely. Rather, it could be involved in physiological processes controlling migratory restlessness and thus enabling birds to perform their nocturnal flights.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1

Fig 1. Immuno-labeling for eCry1b in the retina of European Robins.

Retina of a robin (a) in migratory state, and of one (b) after the end of the migratory period. In (a), there is eCry1b labeling in the ganglion cells (layer 6) and the few displaced ganglion cells (layer 4). eCry1b label is located in the cytosol of the cell; the nuclei, indicated by arrows, show no label. In (b), the labeling in the ganglion cells is very low (see also Fig B in S1 Supporting Information). (c-e) Retinal ganglion cell layer in a robin in migratory state, triple-labeled for (c) eCry1b, (d) NeuN, and (e) DAPI. Practically all cells in the ganglion cell layer express eCry1b. Layers of the retina: 1, photoreceptor outer and inner segments; 2, outer nuclear layer; 3, outer plexiform layer; 4, inner nuclear layer; 5, inner plexiform layer; 6, ganglion cell layer; 7, optic nerve fibre layer. The scale bar is 50 μm for all panels.

Fig 2

Fig 2. Electron-microscopic image of a ganglion cell, and Western blots.

(a) Ganglion cell in the retina of a robin in migratory state. eCry1b labeling is visualized with diaminobenzidine and silver intensification, visible as dark dots (some marked by arrows). Other subcellular components cannot be identified; eCry1b is probably free in the cytosol and also bound to membranes. (b, c) Western blots of the robin retina, indicating eCry1b (b; ~65 kDa) and eCry1a (c; ~70 kDa) in the cytosolic and in the membrane fraction. Both cryptochromes were detected in the same blot; the part showing eCry1a was already published in [15]. F1, cytosolic fraction; F2, membrane fraction; F3; nuclear fraction; F4, cytoskeletal fraction; T, tongue tissue from the same bird as control. (d) Western blot of purified eCry1a and eCry1b that had been treated with the eCry1b antiserum as control for the specificity of the antiserum, indicating that there is no cross-reactivity of the eCry1b antiserum with eCry1a.

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Funded by Deutsche Forschungsgemeinschaft (Grant to RW) and Alfons und Gertrud Kassel Stiftung (Scholarship of CN). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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