The magnetic compass of birds seems to be based on light-dependent radical-pair processes in the eyes. Cryptochromes are currently the only candidate proteins known in vertebrates that may serve as the primary radical-pair-based magnetoreceptor molecules. Previous immunohistochemical studies have suggested that cryptochrome 1a (Cry1a) is localised in the photoreceptor outer segments of the ultraviolet/violet (UV/V) cones, and it has been claimed that differences in Cry1a antibody staining intensities show that Cry1a is activated by light and that this should make Cry1a the most likely magnetoreceptive candidate molecule. Here, we present an independent study of Cry1a distribution within retinae of several bird species, ranging from non-migratory domestic chicken and rock pigeon to night-migratory passerines, using both the previously used antibody and two newly generated antibodies, one against the same epitope as the originally used antibody and one against a different epitope of Cry1a. We confirm the UV/V cone outer segment localisation of Cry1a in all the tested bird species. In some stainings, we found Cry1a immunoreactivity as a distinct punctate pattern throughout the whole length of the UV/V cone outer segments. These dots with a diameter of around 170 nm might suggest that many Cry1a molecules accumulate in distinct spots in the UV/V cone outer segments. However, we did not see any notable difference in Cry1a immunoreactivity between lightand dark-adapted retinae. We find no evidence whatsoever that a C-terminal antibody against Cry1a labels only a light-activated form of the Cry1a protein.

The primary sensory molecule underlying light-dependent magnetic compass orientation in migratory birds has still not been identified. The cryptochromes are the only known class of vertebrate proteins which could mediate this mechanism in the avian retina. Cryptochrome 4 of the night-migratory songbird the European robin (Erithacus rubecula; erCry4) has several of the properties needed to be the primary magnetoreceptor in the avian eye. Here, we report on the identification of a novel isoform of erCry4, which we named erCry4b. Cry4b includes an additional exon of 29 amino acids compared to the previously described form of Cry4, now called Cry4a. When comparing the retinal circadian mRNA expression pattern of the already known isoform erCry4a and the novel erCry4b isoform, we find that erCry4a is stably expressed throughout day and night, whereas erCry4b shows a diurnal mRNA oscillation. The differential characteristics of the two erCry4 isoforms regarding their 24-h rhythmicity in mRNA expression leads us to suggest that they might have different functions. Based on the 24-h expression pattern, erCry4a remains the more likely cryptochrome to be involved in radical-pair-based magnetoreception, but at the present time, an involvement of erCry4b cannot be excluded.

Cryptochromes are photolyase-related blue-light receptors acting as core components of the mammalian circadian clock in the cell nuclei. One or more members of the cryptochrome protein family are also assumed to play a role in avian magnetoreception, but the primary sensory molecule in the retina of migratory birds that mediates light-dependent magnetic compass orientation has still not been identified. The mRNA of cryptochrome 2 (Cry2) has been reported to be located in the cell nuclei of the retina, but Cry2 localisation has not yet been demonstrated at the protein level. Here, we provide evidence that Cry2 protein is located in the photoreceptor inner segments, the outer nuclear layer, the inner nuclear layer and the ganglion cell layer in the retina of night-migratory European robins, homing pigeons and domestic chickens. At the subcellular level, we find Cry2 both in the cytoplasm and the nucleus of cells residing in these layers. This broad nucleic expression rather points to a role for avian Cry2 in the circadian clock and is consistent with a function as a transcription factor, analogous to mammalian Cry2, and speaks against an involvement in magnetoreception.

Visual (and probably also magnetic) signal processing starts at the first synapse, at which photoreceptors contact different types of bipolar cells, thereby feeding information into different processing channels. In the chicken retina, 15 and 22 different bipolar cell types have been identified based on serial electron microscopy and single‐cell transcriptomics, respectively. However, immunohistochemical markers for avian bipolar cells were only anecdotally described so far. Here, we systematically tested 12 antibodies for their ability to label individual bipolar cells in the bird retina and compared the eight most suitable antibodies across distantly related species, namely domestic chicken, domestic pigeon, common buzzard, and European robin, and across retinal regions. While two markers (GNB3 and EGFR) labeled specifically ON bipolar cells, most markers labeled in addition to bipolar cells also other cell types in the avian retina. Staining pattern of four markers (CD15, PKCα, PKCβ, secretagogin) was species‐specific. Two markers (calbindin and secretagogin) showed a different expression pattern in central and peripheral retina. For the chicken and European robin, we found slightly more ON bipolar cell somata in the inner nuclear layer than OFF bipolar cell somata. In contrast, OFF bipolar cells made more ribbon synapses than ON bipolar cells in the inner plexiform layer of these species. Finally, we also analyzed the photoreceptor connectivity of selected bipolar cell types in the European robin retina. In summary, we provide a catalog of bipolar cell markers for different bird species, which will greatly facilitate analyzing the retinal circuitry of birds on a larger scale.

Night-migratory birds use the Earth’s magnetic field to determine the direction in which they want to migrate. Many studies suggest that this “magnetic compass sense” is light dependent and mediated by blue light sensors, called cryptochromes, which are expressed in the retina of night-migratory birds. In this review, we summarize the evidence that the avian retina processes not only visual information but also magnetic compass information. We also review the current knowledge on cryptochrome expression in the bird retina and highlight open questions which we aim to address within the framework of SFB 1372 Magnetoreception and Navigation in Vertebrates.