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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.

Pannexin1 (Panx1) belongs to a class of vertebrate proteins that exhibits sequence homology to innexins, the invertebrate gap junction proteins, and which also shares topological similarities with vertebrate gap junction proteins, the connexins. Unlike gap junctional channels, Panx1 forms single-membrane channels, whose functional role in neuronal circuits is still unsettled. We therefore investigated the subcellular distribution of Panx1 in the mouse retina of wildtype and Panx1-null mice by reverse-transcription polymerase chain reaction (RT-PCR), immunohistochemistry, and electron microscopy. Use of Panx1-deficient mice served as a model to assess the physiological role of Panx1 by electroretinographic recordings and also to ensure the specificity of the anti-Panx1 antibody labeling. Expression of Panx1 was found in type 3a OFF bipolar cells and in dendrites and axonal processes of horizontal cells. Panx1 was also found in horizontal cell dendrites representing the lateral elements of the triad synapse at cone and rod terminals. In vivo electroretinography of Panx1 knockout mice indicated an increased a- and b-wave compared to Panx1 wildtype mice under scotopic conditions. The effect on the b-wave was confirmed by in vitro electroretinograms from the inner retina. These results suggest that Panx1 channels serve as sinks for extracellular current flow making them possible candidates for the mediation of feedback from horizontal cells to photoreceptors.

The identification of the proteins that make up the gap junction channels between rods and cones is of crucial importance to understand the functional role of photoreceptor coupling within the retinal network. In vertebrates, connexin proteins constitute the structural components of gap junction channels. Connexin36 is known to be expressed in cones whereas extensive investigations have failed to identify the corresponding connexin expressed in rods. Using immunoelectron microscopy, we demonstrate that connexin36 (Cx36) is present in gap junctions of cone but not rod photoreceptors in the mouse retina. To identify the rod connexin, we used nested reverse transcriptase polymerase chain reaction and tested retina and photoreceptor samples for messenger RNA (mRNA) expression of all known connexin genes. In addition to connexin36, we detected transcripts for connexin32, connexin43, connexin45, connexin50, and connexin57 in photoreceptor samples. Immunohistochemistry showed that connexin43, connexin45, connexin50, and connexin57 proteins are expressed in the outer plexiform layer. However, none of these connexins was detected at gap junctions between rods and cones as a counterpart of connexin36. Therefore, the sought-after rod protein must be either an unknown connexin sequence, a connexin36 splice product not detected by our antibodies, or a protein from a further gap junction protein family.

Birds seem to use a light-dependent, radical-pair-based magnetic compass. In vertebrates, cryptochromes are the only class of proteins that form radical pairs upon photo-excitation. Therefore, they are currently the only candidate proteins for light-dependent magnetoreception. Cryptochrome 4 (Cry4) is particularly interesting because it has only been found in vertebrates that use a magnetic compass. However, its structure and localization within the retina has remained unknown. Here, we sequenced night-migratory European robin (Erithacus rubecula) Cry4 from the retina and predicted the currently unresolved structure of the erCry4 protein, which suggests that erCry4 should bind Flavin. We also found that Cry1a, Cry1b, and Cry2 mRNA display robust circadian oscillation patterns, whereas Cry4 shows only a weak circadian oscillation. When we compared the relative mRNA expression levels of the cryptochromes during the spring and autumn migratory seasons relative to the non-migratory seasons in European robins and domestic chickens (Gallus gallus), the Cry4 mRNA expression level in European robin retinae, but not in chicken retinae, is significantly higher during the migratory season compared to the non-migratory seasons. Cry4 protein is specifically expressed in the outer segments of the double cones and long-wavelength single cones in European robins and chickens. A localization of Cry4 in double cones seems to be ideal for light-dependent magnetoreception. Considering all of the data presented here, especially including its localization within the European robin retina, its likely binding of Flavin, and its increased expression during the migratory season in the migratory bird but not in chicken, Cry4 could be the magnetoreceptive protein. Günther et al. suggest that Cry4 could be the magnetoreceptive protein in night-migratory songbirds. Cry4 seems to bind Flavin, shows only weak circadian oscillations, is specifically located in double cones and long-wavelength single cones, and is upregulated during the migratory season in night-migratory European robins but not in chicken.

In the vertebrate retina, horizontal cells generate the inhibitory surround of bipolar cells, an essential step in contrast enhancement. For the last decades, the mechanism involved in this inhibitory synaptic pathway has been a major controversy in retinal research. One hypothesis suggests that connexin hemichannels mediate this negative feedback signal; another suggests that feedback is mediated by protons. Mutant zebrafish were generated that lack connexin 55.5 hemichannels in horizontal cells. Whole cell voltage clamp recordings were made from isolated horizontal cells and cones in flat mount retinas. Light-induced feedback from horizontal cells to cones was reduced in mutants. A reduction of feedback was also found when horizontal cells were pharmacologically hyperpolarized but was absent when they were pharmacologically depolarized. Hemichannel currents in isolated horizontal cells showed a similar behavior. The hyperpolarization-induced hemichannel current was strongly reduced in the mutants while the depolarization-induced hemichannel current was not. Intracellular recordings were made from horizontal cells. Consistent with impaired feedback in the mutant, spectral opponent responses in horizontal cells were diminished in these animals. A behavioral assay revealed a lower contrast-sensitivity, illustrating the role of the horizontal cell to cone feedback pathway in contrast enhancement. Model simulations showed that the observed modifications of feedback can be accounted for by an ephaptic mechanism. A model for feedback, in which the number of connexin hemichannels is reduced to about 40%, fully predicts the specific asymmetric modification of feedback. To our knowledge, this is the first successful genetic interference in the feedback pathway from horizontal cells to cones. It provides direct evidence for an unconventional role of connexin hemichannels in the inhibitory synapse between horizontal cells and cones. This is an important step in resolving a long-standing debate about the unusual form of (ephaptic) synaptic transmission between horizontal cells and cones in the vertebrate retina., Author Summary Contrast enhancement is a fundamental feature of our visual system, initiated at the first synaptic connections in the retina. These are the synapses between photoreceptors (rods and cones) and their targets, horizontal cells and bipolar cells. Horizontal cells receive input from many cones and subsequently send a feedback signal to photoreceptors. Bipolar cells, however, receive direct input from only a few photoreceptors, but also receive indirect inhibitory input from surrounding cones via the horizontal cell feedback pathway. This organization induces the classic center/surround organization of bipolar cells and is considered the first step in contrast enhancement. Exactly how horizontal cells send feedback signals to photoreceptors has remained a mystery, however. One hypothesis posits that connexin hemichannels are involved. In this study, we tested this hypothesis using mutant zebrafish that lack connexin hemichannels specifically in horizontal cells. Our electrophysiology experiments showed that feedback is indeed reduced in these mutants, confirming that connexin hemichannels play an important role in feedback from horizontal cells to cones. In addition, we find that these mutant fish have decreased contrast sensitivity at a behavioral level, illustrating that functionally relevant contrast enhancement begins at the first synapse of the visual system.