Localisation of cryptochrome 2 in the avian retina.

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.

Synaptic transmission from horizontal cells to cones is impaired by loss of connexin hemichannels.

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.

Morphology, biochemistry and connectivity of Cluster N and the hippocampal formation in a migratory bird.

The exceptional navigational capabilities of migrating birds are based on the perception and integration of a variety of natural orientation cues. The "Wulst" in the forebrain of night-migratory songbirds contains a brain area named "Cluster N", which is involved in processing directional navigational information derived from the Earth´s magnetic field. Cluster N is medially joined by the hippocampal formation, known to retrieve and utilise navigational information. To investigate the connectivity and neurochemical characteristics of Cluster N and the hippocampal formation of migratory birds, we performed morphological and histochemical analyses based on the expression of calbindin, calretinin, parvalbumin, glutamate receptor type 1 and early growth response protein-1 in the night-migratory Garden warbler (Sylvia borin) and mapped their mutual connections using neuronal tract tracing. The resulting expression patterns revealed regionally restricted neurochemical features, which mapped well onto the hippocampal and hyperpallial substructures known from other avian species. Magnetic field-induced neuronal activation covered caudal parts of the hyperpallium and the medially adjacent hippocampal dorsomedial/dorsolateral subdivisions. Neuronal tract tracings revealed connections between Cluster N and the hippocampal formation with the vast majority originating from the densocellular hyperpallium, either directly or indirectly via the area corticoidea dorsolateralis. Our data indicate that the densocellular hyperpallium could represent a central relay for the transmission of magnetic compass information to the hippocampal formation where it might be integrated with other navigational cues in night-migratory songbirds.

Double-Cone Localization and Seasonal Expression Pattern Suggest a Role in Magnetoreception for European Robin Cryptochrome 4.

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.

Expression and Localization of Connexins in the Outer Retina of the Mouse.

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.

Localisation of the Putative Magnetoreceptive Protein Cryptochrome 1b in the Retinae of Migratory Birds and Homing Pigeons.

Cryptochromes are ubiquitously expressed in various animal tissues including the retina. Some cryptochromes are involved in regulating circadian activity. Cryptochrome proteins have also been suggested to mediate the primary mechanism in light-dependent magnetic compass orientation in birds. Cryptochrome 1b (Cry1b) exhibits a unique carboxy terminus exclusively found in birds so far, which might be indicative for a specialised function. Cryptochrome 1a (Cry1a) is so far the only cryptochrome protein that has been localised to specific cell types within the retina of migratory birds. Here we show that Cry1b, an alternative splice variant of Cry1a, is also expressed in the retina of migratory birds, but it is primarily located in other cell types than Cry1a. This could suggest different functions for the two splice products. Using diagnostic bird-specific antibodies (that allow for a precise discrimination between both proteins), we show that Cry1b protein is found in the retinae of migratory European robins (Erithacus rubecula), migratory Northern Wheatears (Oenanthe oenanthe) and pigeons (Columba livia). In all three species, retinal Cry1b is localised in cell types which have been discussed as potentially well suited locations for magnetoreception: Cry1b is observed in the cytosol of ganglion cells, displaced ganglion cells, and in photoreceptor inner segments. The cytosolic rather than nucleic location of Cry1b in the retina reported here speaks against a circadian clock regulatory function of Cry1b and it allows for the possible involvement of Cry1b in a radical-pair-based magnetoreception mechanism.

Connexin50 couples axon terminals of mouse horizontal cells by homotypic gap junctions.

Horizontal cells in the mouse retina are of the axon-bearing B-type and contribute to the gain control of photoreceptors and to the center-surround organization of bipolar cells by providing feedback and feedforward signals to photoreceptors and bipolar cells, respectively. Horizontal cells form two independent networks, coupled by dendro-dendritic and axo-axonal gap junctions composed of connexin57 (Cx57). In Cx57-deficient mice, occasionally the residual tracer coupling of horizontal cell somata was observed. Also, negative feedback from horizontal cells to photoreceptors, potentially mediated by connexin hemichannels, appeared unaffected. These results point to the expression of a second connexin in mouse horizontal cells. We investigated the expression of Cx50, which was recently identified in axonless A-type horizontal cells of the rabbit retina. In the mouse retina, Cx50-immunoreactive puncta were predominantly localized on large axon terminals of horizontal cells. Electron microscopy did not reveal any Cx50-immunolabeling at the membrane of horizontal cell tips invaginating photoreceptor terminals, ruling out the involvement of Cx50 in negative feedback. Moreover, Cx50 colocalized only rarely with Cx57 on horizontal cell processes, indicating that both connexins form homotypic rather than heterotypic or heteromeric gap junctions. To check whether the expression of Cx50 is changed when Cx57 is lacking, we compared the Cx50 expression in wildtype and Cx57-deficient mice. However, Cx50 expression was unaffected in Cx57-deficient mice. In summary, our results indicate that horizontal cell axon terminals form two independent sets of homotypic gap junctions, a feature which might be important for light adaptation in the retina.

Expression of Pannexin1 in the outer plexiform layer of the mouse retina and physiological impact of its knockout.

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.