Prussian blue technique is prone to yield false negative results in magnetoreception research.

Perls's Prussian blue staining technique has been used in magnetoreception research to screen tissues for iron-rich structures as proxies for putative magnetoreceptor structures based on magnetic particles. However, seemingly promising structural candidates in the upper beak of birds detected with Prussian blue turned out to be either irreproducible or located in non-neuronal cells, which has spurred a controversy that has not been settled yet. Here we identify possible pitfalls in the previous works and apply the Prussian blue technique to tissues implicated in magnetic-particle-based magnetoreception, in an effort to reassess its suitability for staining single-domain magnetite, i.e., the proposed magnetic substrate for the interaction with the external magnetic field. In the upper beak of night-migratory songbirds, we found staining products in great numbers, but not remotely associated with fiber terminals of the traced ophthalmic branch of the trigeminal nerve. Surprisingly, staining products were absent from the lamina propria in the olfactory rosette of rainbow trout where candidate magnetoreceptor structures were identified with different techniques earlier. Critically, magnetosome chains in whole cells of magnetotactic bacteria remained unstained. The failure to label single-domain magnetite in positive control samples is a serious limitation of the technique and suggests that two most influential but antipodal studies conducted previously stood little chances of obtaining correct positive results under the assumption that magnetosome-like particles were present in the tissues. Nonetheless, the staining technique appears suitable to identify tissue contamination with iron-rich fine dust trapped in epithelia already in vivo.

Interaction of G protein-coupled receptor kinases and recoverin isoforms is determined by localization in zebrafish photoreceptors.

The zebrafish retina expresses four recoverin genes (rcv1a, rcv1b, rcv2a and rcv2b) and four opsin kinase genes (grk1a, grk1b, grk7a and grk7b) coding for recoverin and G protein-coupled receptor kinase (opsin kinase) paralogs, respectively. Both protein groups are suggested to form regulatory complexes in rod and cone outer segments, but at present, we lack information about co-localization of recoverin and opsin kinases in zebrafish retinae and which protein-protein interacting pairs form. We analyzed the distribution and co-localization of recoverin and opsin kinase expression in the zebrafish retina. For this purpose, we used custom-tailored monospecific antibodies revealing that the amount of recoverin paralogs in a zebrafish retina can differ by more than one order of magnitude with the highest amount for recoverin 1a and 2b. Further, immunohistochemical labelling showed presence of recoverin 1a in all rod cell compartments, but it only co-localized with opsin kinase 1a in rod outer segments. In contrast, recoverin 2b was only detected in double cones and co-localized with opsin kinases 1b, 7a and 7b. Further, we investigated the interaction between recoverin and opsin kinase variants by surface plasmon resonance spectroscopy indicating interaction of recoverin 1a and recoverin 2b with all opsin kinases. However, binding kinetics for recoverin 1a differed from those observed with recoverin 2b that showed slower association and dissociation processes. Our results indicate diverse recoverin and opsin kinase properties due to differential expression and interaction profiles.

Connexin30.2: In Vitro Interaction with Connexin36 in HeLa Cells and Expression in AII Amacrine Cells and Intrinsically Photosensitive Ganglion Cells in the Mouse Retina.

Electrical coupling via gap junctions is an abundant phenomenon in the mammalian retina and occurs in all major cell types. Gap junction channels are assembled from different connexin subunits, and the connexin composition of the channel confers specific properties to the electrical synapse. In the mouse retina, gap junctions were demonstrated between intrinsically photosensitive ganglion cells and displaced amacrine cells but the underlying connexin remained undetermined. In the primary rod pathway, gap junctions play a crucial role, coupling AII amacrine cells among each other and to ON cone bipolar cells. Although it has long been known that connexin36 and connexin45 are necessary for the proper functioning of this most sensitive rod pathway, differences between homocellular AII/AII gap junctions and AII/ON bipolar cell gap junctions suggested the presence of an additional connexin in AII amacrine cells. Here, we used a connexin30.2-lacZ mouse line to study the expression of connexin30.2 in the retina. We show that connexin30.2 is expressed in intrinsically photosensitive ganglion cells and AII amacrine cells. Moreover, we tested whether connexin30.2 and connexin36-both expressed in AII amacrine cells-are able to interact with each other and are deposited in the same gap junctional plaques. Using newly generated anti-connexin30.2 antibodies, we show in HeLa cells that both connexins are indeed able to interact and may form heteromeric channels: both connexins were co-immunoprecipitated from transiently transfected HeLa cells and connexin30.2 gap junction plaques became significantly larger when co-expressed with connexin36. These data suggest that connexin36 is able to form heteromeric gap junctions with another connexin. We hypothesize that co-expression of connexin30.2 and connexin36 may endow AII amacrine cells with the means to differentially regulate its electrical coupling to different synaptic partners.

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.

2-deoxyribose deprives cultured astrocytes of their glutathione.

High concentrations of 2-deoxy-D-ribose (2dRib) have been reported to cause oxidative stress and to disturb the glutathione (GSH) metabolism of various cell types. Exposure of astrocyte-rich primary cultures to millimolar concentrations of 2dRib or its stereoisomer 2-deoxy-L-ribose, but not the incubation with ribose, 2-deoxyglucose, glucose, fructose or saccharose, lowered the cellular GSH content in a time and concentration dependent manner. After exposure for 4 h to 30 mM 2dRib the cells contained 2dRib in a concentration of about 24 mM. Under these conditions 2dRib did not compromise cell viability and the ability of the cells to synthesise GSH, nor were the cellular ratio of glutathione disulfide (GSSG) to GSH and the extracellular concentrations of GSH or GSSG increased. These data demonstrate that 2dRib deprives viable cultured astrocytes of GSH and suggest that a cellular reaction of GSH with 2dRib or its metabolites is involved in the deprivation of astrocytic GSH.