Quantitative assessment of intraneural vascular alterations in peripheral nerve trauma using high-resolution neurosonography: technical note.

High-resolution neurosonography (HRNS) has become a major imaging modality in assessment of peripheral nerve trauma in the recent years. However, the vascular changes of traumatic lesions have not been quantitatively assessed in HRNS. Here, we describe the vascular-ratio, a novel HRNS-based quantitative parameter for the assessment of intraneural vascular alterations in patients with nerve lesions. N = 9 patients suffering from peripheral nerve trauma were examined clinically, electrophysiologically and with HRNS (SonoSite Exporte, Fuji). Image analyses using Fiji included determination of the established fascicular ratio (FR), the cross-section ratio (CSR), and as an extension, the calculation of a vascular ratio (VR) of the healthy versus damaged nerve and a muscle perfusion ratio (MPR) in comparison to a healthy control group. The mean VR in the healthy part of the affected nerve (14.14%) differed significantly (p < 0.0001) from the damaged part (VR of 43.26%). This coincides with significant differences in the FR and CSR calculated for the damaged part versus the healthy part and the controls. In comparison, there was no difference between VRs determined for the healthy part of the affected nerve and the healthy controls (14.14% / 17.72%). However, the MPR of denervated muscles was significantly decreased compared to the non-affected contralateral controls. VR and MPR serve as additional tools in assessing peripheral nerve trauma. Image analysis and calculation are feasible. Combined with the more morphologic FR and CSR, the VR and MPR provide a more detailed insight into alterations accompanying nerve trauma.

Synaptic Remodeling in the Cone Pathway After Early Postnatal Horizontal Cell Ablation.

The first synapse of the visual pathway is formed by photoreceptors, horizontal cells and bipolar cells. While ON bipolar cells invaginate into the photoreceptor terminal and form synaptic triads together with invaginating horizontal cell processes, OFF bipolar cells make flat contacts at the base of the terminal. When horizontal cells are ablated during retina development, no invaginating synapses are formed in rod photoreceptors. However, how cone photoreceptors and their synaptic connections with bipolar cells react to this insult, is unclear so far. To answer this question, we specifically ablated horizontal cells from the developing mouse retina. Following ablation around postnatal day 4 (P4)/P5, cones initially exhibited a normal morphology and formed flat contacts with OFF bipolar cells, but only few invaginating contacts with ON bipolar cells. From P15 on, synaptic remodeling became obvious with clustering of cone terminals and mislocalized cone somata in the OPL. Adult cones (P56) finally displayed highly branched axons with numerous terminals which contained ribbons and vesicular glutamate transporters. Furthermore, type 3a, 3b, and 4 OFF bipolar cell dendrites sprouted into the outer nuclear layer and even expressed glutamate receptors at the base of newly formed cone terminals. These results indicate that cones may be able to form new synapses with OFF bipolar cells in adult mice. In contrast, cone terminals lost their invaginating contacts with ON bipolar cells, highlighting the importance of horizontal cells for synapse maintenance. Taken together, our data demonstrate that early postnatal horizontal cell ablation leads to differential remodeling in the cone pathway: whereas synapses between cones and ON bipolar cells were lost, new putative synapses were established between cones and OFF bipolar cells. These results suggest that synapse formation and maintenance are regulated very differently between flat and invaginating contacts at cone terminals.

Farnesylation of Zebrafish G-Protein-Coupled Receptor Kinase Using Bio-orthogonal Labeling.

G-protein-coupled receptors are deactivated or desensitized by phosphorylation by respective G-protein-coupled receptor kinases (GRKs). In zebrafish rod and cone photoreceptor cells, four orthologous GRKs are expressed participating in the deactivation of rod and cone opsins. An important feature of GRKs in general is the consensus sites for lipid modification, which would allow the posttranslational attachment of isoprenoids facilitating membrane association and enzymatic performance. Because direct proof is missing for isoprenoid modification of zebrafish GRKs, we used a semichemical approach to study the incorporation of a farnesyl moiety into a GRK and its cellular consequences. The approach involves organic synthesis of a functionalized farnesyl derivative that is suitable for a subsequent alkyne-azide cycloaddition (click reaction). For this purpose, zebrafish GRK was expressed in HEK293 cells and modified in situ with the synthetic farnesyl moiety. Successful farnesylation by an endogenous farnesyltransferase was detected by immunoblotting and immunocytochemistry using a biotin-streptavidin-coupled assay and ligation with a fluorescence dye, respectively. Immunocytochemical detection of farnesylated GRK in different cell compartments indicates the applicability of the approach for studying the transport of cellular components.

Rapid and efficient immunomagnetic isolation of endothelial cells from human peripheral nerves.

Endothelial cells (ECs) have gained an increased scientific focus since they were reported to provide guidance for Schwann cells and subsequently following axons after nerve injuries. However, previous protocols for the isolation of nerve-derived ECs from human nerves are ineffective regarding time and yield. Therefore, we established a novel and efficient protocol for the isolation of ECs from human peripheral nerves by means of immunomagnetic CD31-antibody conjugated Dynabeads and assessed the purity of the isolated cells. The easy-to-follow and time-effective isolation method allows the isolation of > 95% pure ECs. The isolated ECs were shown to express highly specific EC marker proteins and revealed functional properties by formation of CD31 and VE-cadherin positive adherens junctions, as well as ZO-1 positive tight-junctions. Moreover, the formation of capillary EC-tubes was observed in-vitro. The novel protocol for the isolation of human nerve-derived ECs allows and simplifies the usage of ECs in research of the human blood-nerve-barrier and peripheral nerve regeneration. Additionally, a potential experimental application of patient-derived nerve ECs in the in-vitro vascularization of artificial nerve grafts is feasible.

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.

Phosphorylation of Connexin36 near the C-terminus switches binding affinities for PDZ-domain and 14-3-3 proteins in vitro.

Connexin36 (Cx36) is the most abundant connexin in central nervous system neurons. It forms gap junction channels that act as electrical synapses. Similar to chemical synapses, Cx36-containing gap junctions undergo activity-dependent plasticity and complex regulation. Cx36 gap junctions represent multimolecular complexes and contain cytoskeletal, regulatory and scaffolding proteins, which regulate channel conductance, assembly and turnover. The amino acid sequence of mammalian Cx36 harbors a phosphorylation site for the Ca2+/calmodulin-dependent kinase II at serine 315. This regulatory site is homologous to the serine 298 in perch Cx35 and in close vicinity to a PDZ binding domain at the very C-terminal end of the protein. We hypothesized that this phosphorylation site may serve as a molecular switch, influencing the affinity of the PDZ binding domain for its binding partners. Protein microarray and pulldown experiments revealed that this is indeed the case: phosphorylation of serine 298 decreased the binding affinity for MUPP1, a known scaffolding partner of connexin36, and increased the binding affinity for two different 14-3-3 proteins. Although we did not find the same effect in cell culture experiments, our data suggest that phosphorylation of serine 315/298 may serve to recruit different proteins to connexin36/35-containing gap junctions in an activity-dependent manner.

Rod Bipolar Cells Require Horizontal Cells for Invagination Into the Terminals of Rod Photoreceptors.

In the central nervous system, neuronal processing relies on the precisely orchestrated formation of synapses during development. The first synapse of the visual system is a triad synapse, comprising photoreceptors, horizontal cells and bipolar cells. During the second postnatal week, the axon terminal processes of horizontal cells invaginate rod spherules, followed by rod bipolar cell dendrites. Both elements finally oppose the synaptic ribbon (the release site of glutamate). However, it has not been fully elucidated whether horizontal cells are essential for rod bipolar cell dendrites to find their way into the rod terminal. In the present study, we investigated this question by specifically ablating horizontal cells from the early postnatal mouse retina. We monitored the formation of the rod-to-rod bipolar cell synapse during retinal maturation until postnatal day 21. Based on quantitative electron microscopy, we found that without horizontal cells, the dendrites of rod bipolar cells never entered rod terminals. Furthermore, rods displayed significantly fewer and shorter presynaptic ribbons, suggesting that glutamate release is decreased, which coincided with significantly reduced expression of postsynaptic proteins (mGluR6, GPR179) in rod bipolar cells. Collectively, our findings uncover that horizontal cells are indeed necessary guideposts for rod bipolar cells. Whether horizontal cells release diffusible guidance cues or provide structural guidance by expressing specific cell adhesion molecules remains to be seen.

Localization of Retinal Ca2+/Calmodulin-Dependent Kinase II-ß (CaMKII-ß) at Bipolar Cell Gap Junctions and Cross-Reactivity of a Monoclonal Anti-CaMKII-ß Antibody With Connexin36.

Neuronal gap junctions formed by connexin36 (Cx36) and chemical synapses share striking similarities in terms of plasticity. Ca2+/calmodulin-dependent protein kinase II (CaMKII), an enzyme known to induce memory formation at chemical synapses, has recently been described to potentiate electrical coupling in the retina and several other brain areas via phosphorylation of Cx36. The contribution of individual CaMKII isoforms to this process, however, remains unknown. We recently identified CaMKII-ß at electrical synapses in the mouse retina. Now, we set out to identify cell types containing Cx36 gap junctions that also express CaMKII-ß. To ensure precise description, we first tested the specificity of two commercially available antibodies on CaMKII-ß-deficient retinas. We found that a polyclonal antibody was highly specific for CaMKII-ß. However, a monoclonal antibody (CB-ß-1) recognized CaMKII-ß but also cross-reacted with the C-terminal tail of Cx36, making localization analyses with this antibody inaccurate. Using the polyclonal antibody, we identified strong CaMKII-ß expression in bipolar cell terminals that were secretagogin- and HCN1-positive and thus represent terminals of type 5 bipolar cells. In these terminals, a small fraction of CaMKII-ß also colocalized with Cx36. A similar pattern was observed in putative type 6 bipolar cells although there, CaMKII expression seemed less pronounced. Next, we tested whether CaMKII-ß influenced the Cx36 expression in bipolar cell terminals by quantifying the number and size of Cx36-immunoreactive puncta in CaMKII-ß-deficient retinas. However, we found no significant differences between the genotypes, indicating that CaMKII-ß is not necessary for the formation and maintenance of Cx36-containing gap junctions in the retina. In addition, in wild-type retinas, we observed frequent association of Cx36 and CaMKII-ß with synaptic ribbons, i.e., chemical synapses, in bipolar cell terminals. This arrangement resembled the composition of mixed synapses found for example in Mauthner cells, in which electrical coupling is regulated by glutamatergic activity. Taken together, our data imply that CaMKII-ß may fulfill several functions in bipolar cell terminals, regulating both Cx36-containing gap junctions and ribbon synapses and potentially also mediating cross-talk between these two types of bipolar cell outputs.

Fascicular Ratio Pilot Study: High-Resolution Neurosonography-A Possible Tool for Quantitative Assessment of Traumatic Peripheral Nerve Lesions Before and After Nerve Surgery.

BACKGROUND: Clinical and electrophysiological assessments prevail in evaluation of traumatic nerve lesions and their regeneration following nerve surgery in humans. Recently, high-resolution neurosonography (HRNS) and magnetic resonance neurography have gained significant importance in peripheral nerve imaging. The use of the grey-scale-based "fascicular ratio" (FR) was established using both modalities allowing for quantitative assessment. OBJECTIVE: To find out whether FR using HRNS can assess nerve trauma and structural reorganization in correlation to postoperative clinical development. METHODS: Retrospectively, 16 patients with operated traumatic peripheral nerve lesions were included. The control group consisted of 6 healthy volunteers. All imaging was performed with a 15 to 6 MHz ultrasound probe (SonoSite X-Porte; Fujifilm, Tokyo, Japan). FR was calculated using Fiji () on 8-bit-images ("MaxEntropy" using "Auto-Threshold" plug-in). RESULTS: Thirteen of 16 patients required autologous nerve grafting and 3 of 16 extra-intraneural neurolysis. There was no statistical difference between the FR of nonaffected patients' nerve portion with 43.48% and controls with FR 48.12%. The neuromatous nerve portion in grafted patients differed significantly with 85.05%. Postoperatively, FR values returned to normal with a mean of 39.33%. In the neurolyzed patients, FR in the affected portion was 78.54%. After neurolysis, FR returned to healthy values (50.79%). Ten of 16 patients showed clinical reinnervation. CONCLUSION: To our best knowledge, this is the first description of FR using HRNS for quantitative assessment of nerve damage and postoperative structural reorganization. Our results show a significant difference in healthy vs lesioned nerves and a change in recovering nerve portions towards a more "physiological" ratio. Further evaluation in larger patient groups is required.

Analysis of regeneration- and myelination-associated proteins in human neuroma in continuity and discontinuity.

BACKGROUND: Neuromas are pathologic nerve distensions caused by a nerve's response to trauma, resulting in a dysfunctional to non-functional nerve. Depending on the severance of the affected nerve, the resulting neuroma can be differentiated into continuous and stump neuroma. While neuroma formation has been investigated in animal models with enormous regenerative capacity, the search for differences in human response to nerve trauma on a molecular level ultimately seeks to identify reasons for functionally successful versus unsuccessful regeneration after peripheral nerve trauma in man. METHODS: In the present study, the regenerative potential of axons and the capability of Schwann cells (SC) to remyelinate regenerating axons was quantitatively and segmentally analyzed and compared within human neuroma in-continuity and discontinuity. RESULTS: For the stump neuroma and the neuroma in-continuity, there was a significant reduction of the total number of axons (86% stump neuroma and 91% neuroma in-continuity) from the proximal to the distal part of the neuroma, while the amount of fibrotic tissue increased, respectively. Labeling the myelin sheath of regenerating axons revealed a remyelination of regenerating axons by SCs in both neuroma types. The segmented analysis showed no distinct alterations in the number and spatial distribution of regenerating, mature, and myelinated axons between continuous and discontinuous neuroma. CONCLUSIONS: The quantitative and segmented analysis showed no distinct alterations in the number and spatial distribution of regenerating, mature, and myelinated axons between continuous and discontinuous neuroma, while the extensive expression of Gap43 in up to 55% of the human neuroma axons underlines their regenerative capacity independent of whether the neuroma is in continuity or discontinuity. Remyelination of Gap43-positive axons suggests that the capability of SCs to remyelinate regenerating axons is preserved in neuroma tissue.

Control of the Nucleotide Cycle in Photoreceptor Cell Extracts by Retinal Degeneration Protein 3.

Retinal degeneration protein 3 (RD3) is crucial for photoreceptor cell survival and linked to Leber Congenital Amaurosis type 12 (LCA12), a hereditary retinal disease in humans. RD3 inhibits photoreceptor guanylate cyclases GC-E and GC-F and is involved in transport of GCs from the inner to the outer segments. Otherwise, its role in photoreceptor physiology is poorly understood. Here, we describe a new function of RD3. Purified RD3 evoked an increase in guanylate kinase activity, an enzyme that is involved in the nucleotide cycle in photoreceptors. We demonstrate a direct interaction between guanylate kinase and RD3 using back-scattering interferometry and show by immunohistochemistry of mouse retina sections that RD3 and guanylate kinase co-localize in photoreceptor inner segments and to a lesser extent in the outer plexiform layer. Our findings point toward a more complex function of RD3 in photoreceptors. The RD3 - guanylate kinase interaction may also play a role in other cellular systems, while the GC - RD3 interaction is exclusive to photoreceptors.

Synthesis and Evaluation of Two Novel All -trans-Retinoic Acid Conjugates: Biocompatible and Functional Tools for Retina Research.

The vitamin A derivative all- trans-retinoic acid (ATRA) is an important biologically active metabolite that regulates a variety of essential biological processes in particular via gene-regulatory mechanisms. In the retina, ATRA is a light-dependent byproduct of the phototransduction cascade. Here, ATRA is not only needed for proper retinal development, but it also acts as a neuromodulator on horizontal cells, second-order inhibitory neurons in the outer retina, which reveal morphological and physiological changes when the retina is treated with ATRA. There is evidence that gene-regulatory mechanisms may only be partially involved in these neuromodulatory processes and the underlying nontranscriptional mechanisms are still elusive. This is, among other things, due to the lack of appropriately labeled ATRA, which would allow the tracking of ATRA in cells or a given tissue. To overcome this obstacle, we designed, synthesized, and evaluated two conjugates of ATRA, one conjugated with biotin (biotin-ATRA) and one conjugated with diaminoterephthalate fluorophore (DAT-ATRA), as molecular tools for different fields of application. The biocompatibility of both compounds was demonstrated via cell viability assays in cultured N2a-cells. N2a-cells exposed to the compounds showed no significant changes in the viability rate. The functionality of synthesized ATRA-conjugates was verified using retinal tissue derived from adult carp. The binding of ATRA-conjugates to distinct retinal cells was assessed in primary cultures of carp retina. Hereby, horizontal and Müller cells have been identified as specific target cells of the new ATRA compounds. Electron microscopy further confirmed that the new substances are still able to induce synaptic plasticity at horizontal cell dendrites resulting in formation of spine synapses, as it is shown for native ATRA. Taken together, the novel ATRA-conjugates represent biocompatible and functional molecular tools, which may further provide the possibility to track ATRA in neuronal cells and study its modulatory effects in different cell systems.

Eliminating Glutamatergic Input onto Horizontal Cells Changes the Dynamic Range and Receptive Field Organization of Mouse Retinal Ganglion Cells.

In the mammalian retina, horizontal cells receive glutamatergic inputs from many rod and cone photoreceptors and return feedback signals to them, thereby changing photoreceptor glutamate release in a light-dependent manner. Horizontal cells also provide feedforward signals to bipolar cells. It is unclear, however, how horizontal cell signals also affect the temporal, spatial, and contrast tuning in retinal output neurons, the ganglion cells. To study this, we generated a genetically modified mouse line in which we eliminated the light dependency of feedback by deleting glutamate receptors from mouse horizontal cells. This genetic modification allowed us to investigate the impact of horizontal cells on ganglion cell signaling independent of the actual mode of feedback in the outer retina and without pharmacological manipulation of signal transmission. In control and genetically modified mice (both sexes), we recorded the light responses of transient OFF-α retinal ganglion cells in the intact retina. Excitatory postsynaptic currents (EPSCs) were reduced and the cells were tuned to lower temporal frequencies and higher contrasts, presumably because photoreceptor output was attenuated. Moreover, receptive fields of recorded cells showed a significantly altered surround structure. Our data thus suggest that horizontal cells are responsible for adjusting the dynamic range of retinal ganglion cells and, together with amacrine cells, contribute to the center/surround organization of ganglion cell receptive fields in the mouse.SIGNIFICANCE STATEMENT Horizontal cells represent a major neuronal class in the mammalian retina and provide lateral feedback and feedforward signals to photoreceptors and bipolar cells, respectively. The mode of signal transmission remains controversial and, moreover, the contribution of horizontal cells to visual processing is still elusive. To address the question of how horizontal cells affect retinal output signals, we recorded the light responses of transient OFF-α retinal ganglion cells in a newly generated mouse line. In this mouse line, horizontal cell signals were no longer modulated by light. With light response recordings, we show that horizontal cells increase the dynamic range of retinal ganglion cells for contrast and temporal changes and contribute to the center/surround organization of their receptive fields.

HDAC6 inhibition by tubastatin A is protective against oxidative stress in a photoreceptor cell line and restores visual function in a zebrafish model of inherited blindness.

Retinal diseases, such as hereditary retinitis pigmentosa and age-related macular degeneration, are characterized by the progressive loss of photoreceptors. Histone deacetylase 6 (HDAC6) is considered as a stress surveillance factor and a potential target for neuroprotection and regeneration. Overexpression of HDAC6 has been connected to neurodegenerative disorders, and its suppression may provide protection. Here we show that HDAC6 is constitutively present in the mouse retina, and in the cone-like mouse cell line 661W. In 661W cells HDAC6 inhibition by the specific inhibitor tubastatin A (TST) led to the acetylation of α-tubulin, which is a major substrate for HDAC6. After oxidative stress, exerted by hydrogen peroxide, TST promoted cell survival and the upregulation of heat-shock proteins HSP70 and HSP25 by activation of heat-shock transcription factor 1. Furthermore, in response to oxidative stress the redox regulatory protein peroxiredoxin 1 (Prx1) was modulated in 661W cells by HDAC6 inhibition. The peroxide reducing activity of Prx1 is dependent on its acetylation, which is mediated by HDAC6. Pre-incubation with TST prevented the inactivation of Prx1 and its preserved activity may exert protective effects in photoreceptor cells. To determine whether TST treatment has a therapeutic effect on visual function, the dyeucd6 zebrafish model of inherited sight loss was utilized. Zebrafish have developed as a suitable model system for pharmacological testing. In vivo application of TST caused the hyperacetylation of α-tubulin, indicating that HDAC6 is active in this model. Furthermore, TST was sufficient to rescue visual function and retinal morphology. Hence, HDAC6 inhibition and the regulation of peroxiredoxin activity may play a significant role in protecting retinal cells and in particular photoreceptors, which are exposed to high levels of reactive oxygen species derived from oxidative stress-induced injuries.

Differential Distribution of Retinal Ca2+/Calmodulin-Dependent Kinase II (CaMKII) Isoforms Indicates CaMKII-ß and -δ as Specific Elements of Electrical Synapses Made of Connexin36 (Cx36).

AII amacrine cells are essential interneurons of the primary rod pathway and transmit rod-driven signals to ON cone bipolar cells to enable scotopic vision. Gap junctions made of connexin36 (Cx36) mediate electrical coupling among AII cells and between AII cells and ON cone bipolar cells. These gap junctions underlie a remarkable degree of plasticity and are modulated by different signaling cascades. In particular, Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been characterized as an important regulator of Cx36, capable of potentiating electrical coupling in AII cells. However, it is unclear which CaMKII isoform mediates this effect. To obtain a more detailed understanding of the isoform composition of CaMKII at retinal gap junctions, we analyzed the retinal distribution of all four CaMKII isoforms using confocal microscopy. These experiments revealed a differential distribution of CaMKII isoforms: CaMKII-α was strongly expressed in starburst amacrine cells, which are known to lack electrical coupling. CaMKII-ß was abundant in OFF bipolar cells, which form electrical synapses in the outer and the inner retina. CaMKII-γ was diffusely distributed across the entire retina and could not be assigned to a specific cell type. CaMKII-δ labeling was evident in bipolar and AII amacrine cells, which contain the majority of Cx36-immunoreactive puncta in the inner retina. We double-labeled retinas for Cx36 and the four CaMKII isoforms and revealed that the composition of the CaMKII enzyme differs between gap junctions in the outer and the inner retina: in the outer retina, only CaMKII-ß colocalized with Cx36-containing gap junctions, whereas in the inner retina, CaMKII-ß and -δ colocalized with Cx36. This finding suggests that gap junctions in the inner and the outer retina may be regulated differently although they both contain the same connexin. Taken together, our study identifies CaMKII-ß and -δ as Cx36-specific regulators in the mouse retina with CaMKII-δ regulating the primary rod pathway.

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.

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.

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.

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.

Identification of a common non-apoptotic cell death mechanism in hereditary retinal degeneration.

Cell death in neurodegenerative diseases is often thought to be governed by apoptosis; however, an increasing body of evidence suggests the involvement of alternative cell death mechanisms in neuronal degeneration. We studied retinal neurodegeneration using 10 different animal models, covering all major groups of hereditary human blindness (rd1, rd2, rd10, Cngb1 KO, Rho KO, S334ter, P23H, Cnga3 KO, cpfl1, Rpe65 KO), by investigating metabolic processes relevant for different forms of cell death. We show that apoptosis plays only a minor role in the inherited forms of retinal neurodegeneration studied, where instead, a non-apoptotic degenerative mechanism common to all mutants is of major importance. Hallmark features of this pathway are activation of histone deacetylase, poly-ADP-ribose-polymerase, and calpain, as well as accumulation of cyclic guanosine monophosphate and poly-ADP-ribose. Our work thus demonstrates the prevalence of alternative cell death mechanisms in inherited retinal degeneration and provides a rational basis for the design of mutation-independent treatments.