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.

Zebrafish connexin 79.8 (Gja8a): A lens connexin used as an electrical synapse in some neurons.

In the mammalian central nervous system, a remarkably small number of connexins is used in electrical synapses, with the majority formed from Cx36. A larger number has been detected in teleosts, with some seeming to serve restricted roles. Here, we report the discovery of a new connexin expressed in the zebrafish lens and a limited set of neurons. Zebrafish cx79.8 (gja8a), previously annotated incorrectly as cx50.5 based on a partial cDNA sequence, is a homologue of mammalian Cx50 (Gja8). We examined its expression through transgenic promoter-reporter constructs, in situ hybridization, and immunolabeling, and examined regulation of coupling in transfected HeLa cells. cx79.8 was expressed most strongly in the lens, but expression was also found in several groups of neurons in the cerebellum and related areas at the midbrain-hindbrain boundary, in cone photoreceptors, and in neurons in the retinal inner nuclear and ganglion cell layers. Labeling in the retina with antibodies against two C-terminal epitopes revealed numerous small punctate spots in the inner plexiform layer and along the somata of cones. Abundant gap junctions were labeled in the outer 1/3 of the lens, but were absent from the center, suggesting that the epitopes or the entire protein was absent from the center. Cx79.8 tracer coupling was strongly regulated by phosphorylation, and was extremely low in control conditions in HeLa cells due to protein phosphatase 2A activity. These properties allow coupling to be strongly restricted in situ, a frequently observed property for electrical synapses. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 548-561, 2017.

A Novel Method Combining Vitreous Aspiration and Intravitreal AAV2/8 Injection Results in Retina-Wide Transduction in Adult Mice.

PURPOSE: Gene therapies to treat eye disorders have been extensively studied in the past 20 years. Frequently, adeno-associated viruses were applied to the subretinal or intravitreal space of the eye to transduce retinal cells with nucleotide sequences of therapeutic potential. In this study we describe a novel intravitreal injection procedure that leads to a reproducible adeno-associated virus (AAV)2/8-mediated transduction of more than 70% of the retina. METHODS: Prior to a single intravitreal injection of a enhanced green fluorescent protien (GFP)-expressing viral suspension, we performed an aspiration of vitreous tissue from wild-type C57Bl/6J mice. One and one-half microliters of AAV2/8 suspension was injected. Funduscopy, optical coherence tomography (OCT), laser scanning microscopy of retinal flat mounts, cryosections of eye cups, and ERG recordings verified the efficacy and safety of the method. RESULTS: The combination of vitreous aspiration and intravitreal injection resulted in an almost complete transduction of the retina in approximately 60% of the eyes and showed transduced cells in all retinal layers. Photoreceptors and RPE cells were predominantly transduced. Eyes presented with well-preserved retinal morphology. Electroretinographic recordings suggested that the new combination of techniques did not cause significant alterations of the retinal physiology. CONCLUSIONS: We show a novel application technique of AAV2/8 to the vitreous of mice that leads to widespread transduction of the retina. The results of this study have implications for virus-based gene therapies and basic science; for example, they might provide an approach to apply gene replacement strategies or clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in vivo. It may further help to develop similar techniques for larger animal models or humans.

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.