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Complex sphingolipids are strongly expressed in neuronal tissue and contain ceramides in their backbone. Ceramides are synthesized by six ceramide synthases (CerS1-6). Although it is known that each tissue has a unique profile of ceramide synthase expression and ceramide synthases are implicated in several neurodegenerative disorders, the expression of ceramide synthase isoforms has not been investigated in the retina. Here we demonstrate CerS1, CerS2 and CerS4 expression in mouse retina and cornea, with CerS4 ubiquitously expressed in all retinal neurons and Müller cells. To test whether ceramide synthase deficiency affects retinal function, we compared electroretinograms and retina morphology between wild-type and CerS1-, CerS2- and CerS4-deficient mice. Electroretinograms were strongly reduced in amplitude in ceramide synthase-deficient mice, suggesting that signalling in the outer retina is affected. However, the number of photoreceptors and cone outer segment length were unaltered and no changes in retinal layer thickness or synaptic structures were found. Mass spectrometric analyses of ceramides, hexosyl-ceramides and sphingomyelins showed that C20 to C24 acyl-containing species were decreased whereas C16-containing species were increased in the retina of ceramide synthase-deficient mice. Similar but smaller changes were also found in the cornea. Thus, we hypothesize that the replacement of very long-chain fatty acyl residues by shorter C16 residues may affect the electrical properties of retina and cornea, and alter receptor-mediated signal transduction, vesicle-mediated synaptic transmission or corneal light transmission. Future studies need to identify the molecular targets of ceramides or derived sphingolipids in light signal transduction and transmission in the eye.

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.

Amacrine cells comprise ∼ 30 morphological types in the mammalian retina. The synaptic connectivity and function of a few γ-aminobutyric acid (GABA)ergic wide-field amacrine cells have recently been studied; however, with the exception of the rod pathway-specific AII amacrine cell, the connectivity of glycinergic small-field amacrine cells has not been investigated in the mouse retina. Here, we studied the morphology and connectivity pattern of the small-field A8 amacrine cell. A8 cells in mouse retina are bistratified with lobular processes in the ON sublamina and arboreal dendrites in the OFF sublamina of the inner plexiform layer. The distinct bistratified morphology was first visible at postnatal day 8, reaching the adult shape at P13, around eye opening. The connectivity of A8 cells to bipolar cells and ganglion cells was studied by double and triple immunolabeling experiments by using various cell markers combined with synaptic markers. Our data suggest that A8 amacrine cells receive glutamatergic input from both OFF and ON cone bipolar cells. Furthermore, A8 cells are coupled to ON cone bipolar cells by gap junctions, and provide inhibitory input via glycine receptor (GlyR) subunit α1 to OFF cone bipolar cells and to ON A-type ganglion cells. Measurements of spontaneous glycinergic postsynaptic currents and GlyR immunolabeling revealed that A8 cells express GlyRs containing the α2 subunit. The results show that the bistratified A8 cell makes very similar synaptic contacts with cone bipolar cells as the rod pathway-specific AII amacrine cell. However, unlike AII cells, A8 amacrine cells provide glycinergic input to ON A-type ganglion cells.

Mammalian retinas comprise a variety of interneurons, among which amacrine cells represent the largest group, with more than 30 different cell types each exhibiting a rather distinctive morphology and carrying out a unique function in retinal processing. However, many amacrine types have not been studied systematically because, in particular, amacrine cells with large dendritic fields, i.e. wide-field amacrine cells, have a low abundance and are therefore difficult to target. Here, we used a transgenic mouse line expressing the coding sequence of enhanced green fluorescent protein under the promoter for choline acetyltransferase (ChAT-EGFP mouse) and characterized a single wide-field amacrine cell population monostratifying in layer 2/3 of the inner plexiform layer (WA-S2/3 cell). Somata of WA-S2/3 cells are located either in the inner nuclear layer or are displaced to the ganglion cell layer and exhibit a low cell density. Using immunohistochemistry, we show that WA-S2/3 cells are presumably GABAergic but may also release acetylcholine as their somata are weakly positive for ChAT. Two-photon-guided patch-clamp recordings from intact retinas revealed WA-S2/3 cells to be ON-OFF cells with a homogenous receptive field even larger than the dendritic field. The large spatial extent of the receptive field is most likely due to the extensive homologous and heterologous coupling among WA-S2/3 cells and to other amacrine cells, respectively, as indicated by tracer injections. In summary, we have characterized a novel type of GABAergic ON-OFF wide-field amacrine cell which is ideally suited to providing long-range inhibition to ganglion cells due to its strong coupling.

Complexins (Cplxs) regulate the speed and Ca(2+)-sensitivity of synaptic vesicle fusion. It has been shown that all four known Cplxs are present at mouse retinal synapses--at conventional amacrine cell synapses (Cplx 1 to Cplx 3) and at photoreceptor and bipolar cell ribbon synapses (Cplx 3 and Cplx 4) [K. Reim et al. (2005) J. Cell Biol., 169, 669-680]. Electroretinographic recordings in Cplx 3/Cplx 4 double-knockout (DKO) mice showed perturbed transmission in the outer plexiform layer, and possible changes in the inner plexiform layer [K. Reim et al. (2009) J. Cell Sci., 122, 1352-1361]. In the present study, we examined the effects of the absence of Cplx 3 and Cplx 4 on ganglion cell responses. We report that the lack of Cplx 3 and Cplx 4 differentially impacts the ON and OFF pathways. Under photopic conditions, the responses in the cone OFF pathway are largely unaffected, whereas the responses in the cone ON pathway are diminished in Cplx 3/Cplx 4 DKO mice. Under scotopic conditions, both ON and OFF response rates are reduced and high-sensitivity OFF responses are missing in Cplx 3/Cplx 4 DKO mice. The electrophysiological findings are corroborated by new immunocytochemical findings. We now show that rod spherules contain only Cplx 4. However, both Cplx 3 and Cplx 4 co-localize in cone pedicles. In the inner plexiform layer, Cplx 3 is present in rod bipolar cell terminals and in amacrine cell processes. Most importantly, Cplx 3 is localized in the lobular appendages of AII amacrine cells, the sites of signal transmission from the primary rod pathway into the OFF pathway in the inner plexiform layer.

In the mouse retina, connexin45 (Cx45) participates in the gap junction between ON cone bipolar cells and AII amacrine cells, which constitutes an essential element of the primary rod pathway. Although it has been shown that Cx45 is also expressed in OFF bipolar cells, its subcellular localization and functional role in these cells are unknown. Here, we analyzed the localization of Cx45 on OFF bipolar cells in the mouse retina. For this, we used wild-type mice and a transgenic mouse line that expressed, in addition to native Cx45, a fusion protein consisting of Cx45 and the enhanced green fluorescent protein (EGFP). Cx45-EGFP expression generates an EGFP signal at gap junctions containing Cx45. Combining immunohistochemistry with intracellular injections, we found that Cx45 was present on dendrites and axon terminals of all OFF bipolar cell types. Cx45 was not found at intersections of two terminal processes of the same type, suggesting that Cx45 might not form gap junctions between axon terminals of the same OFF bipolar cell type but rather might connect OFF bipolar cells to amacrine or ganglion cells. In OFF bipolar cell dendrites, Cx45 was found predominantly in the proximal outer plexiform layer (OPL), well below the cone pedicles. Cx45 did not colocalize with Cx36, which is found predominantly in the distal OPL. We conclude that Cx45 is expressed on OFF bipolar cell dendrites, presumably forming gap junctions with cells of the same type, and on OFF bipolar cell axon terminals, presumably forming heterologous gap junctions with other retinal neurons. J. Comp. Neurol. 519:433–450, 2011. © 2010 Wiley-Liss, Inc.

Organic semiconductors are emerging as promising candidates for novel electrically self‐sufficient photovoltaic prosthetics for neurostimulation, especially for restoration of light sensitivity in degenerate retina. Considering future applications, it is essential to gain fundamental insight into the signaling mechanisms at the organic photosensor–electrolyte–neuron interface. Particularly, targeting voltage‐gated ion channels by a pure photocapacitive stimulation is a preferred therapeutic approach as it avoids redox reactions involved in Faradaic charge injection. The present study investigates whether single neuroblastoma (N2A) cells, grown on a photosensor based on a small molecular squaraine:fullerene photoactive layer blend, optionally covered with silicon dioxide, can be activated by photocapacitive stimulation. Indeed, upon pulsed illumination, a rapid transient photocurrent strongly depolarizes the membrane potential and subsequently activates fast‐responding voltage‐gated sodium channels. The dielectric top coating on the organic layer ensures sufficient capacitive charge injection efficiency while maintaining the rapid response of the device. Due to the high irradiance level required for photocapacitive stimulation, another slower, independent, and unintended, nonelectrical signaling pathway is identified. This activates voltage‐gated potassium channels, presumably by photothermal effects. The present study provides the basis for further improvements on standalone photovoltaic neurostimulating platforms based on organic photoactive layers.

Mouse horizontal cells are coupled by gap junctions composed of connexin57. These gap junctions are regulated by ambient light via multiple neuromodulators including dopamine. In order to analyze the distribution and structure of horizontal cell gap junctions in the mouse retina, and examine the effects of light adaptation on gap junction density, we developed antibodies that detect mouse retinal connexin57. Using immunohistochemistry in retinal slices, flatmounted retinas, and dissociated retinal cells, we showed that connexin57 is expressed in the dendrites and axon terminal processes of mouse horizontal cells. No staining was found in retinas of connexin57-deficient mice. Significantly more connexin57-positive puncta were found in the distal than in the proximal outer plexiform layer, indicating a higher level of expression in axon terminal processes than in the dendrites. We also examined the gap junctions using immunoelectron microscopy and showed that connexin57 does not form hemichannels in the horizontal cell dendritic tips. Light adaptation resulted in a significant increase in the number of connexin57-immunoreactive plaques in the outer plexiform layer, consistent with previously reported effects of light adaptation on connexin57 expression in the mouse retina. This study shows for the first time the detailed location of connexin57 expression within mouse horizontal cells, and provides the first ultrastructural data on mouse horizontal cell gap junctions. J. Comp. Neurol. 513:363–374

The primary rod pathway in mammals contains gap junctions between AII amacrine cells and ON cone bipolar cells which relay the rod signal into the cone pathway under scotopic conditions. Two gap junctional proteins, connexin36 (Cx36) and connexin45 (Cx45), appear to play a pivotal role in this pathway because lack of either protein leads to an impairment of visual transmission under scotopic conditions. To investigate whether these connexins form heterotypic gap junctions between ON cone bipolar and AII amacrine cells, we used newly developed Cx45 antibodies and studied the cellular and subcellular distribution of this protein in the mouse retina. Specificity of the Cx45 antibodies was determined, among others, by Western blot and immunostaining of mouse heart, where Cx45 is abundantly expressed. In mouse retina, Cx45 immunosignals were detected in both plexiform layers and the ganglion cell layer. Double staining for Cx45 and Cx36 revealed a partial overlap in the punctate patterns in the ON sublamina of the inner plexiform layer of the retina. We quantified the distributions of these two connexins in the ON sublamina, and detected 30% of the Cx45 signals to be co-localized with or in close apposition to Cx36 signals. Combining immunostaining and intracellular dye injection revealed an overlap or tight association of Cx36 and Cx45 signals on the terminals of injected AII amacrine and two types of ON cone bipolar cells. Our results provide direct evidence for heterotypic gap junctions composed of Cx36 and Cx45 between AII amacrine and certain types of ON cone bipolar cells.