Intralumenal docking of connexin 36 channels in the ER isolates mistrafficked protein.

The intracellular domains of connexins are essential for the assembly of gap junctions. For connexin 36 (Cx36), the major neuronal connexin, it has been shown that a dysfunctional PDZ-binding motif interferes with electrical synapse formation. However, it is still unknown how this motif coordinates the transport of Cx36. In the present study, we characterize a phenotype of Cx36 mutants that lack a functional PDZ-binding motif using HEK293T cells as an expression system. We provide evidence that an intact PDZ-binding motif is critical for proper endoplasmic reticulum (ER) export of Cx36. Removing the PDZ-binding motif of Cx36 results in ER retention and the formation of multimembrane vesicles containing gap junction-like connexin aggregates. Using a combination of site-directed mutagenesis and electron micrographs, we reveal that these vesicles consist of Cx36 channels that docked prematurely in the ER. Our data suggest a model in which ER-retained Cx36 channels reshape the ER membrane into concentric whorls that are released into the cytoplasm.

Immunohistochemical characterization of bipolar cells in four distantly related avian species.

Visual (and probably also magnetic) signal processing starts at the first synapse, at which photoreceptors contact different types of bipolar cells, thereby feeding information into different processing channels. In the chicken retina, 15 and 22 different bipolar cell types have been identified based on serial electron microscopy and single-cell transcriptomics, respectively. However, immunohistochemical markers for avian bipolar cells were only anecdotally described so far. Here, we systematically tested 12 antibodies for their ability to label individual bipolar cells in the bird retina and compared the eight most suitable antibodies across distantly related species, namely domestic chicken, domestic pigeon, common buzzard, and European robin, and across retinal regions. While two markers (GNB3 and EGFR) labeled specifically ON bipolar cells, most markers labeled in addition to bipolar cells also other cell types in the avian retina. Staining pattern of four markers (CD15, PKCα, PKCß, secretagogin) was species-specific. Two markers (calbindin and secretagogin) showed a different expression pattern in central and peripheral retina. For the chicken and European robin, we found slightly more ON bipolar cell somata in the inner nuclear layer than OFF bipolar cell somata. In contrast, OFF bipolar cells made more ribbon synapses than ON bipolar cells in the inner plexiform layer of these species. Finally, we also analyzed the photoreceptor connectivity of selected bipolar cell types in the European robin retina. In summary, we provide a catalog of bipolar cell markers for different bird species, which will greatly facilitate analyzing the retinal circuitry of birds on a larger scale.

Morphology of the "prefrontal" nidopallium caudolaterale in the long-distance night-migratory Eurasian blackcap (Sylvia atricapilla).

Migrating birds have developed remarkable navigational capabilities to successfully master biannual journeys between their breeding and wintering grounds. To reach their intended destination, they need to calculate navigational goals from a large variety of natural directional and positional cues to set a meaningful motor output command. One brain area, which has been associated with such executive functions, is the nidopallium caudolaterale (NCL), which, due to its striking similarities in terms of neurochemistry, connectivity and function, is considered analogous to the mammalian prefrontal cortex. To establish a baseline for further analyses elucidating the neuronal correlates underlying avian navigation, we performed quantitative and qualitative analyses of dopaminergic fibres in the brains of long-distance night-migratory Eurasian blackcaps (Sylvia atricapilla). We identified four regions in the caudal telencephalon, each of which was characterized by its specific dopaminergic innervation pattern. At least three of them presumably constitute subareas of the NCL in Eurasian blackcaps and could thus be involved in integrating navigational input from different sensory systems. The observed heterogeneity and parcellation of the NCL subcompartments in this migratory species could be a consequence of the special demands related to navigation.

Double cones in the avian retina form an oriented mosaic which might facilitate magnetoreception and/or polarized light sensing.

To navigate between breeding and wintering grounds, night-migratory songbirds are aided by a light-dependent magnetic compass sense and maybe also by polarized light vision. Although the underlying mechanisms for magnetoreception and polarized light sensing remain unclear, double cone photoreceptors in the avian retina have been suggested to represent the primary sensory cells. To use these senses, birds must be able to separate the directional information from the Earth's magnetic field and/or light polarization from variations in light intensity. Theoretical considerations suggest that this could be best achieved if neighbouring double cones were oriented in an ordered pattern. Therefore, we investigate the orientation patterns of double cones in European robins (Erithacus rubecula) and domestic chickens (Gallus gallus domesticus). We used whole-mounted retinas labelled with double cone markers to quantify the orientations of individual double cones in relation to their nearest neighbours. In both species, our data show that the double cone array is highly ordered: the angles between neighbouring double cones were more likely to be 90°/-90° in the central retina and 180°/0° in the peripheral retina, respectively. The observed regularity in double cone orientation could aid the cells' putative function in light-dependent magnetoreception and/or polarized light sensing.

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.

Retinal horizontal cells use different synaptic sites for global feedforward and local feedback signaling.

In the outer plexiform layer (OPL) of the mammalian retina, cone photoreceptors (cones) provide input to more than a dozen types of cone bipolar cells (CBCs). In the mouse, this transmission is modulated by a single horizontal cell (HC) type. HCs perform global signaling within their laterally coupled network but also provide local, cone-specific feedback. However, it is unknown how HCs provide local feedback to cones at the same time as global forward signaling to CBCs and where the underlying synapses are located. To assess how HCs simultaneously perform different modes of signaling, we reconstructed the dendritic trees of five HCs as well as cone axon terminals and CBC dendrites in a serial block-face electron microscopy volume and analyzed their connectivity. In addition to the fine HC dendritic tips invaginating cone axon terminals, we also identified "bulbs," short segments of increased dendritic diameter on the primary dendrites of HCs. These bulbs are in an OPL stratum well below the cone axon terminal base and make contacts with other HCs and CBCs. Our results from immunolabeling, electron microscopy, and glutamate imaging suggest that HC bulbs represent GABAergic synapses that do not receive any direct photoreceptor input. Together, our data suggest the existence of two synaptic strata in the mouse OPL, spatially separating cone-specific feedback and feedforward signaling to CBCs. A biophysical model of a HC dendritic branch and voltage imaging support the hypothesis that this spatial arrangement of synaptic contacts allows for simultaneous local feedback and global feedforward signaling by HCs.

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.

Double Cones and the Diverse Connectivity of Photoreceptors and Bipolar Cells in an Avian Retina.

Double cones are the most common photoreceptor cell type in most avian retinas, but their precise functions remain a mystery. Among their suggested functions are luminance detection, polarized light detection, and light-dependent, radical pair-based magnetoreception. To better understand the function of double cones, it will be crucial to know how they are connected to the neural network in the avian retina. Here we use serial sectioning, multibeam scanning electron microscopy to investigate double-cone anatomy and connectivity with a particular focus on their contacts to other photoreceptor and bipolar cells in the chicken retina. We found that double cones are highly connected to neighboring double cones and with other photoreceptor cells through telodendria-to-terminal and telodendria-to-telodendria contacts. We also identified 15 bipolar cell types based on their axonal stratifications, photoreceptor contact pattern, soma position, and dendritic and axonal field mosaics. Thirteen of these 15 bipolar cell types contacted at least one or both members of the double cone. All bipolar cells were bistratified or multistratified. We also identified surprising contacts between other cone types and between rods and cones. Our data indicate a much more complex connectivity network in the outer plexiform layer of the avian retina than originally expected.SIGNIFICANCE STATEMENT Like in humans, vision is one of the most important senses for birds. Here, we present the first serial section multibeam scanning electron microscopy dataset from any bird retina. We identified many previously undescribed rod-to-cone and cone-to-cone connections. Surprisingly, of the 15 bipolar cell types we identified, 11 received input from rods and 13 of 15 received at least part of their input from double cones. Therefore, double cones seem to play many different and important roles in avian retinal processing, and the neural network and thus information processing in the outer retina are much more complex than previously expected. These fundamental findings will be very important for several fields of science, including vertebrate vision, avian magnetoreception, and comparative neuroanatomy.

Expression of cell markers and transcription factors in the avian retina compared with that in the marmoset retina.

In the vertebrate retina, amacrine and ganglion cells represent the most diverse cell classes. They can be classified into different cell types by several features, such as morphology, light responses, and gene expression profile. Although birds possess high visual acuity (similar to primates that we used here for comparison) and tetrachromatic color vision, data on the expression of transcription factors in retinal ganglion cells of birds are largely missing. In this study, we tested various transcription factors, known to label subpopulations of cells in mammalian retinae, in two avian species: the common buzzard (Buteo buteo), a raptor with exceptional acuity, and the domestic pigeon (Columba livia domestica), a good navigator and widely used model for visual cognition. Staining for the transcription factors Foxp2, Satb1 and Satb2 labeled most ganglion cells in the avian ganglion cell layer. CtBP2 was established as marker for displaced amacrine cells, which allowed us to reliably distinguish ganglion cells from displaced amacrine cells and assess their densities in buzzard and pigeon. When we additionally compared the temporal and central fovea of the buzzard with the fovea of primates, we found that the cellular organization in the pits was different in primates and raptors. In summary, we demonstrate that the expression of transcription factors is a defining feature of cell types not only in the retina of mammals but also in the retina of birds. The markers, which we have established, may provide useful tools for more detailed studies on the retinal circuitry of these highly visual animals.

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.

A novel isoform of cryptochrome 4 (Cry4b) is expressed in the retina of a night-migratory songbird.

The primary sensory molecule underlying light-dependent magnetic compass orientation in migratory birds has still not been identified. The cryptochromes are the only known class of vertebrate proteins which could mediate this mechanism in the avian retina. Cryptochrome 4 of the night-migratory songbird the European robin (Erithacus rubecula; erCry4) has several of the properties needed to be the primary magnetoreceptor in the avian eye. Here, we report on the identification of a novel isoform of erCry4, which we named erCry4b. Cry4b includes an additional exon of 29 amino acids compared to the previously described form of Cry4, now called Cry4a. When comparing the retinal circadian mRNA expression pattern of the already known isoform erCry4a and the novel erCry4b isoform, we find that erCry4a is stably expressed throughout day and night, whereas erCry4b shows a diurnal mRNA oscillation. The differential characteristics of the two erCry4 isoforms regarding their 24-h rhythmicity in mRNA expression leads us to suggest that they might have different functions. Based on the 24-h expression pattern, erCry4a remains the more likely cryptochrome to be involved in radical-pair-based magnetoreception, but at the present time, an involvement of erCry4b cannot be excluded.

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.

Corrigendum: Gap Junctions in A8 Amacrine Cells Are Made of Connexin36 but Are Differently Regulated Than Gap Junctions in AII Amacrine Cells.

[This corrects the article DOI: 10.3389/fnmol.2019.00099.].

Gap Junctions in A8 Amacrine Cells Are Made of Connexin36 but Are Differently Regulated Than Gap Junctions in AII Amacrine Cells.

In the mammalian retina, amacrine cells represent the most diverse cell class and are involved in the spatio-temporal processing of visual signals in the inner plexiform layer. They are connected to bipolar, other amacrine and ganglion cells, forming complex networks via electrical and chemical synapses. The small-field A8 amacrine cell was shown to receive non-selective glutamatergic input from OFF and ON cone bipolar cells at its bistratified dendrites in sublamina 1 and 4 of the inner plexiform layer. Interestingly, it was also shown to form electrical synapses with ON cone bipolar cells, thus resembling the rod pathway-specific AII amacrine cell. In contrast to the AII cell, however, the electrical synapses of A8 cells are poorly understood. Therefore, we made use of the Ier5-GFP mouse line, in which A8 cells are labeled by GFP, to study the gap junction composition and frequency in A8 cells. We found that A8 cells form <20 gap junctions per cell and these gap junctions consist of connexin36. Connexin36 is present at both OFF and ON dendrites of A8 cells, preferentially connecting A8 cells to type 1 OFF and type 6 and 7 ON bipolar cells and presumably other amacrine cells. Additionally, we show that the OFF dendrites of A8 cells co-stratify with the processes of dopaminergic amacrine cells from which they may receive GABAergic input via GABAA receptor subunit α3. As we found A8 cells to express dopamine receptor D1 (but not D2), we also tested whether A8 cell coupling is modulated by D1 receptor agonists and antagonists as was shown for the coupling of AII cells. However, this was not the case. In summary, our data suggests that A8 coupling is differently regulated than AII cells and may even be independent of ambient light levels and serve signal facilitation rather than providing a separate neuronal pathway.

Phenotyping of Gap-Junctional Coupling in the Mouse Retina.

In the mammalian retina, gap junctions, made of connexin proteins, are found in all neuronal cell types and are important for the transmission of rod photoreceptor signals, spike synchronization, noise reduction, and signal averaging. There are several methods available to assess gap junctional coupling in the retina: simultaneous electrical recordings from two adjacent cells, cut-loading, and intracellular injection of gap junction-permeable tracers. Here, we focus on the latter as it allows precise targeting of the cell of interest and is suitable to assess tracer coupling in a wide variety of retinal cell types, e.g., horizontal cells, amacrine cells, and ganglion cells. Tracer coupling experiments are usually performed in the intact retina and can provide information on the extent of coupling, the identity of synaptic partners, and (when combined with immunohistochemistry or pharmacology) the underlying connexin or the regulation of gap junctions.

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.

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.

Photoelectrical Stimulation of Neuronal Cells by an Organic Semiconductor-Electrolyte Interface.

As a step toward the realization of neuroprosthetics for vision restoration, we follow an electrophysiological patch-clamp approach to study the fundamental photoelectrical stimulation mechanism of neuronal model cells by an organic semiconductor-electrolyte interface. Our photoactive layer consisting of an anilino-squaraine donor blended with a fullerene acceptor is supporting the growth of the neuronal model cell line (N2A cells) without an adhesion layer on it and is not impairing cell viability. The transient photocurrent signal upon illumination from the semiconductor-electrolyte layer is able to trigger a passive response of the neuronal cells under physiological conditions via a capacitive coupling mechanism. We study the dynamics of the capacitive transmembrane currents by patch-clamp recordings and compare them to the dynamics of the photocurrent signal and its spectral responsivity. Furthermore, we characterize the morphology of the semiconductor-electrolyte interface by atomic force microscopy and study the stability of the interface in dark and under illuminated conditions.