In Vitro Cytotoxicity of D18 and Y6 as Potential Organic Photovoltaic Materials for Retinal Prostheses.

Millions of people worldwide are diagnosed with retinal dystrophies such as retinitis pigmentosa and age-related macular degeneration. A retinal prosthesis using organic photovoltaic (OPV) semiconductors is a promising therapeutic device to restore vision to patients at the late onset of the disease. However, an appropriate cytotoxicity approach has to be employed on the OPV materials before using them as retinal implants. In this study, we followed ISO standards to assess the cytotoxicity of D18, Y6, PFN-Br and PDIN individually, and as mixtures of D18/Y6, D18/Y6/PFN-Br and D18/Y6/PDIN. These materials were proven for their high performance as organic solar cells. Human RPE cells were put in direct and indirect contact with these materials to analyze their cytotoxicity by the MTT assay, apoptosis by flow cytometry, and measurements of cell morphology and proliferation by immunofluorescence. We also assessed electrophysiological recordings on mouse retinal explants via microelectrode arrays (MEAs) coated with D18/Y6. In contrast to PFN-Br and PDIN, all in vitro experiments show no cytotoxicity of D18 and Y6 alone or as a D18/Y6 mixture. We conclude that D18/Y6 is safe to be subsequently investigated as a retinal prosthesis.

Cyclosporine A Protects Retinal Explants against Hypoxia.

The retina is a complex neurological tissue and is extremely sensitive to an insufficient supply of oxygen. Hypoxia plays a major role in several retinal diseases, and often results in the loss of cells that are essential for vision. Cyclosporine A (CsA) is a widely used immunosuppressive drug. Furthermore, treatment with CsA has neuroprotective effects in several neurologic disorders. No data are currently available on the tolerated concentration of CsA when applied to the retina. To reveal the most effective dose, retinal explants from rat eyes were exposed to different CsA concentrations (1-9 µg/mL). Immunohistochemistry with brain-specific homeobox/POU domain protein 3a (Brn3a) and TUNEL staining was performed to determine the percentage of total and apoptotic retinal ganglion cells (RGCs), as well as the responses of micro- and macroglial cells. Furthermore, optical coherence tomography (OCT) scans were performed to measure the changes in retinal thickness, and recordings with multielectrode array (MEA) were performed to evaluate spontaneous RGC spiking. To examine the neuroprotective effects, retinas were subjected to a hypoxic insult by placing them in a nitrogen-streamed hypoxic chamber prior to CsA treatment. In the biocompatibility tests, the different CsA concentrations had no negative effect on RGCs and microglia. Neuroprotective effects after a hypoxic insult on RGCs was demonstrated at a concentration of 9 µg/mL CsA. CsA counteracted the hypoxia-induced loss of RGCs, reduced the percentage of TUNEL+ RGCs, and prevented a decrease in retinal thickness. Taken together, the results of this study suggest that CsA can effectively protect RGCs from hypoxia, and the administered concentrations were well tolerated. Further in vivo studies are needed to determine whether local CsA treatment may be a suitable option for hypoxic retinal diseases.

Hypothermia protects retinal ganglion cells against hypoxia-induced cell death in a retina organ culture model.

BACKGROUND: Hypoxia contributes to retinal damage in several retinal diseases, including central retinal artery occlusion, with detrimental consequences like painless, monocular loss of vision. Currently, the treatment options are severely limited due to the short therapy window, as the neuronal cells, especially the retinal ganglion cells (RGCs), are irreversibly damaged within the first few hours. Hypothermia might be a possible treatment option or at least might increase the therapy window. METHODS: To investigate the neuroprotective effect of hypothermia after retinal hypoxia, an easy-to-use ex vivo retinal hypoxia organ culture model developed in our laboratory was used that reliably induced retinal damage on a structural, molecular and functional level. The neuroprotective effect of hypothermia after retinal hypoxia was analysed using optical coherence tomography scans, histological stainings, quantitative real-time polymerase chain reaction, western blotting and microelectrode array recordings. RESULTS: Two different hypothermic temperatures (30°C and 20°C) were evaluated, both exhibited strong neuroprotective effects. Most importantly, hypothermia increased RGC survival after retinal hypoxia. Furthermore, hypothermia counteracted the hypoxia-induced RGC death, reduced macroglia activation, attenuated retinal thinning and protected from loss of spontaneous RGC activity. CONCLUSIONS: These results indicate that already a mild reduction in temperature protects the RGCs against damage and could function as a promising therapeutic option for hypoxic diseases.

Subretinal electrical stimulation reveals intact network activity in the blind mouse retina.

Retinal degeneration (rd) leads to progressive photoreceptor cell death, resulting in vision loss. Stimulation of the inner-retinal neurons by neuroprosthetic implants is one of the clinically approved vision-restoration strategies, providing basic visual percepts to blind patients. However, little is understood as to what degree the degenerating retinal circuitry and the resulting aberrant hyperactivity may prevent the stimulation of physiological electrical activity. Therefore, we electrically stimulated ex vivo retinas from wild-type (wt; C57BL/6J) and blind (rd10 and rd1) mice using an implantable subretinal microchip and simultaneously recorded and analyzed the retinal ganglion cell (RGC) output with a flexible microelectrode array. We found that subretinal anodal stimulation of the rd10 retina and wt retina evoked similar spatiotemporal RGC-spiking patterns. In both retinas, electrically stimulated ON and a small percentage of OFF RGC responses were detected. The spatial selectivity of the retinal network to electrical stimuli reveals an intact underlying network with a median receptive-field center of 350 µm in both retinas. An antagonistic surround is activated by stimulation with large electrode fields. However, in rd10 and to a higher percentage, in rd1 retinas, rhythmic and spatially unconfined RGC patterns were evoked by anodal or by cathodal electrical stimuli. Our findings demonstrate that the surviving retinal circuitry in photoreceptor-degenerated retinas is preserved in a way allowing for the stimulation of temporally diverse and spatially confined RGC activity. Future vision restoration strategies can build on these results but need to avoid evoking the easily inducible rhythmic activity in some retinal circuits.

Daylight vision repair by cell transplantation.

Human daylight vision depends on cone photoreceptors and their degeneration results in visual impairment and blindness as observed in several eye diseases including age-related macular degeneration, cone-rod dystrophies, or late stage retinitis pigmentosa, with no cure available. Preclinical cell replacement approaches in mouse retina have been focusing on rod dystrophies, due to the availability of sufficient donor material from the rod-dominated mouse retina, leaving the development of treatment options for cone degenerations not well studied. Thus, an abundant and traceable source for donor cone-like photoreceptors was generated by crossing neural retina leucine zipper-deficient (Nrl(-/-) ) mice with an ubiquitous green fluorescent protein (GFP) reporter line resulting in double transgenic tg(Nrl(-/-); aGFP) mice. In Nrl(-/-) retinas, all rods are converted into cone-like photoreceptors that express CD73 allowing their enrichment by CD73-based magnetic activated cell sorting prior transplantation into the subretinal space of adult wild-type, cone-only (Nrl(-/-)), or cone photoreceptor function loss 1 (Cpfl1) mice. Donor cells correctly integrated into host retinas, acquired mature photoreceptor morphology, expressed cone-specific markers, and survived for up to 6 months, with significantly increased integration rates in the cone-only Nrl(-/-) retina. Individual retinal ganglion cell recordings demonstrated the restoration of photopic responses in cone degeneration mice following transplantation suggesting, for the first time, the feasibility of daylight vision repair by cell replacement in the adult mammalian retina.

Avoidance of axonal stimulation with sinusoidal epiretinal stimulation.

Objective.Neuromodulation, particularly electrical stimulation, necessitates high spatial resolution to achieve artificial vision with high acuity. In epiretinal implants, this is hindered by the undesired activation of distal axons. Here, we investigate focal and axonal activation of retinal ganglion cells (RGCs) in epiretinal configuration for different sinusoidal stimulation frequencies.Approach.RGC responses to epiretinal sinusoidal stimulation at frequencies between 40 and 100 Hz were tested inex-vivophotoreceptor degenerated (rd10) isolated retinae. Experiments were conducted using a high-density CMOS-based microelectrode array, which allows to localize RGC cell bodies and axons at high spatial resolution.Main results.We report current and charge density thresholds for focal and distal axon activation at stimulation frequencies of 40, 60, 80, and 100 Hz for an electrode size with an effective area of 0.01 mm2. Activation of distal axons is avoided up to a stimulation amplitude of 0.23µA (corresponding to 17.3µC cm-2) at 40 Hz and up to a stimulation amplitude of 0.28µA (14.8µC cm-2) at 60 Hz. The threshold ratio between focal and axonal activation increases from 1.1 for 100 Hz up to 1.6 for 60 Hz, while at 40 Hz stimulation frequency, almost no axonal responses were detected in the tested intensity range. With the use of synaptic blockers, we demonstrate the underlying direct activation mechanism of the ganglion cells. Finally, using high-resolution electrical imaging and label-free electrophysiological axon tracking, we demonstrate the extent of activation in axon bundles.Significance.Our results can be exploited to define a spatially selective stimulation strategy avoiding axonal activation in future retinal implants, thereby solving one of the major limitations of artificial vision. The results may be extended to other fields of neuroprosthetics to achieve selective focal electrical stimulation.

Assessment of chemotherapeutic effects on cancer cells using adhesion noise spectroscopy.

With cancer as one of the leading causes of death worldwide, there is a need for the development of accurate, cost-effective, easy-to-use, and fast drug-testing assays. While the NCI 60 cell-line screening as the gold standard is based on a colorimetric assay, monitoring cells electrically constitutes a label-free and non-invasive tool to assess the cytotoxic effects of a chemotherapeutic treatment on cancer cells. For decades, impedance-based cellular assays extensively investigated various cell characteristics affected by drug treatment but lack spatiotemporal resolution. With progress in microelectrode fabrication, high-density Complementary Metal Oxide Semiconductor (CMOS)-based microelectrode arrays (MEAs) with subcellular resolution and time-continuous recording capability emerged as a potent alternative. In this article, we present a new cell adhesion noise (CAN)-based electrical imaging technique to expand CMOS MEA cell-biology applications: CAN spectroscopy enables drug screening quantification with single-cell spatial resolution. The chemotherapeutic agent 5-Fluorouracil exerts a cytotoxic effect on colorectal cancer (CRC) cells hampering cell proliferation and lowering cell viability. For proof-of-concept, we found sufficient accuracy and reproducibility for CAN spectroscopy compared to a commercially available standard colorimetric biological assay. This label-free, non-invasive, and fast electrical imaging technique complements standardized cancer screening methods with significant advances over established impedance-based approaches.

A comprehensive small interfering RNA screen identifies signaling pathways required for gephyrin clustering.

The postsynaptic scaffold protein gephyrin is clustered at inhibitory synapses and serves for the stabilization of GABA(A) receptors. Here, a comprehensive kinome-wide siRNA screen in a human HeLa cell-based model for gephyrin clustering was used to identify candidate protein kinases implicated in the stabilization of gephyrin clusters. As a result, 12 hits were identified including FGFR1 (FGF receptor 1), TrkB, and TrkC as well as components of the MAPK and mammalian target of rapamycin (mTOR) pathways. For confirmation, the impact of these hits on gephyrin clustering was analyzed in rat primary hippocampal neurons. We found that brain-derived neurotrophic factor (BDNF) acts on gephyrin clustering through MAPK signaling, and this process may be controlled by the MAPK signaling antagonist sprouty2. BDNF signaling through phosphatidylinositol 3-kinase (PI3K)-Akt also activates mTOR and represses GSK3ß, which was previously shown to reduce gephyrin clustering. Gephyrin is associated with inactive mTOR and becomes released upon BDNF-dependent mTOR activation. In primary neurons, a reduction in the number of gephyrin clusters due to manipulation of the BDNF-mTOR signaling is associated with reduced GABA(A) receptor clustering, suggesting functional impairment of GABA signaling. Accordingly, application of the mTOR antagonist rapamycin leads to disinhibition of neuronal networks as measured on microelectrode arrays. In conclusion, we provide evidence that BDNF regulates gephyrin clustering via MAPK as well as PI3K-Akt-mTOR signaling.

Network oscillations in rod-degenerated mouse retinas.

In the mammalian retina, excitatory and inhibitory circuitries enable retinal ganglion cells (RGCs) to signal the occurrence of visual features to higher brain areas. This functionality disappears in certain diseases of retinal degeneration because of the progressive loss of photoreceptors. Recent work in a mouse model of retinal degeneration (rd1) found that, although some intraretinal circuitry is preserved and RGCs maintain characteristic physiological properties, they exhibit increased and aberrant rhythmic activity. Here, extracellular recordings were made to assess the degree of aberrant activity in adult rd1 retinas and to investigate the mechanism underlying such behavior. A multi-transistor array with thousands of densely packed sensors allowed for simultaneous recordings of spiking activity in populations of RGCs and of local field potentials (LFPs). The majority of identified RGCs displayed rhythmic (7-10 Hz) but asynchronous activity. The spiking activity correlated with the LFPs, which reflect an average synchronized excitatory input to the RGCs. LFPs initiated from random positions and propagated across the retina. They disappeared when ionotrophic glutamate receptors or electrical synapses were blocked. They persisted in the presence of other pharmacological blockers, including TTX and inhibitory receptor antagonists. Our results suggest that excitation-transmitted laterally through a network of electrically coupled interneurons-leads to large-scale retinal network oscillations, reflected in the rhythmic spiking of most rd1 RGCs. This result may explain forms of photopsias reported by blind patients, while the mechanism involved should be considered in future treatment strategies targeting the disease of retinitis pigmentosa.

Electrical stimulation of retinal neurons in epiretinal and subretinal configuration using a multicapacitor array.

Electrical stimulation of retinal neurons offers the possibility of partial restoration of visual function. Challenges in neuroprosthetic applications are the long-term stability of the metal-based devices and the physiological activation of retinal circuitry. In this study, we demonstrate electrical stimulation of different classes of retinal neurons with a multicapacitor array. The array--insulated by an inert oxide--allows for safe stimulation with monophasic anodal or cathodal current pulses of low amplitude. Ex vivo rabbit retinas were interfaced in either epiretinal or subretinal configuration to the multicapacitor array. The evoked activity was recorded from ganglion cells that respond to light increments by an extracellular tungsten electrode. First, a monophasic epiretinal cathodal or a subretinal anodal current pulse evokes a complex burst of action potentials in ganglion cells. The first action potential occurs within 1 ms and is attributed to direct stimulation. Within the next milliseconds additional spikes are evoked through bipolar cell or photoreceptor depolarization, as confirmed by pharmacological blockers. Second, monophasic epiretinal anodal or subretinal cathodal currents elicit spikes in ganglion cells by hyperpolarization of photoreceptor terminals. These stimuli mimic the photoreceptor response to light increments. Third, the stimulation symmetry between current polarities (anodal/cathodal) and retina-array configuration (epi/sub) is confirmed in an experiment in which stimuli presented at different positions reveal the center-surround organization of the ganglion cell. A simple biophysical model that relies on voltage changes of cell terminals in the transretinal electric field above the stimulation capacitor explains our results. This study provides a comprehensive guide for efficient stimulation of different retinal neuronal classes with low-amplitude capacitive currents.

High spatial resolution artificial vision inferred from the spiking output of retinal ganglion cells stimulated by optogenetic and electrical means.

With vision impairment affecting millions of people world-wide, various strategies aiming at vision restoration are being undertaken. Thanks to decades of extensive research, electrical stimulation approaches to vision restoration began to undergo clinical trials. Quite recently, another technique employing optogenetic therapy emerged as a possible alternative. Both artificial vision restoration strategies reported poor spatial resolution so far. In this article, we compared the spatial resolution inferred ex vivo under ideal conditions using a computational model analysis of the retinal ganglion cell (RGC) spiking activity. The RGC spiking was stimulated in epiretinal configuration by either optogenetic or electrical means. RGCs activity was recorded from the ex vivo retina of transgenic late-stage photoreceptor-degenerated mice (rd10) using a high-density Complementary Metal Oxide Semiconductor (CMOS) based microelectrode array. The majority of retinal samples were stimulated by both, optogenetic and electrical stimuli using a spatial grating stimulus. A population-level analysis of the spiking activity of identified RGCs was performed and the spatial resolution achieved through electrical and optogenetic photo-stimulation was inferred using a support vector machine classifier. The best f1 score of the classifier for the electrical stimulation in epiretinal configuration was 86% for 32 micron wide gratings and increased to 100% for 128 microns. For optogenetically activated cells, we obtained high f1 scores of 82% for 10 microns grid width for a photo-stimulation frequency of 2.5 Hz and 73% for a photo-stimulation frequency of 10 Hz. A subsequent analysis, considering only the RGCs modulated in both electrical and optogenetic stimulation protocols revealed no significant difference in the prediction accuracy between the two stimulation modalities. The results presented here indicate that a high spatial resolution can be achieved for electrical or optogenetic artificial stimulation using the activated retinal ganglion cell output.

An optoelectronic neural interface approach for precise superposition of optical and electrical stimulation in flexible array structures.

Optical stimulation of genetically modified nerve cells has become one of the state-of-the-art methods in neuroscience. This so-called optogenetic approach allows cell-type specific activation in comparison to more generalized electrical stimulation. Combinations of both stimulation modalities would be desirable to investigate effects in detail and specify differences. This work presents the design of a miniaturized optoelectronic device that allows optical and electrical activation at the same spot. Indium tin oxide (ITO), which is transparent to visible light, has been chosen as electrode material. Light emitting diodes were assembled on a polyimide substrate with integrated interconnection lines, directly behind the electrodes to compare optical with electrical stimulation. The optical transparency of the ITO-polyimide layer stack was investigated and showed sufficient transmission in the required wavelength range. ITO electrodes with diameters up to 1000 µm were electrochemically characterized using electrical impedance spectroscopy (EIS). Several diameters did show comparable results to platinum, a commonly used electrode material. Fully assembled devices were used in combination an ex vivo setting with genetically modified retina to demonstrate the functionality of this approach. Retinal ganglion cells were excited by both, optical and electrical stimulation at the same spot and signals were recorded via standard microelectrode arrays (MEA) as reference. The simultaneous stimulation and recording of directly evoked action potentials indicates a similar mode of action of the two stimulation modalities. Further engineering work is needed to transfer the presented and proven concept into devices for chronic implantation, might it be in animal or first-in-human studies.

Transplanted human cones incorporate into the retina and function in a murine cone degeneration model.

Once human photoreceptors die, they do not regenerate, thus, photoreceptor transplantation has emerged as a potential treatment approach for blinding diseases. Improvements in transplant organization, donor cell maturation, and synaptic connectivity to the host will be critical in advancing this technology for use in clinical practice. Unlike the unstructured grafts of prior cell-suspension transplantations into end-stage degeneration models, we describe the extensive incorporation of induced pluripotent stem cell (iPSC) retinal organoid-derived human photoreceptors into mice with cone dysfunction. This incorporative phenotype was validated in both cone-only as well as pan-photoreceptor transplantations. Rather than forming a glial barrier, Müller cells extended throughout the graft, even forming a series of adherens junctions between mouse and human cells, reminiscent of an outer limiting membrane. Donor-host interaction appeared to promote polarization as well as the development of morphological features critical for light detection, namely the formation of inner and well-stacked outer segments oriented toward the retinal pigment epithelium. Putative synapse formation and graft function were evident at both structural and electrophysiological levels. Overall, these results show that human photoreceptors interacted readily with a partially degenerated retina. Moreover, incorporation into the host retina appeared to be beneficial to graft maturation, polarization, and function.

A flexible protruding microelectrode array for neural interfacing in bioelectronic medicine.

Recording neural signals from delicate autonomic nerves is a challenging task that requires the development of a low-invasive neural interface with highly selective, micrometer-sized electrodes. This paper reports on the development of a three-dimensional (3D) protruding thin-film microelectrode array (MEA), which is intended to be used for recording low-amplitude neural signals from pelvic nervous structures by penetrating the nerves transversely to reduce the distance to the axons. Cylindrical gold pillars (Ø 20 or 50 µm, ~60 µm height) were fabricated on a micromachined polyimide substrate in an electroplating process. Their sidewalls were insulated with parylene C, and their tips were optionally modified by wet etching and/or the application of a titanium nitride (TiN) coating. The microelectrodes modified by these combined techniques exhibited low impedances (~7 kΩ at 1 kHz for Ø 50 µm microelectrode with the exposed surface area of ~5000 µm²) and low intrinsic noise levels. Their functionalities were evaluated in an ex vivo pilot study with mouse retinae, in which spontaneous neuronal spikes were recorded with amplitudes of up to 66 µV. This novel process strategy for fabricating flexible, 3D neural interfaces with low-impedance microelectrodes has the potential to selectively record neural signals from not only delicate structures such as retinal cells but also autonomic nerves with improved signal quality to study neural circuits and develop stimulation strategies in bioelectronic medicine, e.g., for the control of vital digestive functions.

Sharpening of directional selectivity from neural output of rabbit retina.

The estimation of motion direction from time varying retinal images is a fundamental task of visual systems. Neurons that selectively respond to directional visual motion are found in almost all species. In many of them already in the retina direction selective neurons signal their preferred direction of movement. Scientific evidences suggest that direction selectivity is carried from the retina to higher brain areas. Here we adopt a simple integrate-and-fire neuron model, inspired by recent work of Casti et al. (2008), to investigate how directional selectivity changes in cells postsynaptic to directional selective retinal ganglion cells (DSRGC). Our model analysis shows that directional selectivity in the postsynaptic cells increases over a wide parameter range. The degree of directional selectivity positively correlates with the probability of burst-like firing of presynaptic DSRGCs. Postsynaptic potentials summation and spike threshold act together as a temporal filter upon the input spike train. Prior to the intricacy of neural circuitry between retina and higher brain areas, we suggest that sharpening is a straightforward result of the intrinsic spiking pattern of the DSRGCs combined with the summation of excitatory postsynaptic potentials and the spike threshold in postsynaptic neurons.

Spatial and temporal resolution of optogenetically recovered vision in ChR2-transduced mouse retina.

Objective.Retinal ganglion cells (RGCs) represent an attractive target in vision restoration strategies, because they undergo little degeneration compared to other retinal neurons. Here we investigated the temporal and spatial resolution in adult photoreceptor-degenerated (rd10) mouse retinas, where RGCs have been transduced with the optogenetic actuator channelrhodopsin-2 (ChR2).Approach.The RGC spiking activity was recorded continuously with a CMOS-based microelectrode array during a variety of photostimulation protocols. The temporal resolution was assessed through Gaussian white noise stimuli and evaluated using a linear-nonlinear-Poisson model. Spatial sensitivity was assessed upon photostimulation with single rectangular pulses stepped across the retina and upon stimulation with alternating gratings of different spatial frequencies. Spatial sensitivity was estimated using logistic regression or by evaluating the spiking activity of independent RGCs.Main results.The temporal resolution after photostimulation displayed an about ten times faster kinetics as compared to physiological filters in wild-type RGCs. The optimal spatial resolution estimated using the logistic regression model was 10µm and 87µm based on the population response. These values correspond to an equivalent acuity of 1.7 or 0.2 cycles per degree, which is better than expected from the size of RGCs' optogenetic receptive fields.Significance.The high temporal and spatial resolution obtained by photostimulation of optogenetically transduced RGCs indicate that high acuity vision restoration may be obtained by photostimulation of appropriately modified RGCs in photoreceptor-degenerated retinas.

Discrimination of simple objects decoded from the output of retinal ganglion cells upon sinusoidal electrical stimulation.

Objective. Most neuroprosthetic implants employ pulsatile square-wave electrical stimuli, which are significantly different from physiological inter-neuronal communication. In case of retinal neuroprosthetics, which use a certain type of pulsatile stimuli, reliable object and contrast discrimination by implanted blind patients remained challenging. Here we investigated to what extent simple objects can be discriminated from the output of retinal ganglion cells (RGCs) upon sinusoidal stimulation.Approach. Spatially confined objects were formed by different combinations of 1024 stimulating microelectrodes. The RGC activity in theex vivoretina of photoreceptor-degenerated mouse, of healthy mouse or of primate was recorded simultaneously using an interleaved recording microelectrode array implemented in a CMOS-based chip.Main results. We report that application of sinusoidal electrical stimuli (40 Hz) in epiretinal configuration instantaneously and reliably modulates the RGC activity in spatially confined areas at low stimulation threshold charge densities (40 nC mm-2). Classification of overlapping but spatially displaced objects (1° separation) was achieved by distinct spiking activity of selected RGCs. A classifier (regularized logistic regression) discriminated spatially displaced objects (size: 5.5° or 3.5°) with high accuracy (90% or 62%). Stimulation with low artificial contrast (10%) encoded by different stimulus amplitudes generated RGC activity, which was classified with an accuracy of 80% for large objects (5.5°).Significance. We conclude that time-continuous smooth-wave stimulation provides robust, localized neuronal activation in photoreceptor-degenerated retina, which may enable future artificial vision at high temporal, spatial and contrast resolution.

HDAC inhibition ameliorates cone survival in retinitis pigmentosa mice.

Cone photoreceptor cell death in inherited retinal diseases, such as Retinitis Pigmentosa (RP), leads to the loss of high acuity and color vision and, ultimately to blindness. In RP, a vast number of mutations perturb the structure and function of rod photoreceptors, while cones remain initially unaffected. Extensive rod loss in advanced stages of the disease triggers cone death by a mechanism that is still largely unknown. Here, we show that secondary cone cell death in animal models for RP is associated with increased activity of histone deacetylates (HDACs). A single intravitreal injection of an HDAC inhibitor at late stages of the disease, when the majority of rods have already degenerated, was sufficient to delay cone death and support long-term cone survival in two mouse models for RP, affected by mutations in the phosphodiesterase 6b gene. Moreover, the surviving cones remained light-sensitive, leading to an improvement in visual function. RNA-seq analysis of protected cones demonstrated that HDAC inhibition initiated multi-level protection via regulation of different pro-survival pathways, including MAPK, PI3K-Akt, and autophagy. This study suggests a unique opportunity for targeted pharmacological protection of secondary dying cones by HDAC inhibition and creates hope to maintain vision in RP patients even in advanced disease stages.

Electrical Imaging of Light-Induced Signals Across and Within Retinal Layers.

The mammalian retina processes sensory signals through two major pathways: a vertical excitatory pathway, which involves photoreceptors, bipolar cells, and ganglion cells, and a horizontal inhibitory pathway, which involves horizontal cells, and amacrine cells. This concept explains the generation of an excitatory center-inhibitory surround sensory receptive fields-but fails to explain the modulation of the retinal output by stimuli outside the receptive field. Electrical imaging of light-induced signal propagation at high spatial and temporal resolution across and within different retinal layers might reveal mechanisms and circuits involved in the remote modulation of the retinal output. Here we took advantage of a high-density complementary metal oxide semiconductor-based microelectrode array and investigated the light-induced propagation of local field potentials (LFPs) in vertical mouse retina slices. Surprisingly, the LFP propagation within the different retinal layers depends on stimulus duration and stimulus background. Application of the same spatially restricted light stimuli to flat-mounted retina induced ganglion cell activity at remote distances from the stimulus center. This effect disappeared if a global background was provided or if gap junctions were blocked. We hereby present a neurotechnological approach and demonstrated its application, in which electrical imaging evaluates stimulus-dependent signal processing across different neural layers.

Probing and predicting ganglion cell responses to smooth electrical stimulation in healthy and blind mouse retina.

Retinal implants are used to replace lost photoreceptors in blind patients suffering from retinopathies such as retinitis pigmentosa. Patients wearing implants regain some rudimentary visual function. However, it is severely limited compared to normal vision because non-physiological stimulation strategies fail to selectively activate different retinal pathways at sufficient spatial and temporal resolution. The development of improved stimulation strategies is rendered difficult by the large space of potential stimuli. Here we systematically explore a subspace of potential stimuli by electrically stimulating healthy and blind mouse retina in epiretinal configuration using smooth Gaussian white noise delivered by a high-density CMOS-based microelectrode array. We identify linear filters of retinal ganglion cells (RGCs) by fitting a linear-nonlinear-Poisson (LNP) model. Our stimulus evokes spatially and temporally confined spiking responses in RGC which are accurately predicted by the LNP model. Furthermore, we find diverse shapes of linear filters in the linear stage of the model, suggesting diverse preferred electrical stimuli of RGCs. The linear filter base identified by our approach could provide a starting point of a model-guided search for improved stimuli for retinal prosthetics.