Classification of pseudocalcium visual responses from mouse retinal ganglion cells.

Recently, a detailed catalog of 32 retinal ganglion cell (RGC) visual response patterns in mouse has emerged. However, the 10,000 samples required for this catalog-based on fluorescent signals from a calcium indicator dye-are much harder to acquire from the extracellular spike train recordings underlying our bionic vision research. Therefore, we sought to convert spike trains into pseudocalcium signals so that our data could be directly matched to the 32 predefined, calcium signal-based groups. A microelectrode array (MEA) was used to record spike trains from mouse RGCs of 29 retinas. Visual stimuli were adapted from the Baden et al. study; including moving bars, full-field contrast and temporal frequency chirps, and black-white and UV-green color flashes. Spike train histograms were converted into pseudocalcium traces with an OGB-1 convolution kernel. Response features were extracted using sparse principal components analysis to match each RGC to one of the 32 RGC groups. These responses mapped onto of the 32 previously described groups; however, some of the groups remained unmatched. Thus, adaptation of the Baden et al. methodology for MEA recordings of spike trains instead of calcium recordings was partially successful. Different classification methods, however, will be needed to define clear RGC groups from MEA data for our bionic vision research. Nevertheless, others may pursue a pseudocalcium approach to reconcile spike trains with calcium signals. This work will help to guide them on the limitations and potential pitfalls of such an approach.

A case of X-linked retinoschisis with atypical fundus appearance.

PURPOSE: Mutations in the RS1 gene are known to cause retinoschisis, an X-linked hereditary retinal degeneration. Here, we present a case of atypical retinoschisis with clinical findings of retinoschisis and retinitis pigmentosa. METHODS: This report is an observational case report. The detailed ophthalmological examinations included visual field determination, multimodal imaging and electrophysiological recordings. Targeted next-generation sequencing of a retinal disease gene panel was performed. RESULTS: The 55-year-old male, highly hyperopic patient, presented with a best-corrected Snellen visual acuity of 20/100 in the right eye and 20/400 in the left eye. In the kinetic visual field, there was a superior scotoma, as well as a ring scotoma in the inferior hemisphere in the right eye and a concentric visual field constriction to 10° in the left eye. Funduscopy revealed marked pigmentary changes (i.e. bone spicules) in the mid-periphery bilaterally and symmetrically, as well as two small intra-retinal haemorrhages in the left eye. Full-field electroretinography recordings showed extinguished rod and cone responses. Diagnostic-genetic testing revealed a hemizygous missense mutation in the RS1 gene (c.305G > A; p.Arg102Gln) was identified. CONCLUSION: We present a case of atypical retinoschisis with clinical findings of retinitis pigmentosa.

Hypotrichosis with cone-rod dystrophy in a patient with cadherin 3 (CDH3) mutation.

PURPOSE: To investigate a very rare case of hypotrichosis with cone-rod dystrophy caused by a P-cadherin CDH3 mutation. METHODS: A 16-year-old Syrian girl was examined at age 9 and 14 years with an ophthalmological examination, fundus imaging, OCT and electrophysiological recordings (ERG and PERG). A disease-targeted gene panel sequencing was performed. RESULTS: Fundus images showed pigmentations at the posterior eye pole to the mid periphery, as well as vessel tortuosity. OCT images revealed a loss of the outer retinal segments and IS/OS in the central macula. The scotopic and photopic ERGs showed moderately reduced amplitudes at age 9 years that became severely reduced at age of 14 years. The PERG was undetectable at age 9 years. In color vision testing, protan-deutan confusion errors occurred. Gene panel analysis revealed one homozygous mutation in CDH3 (c.1508G>A; p.Arg503His). CONCLUSION: This case shows that a CDH3 mutation besides macula dystrophy can cause widespread cone-rod dystrophy with hypotrichosis without any other pathology besides hypoplastic nails. This points to a common pathway of hair growth and photoreceptor development that can be disturbed by a CDH3 mutation (c.1508G>A; p.Arg503His) located in the EC4 repeat region of the gene.

Retinal dystrophies with bull's-eye maculopathy along with negative ERGs.

PURPOSE: The aim of this study was to examine the ophthalmological characteristics and genotypes of patients with congenital retinal pathologies, who display a bull's-eye maculopathy in the fundus, along with a negative scotopic electroretinogram. METHODS: We analysed the results of five patients showing both a bull's-eye maculopathy, as well as a negative scotopic ERG evoked by a bright flash. Their median age was 39 years (range 11-63 years): three males and two females. All underwent a comprehensive examination with determination of distant visual acuity (ETDRS) and recording of the full-field ERG (scotopic and photopic). Fundus, OCT, and FAF images were obtained, the kinetic visual field was determined, and colour vision (D-15) was tested in most patients. Targeted gene panel sequencing was performed on peripheral blood. RESULTS: One patient carried a homozygous ABCA4 mutation and an additional heterozygous variant in CRX. Two of the five patients were shown to have a heterozygous mutation in the CRX gene, one of whom had an additional heterozygous ABCA4 mutation. Two patients had the common heterozygous mutation c.2413G>A;p.Arg838His in GUCY2D. In all of the patients, there was a reduction in the amplitude of the b-wave with a regular a-wave amplitude in the scotopic bright-flash ERG. CONCLUSIONS: The five patients with bull's-eye maculopathy along with a negative ERG had differing genotypes. Mutations were found in the CRX gene (2 patients), the ABCA4 gene (1 patient), and the GUCY2D gene (2 patients).

The importance of electrode position in visual electrophysiology.

PURPOSE: The DTL fibre electrode is commonly used to record the electric potentials elicited by stimulation of the retina. Two positions are commonly used: it is placed either on the cornea along the lower lid or in the conjunctival fornix. The PERG and OPs have previously been examined and compared under both conditions. The aim of this study was to examine the ERG, flicker response and on-off responses with differing electrode positions. METHODS: Before recruitment, all subjects underwent an ophthalmological examination. We enrolled 13 normal control subjects into the study aged 13-64 years, all with a visual acuity of ≥1.0. We recorded scotopic and photopic ERGs, flicker and on-off responses, for both electrode positions. On the first day, one eye had the electrode placed on the cornea along the lower lid and the other eye had it positioned in the conjunctival sac. On a second day, the recordings were repeated with the alternative electrode placements. RESULTS: ERG, on-off and flicker responses were all smaller by between 20 and 25% when the DTL electrode was positioned in the conjunctival sac, compared to when it was positioned on the cornea, as did the scatter in the data points. This indicates that there is no advantage clinically for one or the other placement. CONCLUSIONS: Our results confirm other reports examining the effect of electrode position on electrophysiological potentials. When recording with the DTL electrode, it is important to ensure that it is placed at the same position in repeat recordings or in multicentre trials and that it is stable and does not move during recording.

Transfer characteristics of subretinal visual implants: corneally recorded implant responses.

PURPOSE: The subretinal Alpha IMS visual implant is a CE-approved medical device for restoration of visual functions in blind patients with end-stage outer retina degeneration. We present a method to test the function of the implant objectively in vivo using standard electroretinographic equipment and to assess the devices' parameter range for an optimal perception. METHODS: Subretinal implant Alpha IMS (Retina Implant AG, Reutlingen, Germany) consists of 1500 photodiode-amplifier-electrode units and is implanted surgically into the subretinal space in blind retinitis pigmentosa patients. The voltages that regulate the amplifiers' sensitivity (V gl) and gain (V bias), related to the perception of contrast and brightness, respectively, are adjusted manually on a handheld power supply device. Corneally recorded implant responses (CRIR) to full-field illumination with long duration flashes in various implant settings for brightness gain (V bias) and amplifiers' sensitivity (V gl) are measured using electroretinographic setup with a Ganzfeld bowl in a protocol of increasing stimulus luminances up to 1000 cd/m2. RESULTS: CRIRs are a meaningful tool for assessing the transfer characteristic curves of the electronic implant in vivo monitoring the implants' voltage output as a function of log luminance in a sigmoidal shape. Changing the amplifiers' sensitivity (V gl) shifts the curve left or right along the log luminance axis. Adjustment of the gain (V bias) changes the maximal output. Contrast perception is only possible within the luminance range of the increasing slope of the function. CONCLUSIONS: The technical function of subretinal visual implants can be measured objectively using a standard electroretinographic setup. CRIRs help the patient to optimise the perception by adjusting the gain and luminance range of the device and are a useful tool for clinicians to objectively assess the function of subretinal visual implants in vivo.

Electrophysiology and colour: a comparison of methods to evaluate inner retinal function.

PURPOSE: Several methods are routinely used in the clinic to diagnose and monitor diseases of inner retinal function. In this study, we compare four such methods in patients with diabetes and glaucoma, to determine correlations between their results and to determine which method is most sensitive for detecting disease. METHODS: Twenty control subjects, 12 patients with early glaucoma and eight patients with diabetes mellitus, were enrolled in the study. All underwent four examinations: transient pattern electroretinogram (PERG), multifocal pattern electroretinogram (mfPERG), chromatic contrast threshold measurements (protan and tritan), and blue-on-yellow short-wavelength automated perimetry (SWAP). RESULTS: For the total cohort of 40 subjects, the results show a significant correlation between the amplitudes of the PERG and those of the mfPERG, as well as between the tritan contrast thresholds and the SWAP MD. Furthermore, ROC analyses reveal that colour contrast thresholds could significantly distinguish between the patient and the control group. Glaucoma patients alone could also be distinguished. CONCLUSIONS: We conclude that the methods compared in this study show correlations between their results if they are testing same pathway or underling cells, and that the colour contrast threshold is the most sensitive method to detect early functional deficits in diabetic and glaucoma patients.

[Personalized molecular medicine: new paradigms in the treatment of cochlear implant and cancer patients]. / Molekulare personalisierte Medizin: Paradigmenwechsel in der Krankenversorgung von Cochlear-Implant- und Tumorpatienten.

OBJECTIVES: To evaluate present options for the indication of cochlear implants (CI) and new forms of treatment for head and neck cancer, melanomas and basal cell carcinomas, with emphasis on future perspectives. METHODS: A literature search was performed in the PubMed database. Search parameters were "personalized medicine", "individualized medicine" and "molecular medicine". RESULTS: Personalized medicine based on molecular-genetic evaluation of functional proteins such as otoferlin, connexin 26 and KCNQ4 or the Usher gene is becoming increasingly important for the indication of CI in the context of infant deafness. Determination of HER2/EGFR mutations in the epithelial growth factor receptor (EGFR) gene may be an important prognostic parameter for therapeutic decisions in head and neck cancer patients. In basal cell carcinoma therapy, mutations in the Hedgehog (PCTH1) and Smoothened (SMO) pathways strongly influence the indication of therapeutic Hedgehog inhibition, e.g. using small molecules. Analyses of c-Kit receptor, BRAF-600E and NRAS mutations are required for specific molecular therapy of metastasizing melanomas. The significant advances in the field of specific molecular therapy are best illustrated by the availability of the first gene therapeutic procedures for treatment of RPE65-induced infantile retinal degradation. CONCLUSION: The aim of personalized molecular medicine is to identify patients who will respond particularly positively or negatively (e.g. in terms of adverse side effects) to a therapy using the methods of molecular medicine. This should allow a specific therapy to be successfully applied or preclude its indication in order to avoid serious adverse side effects. This approach serves to stratify patients for adequate treatment.

Improvements for recording retinal function with Microelectrode Arrays.

A microelectrode array (MEA) is a configuration of multiple electrodes that enables the concurrent targeting of multiple sites for extracellular recording and stimulation. By utilizing light pulses or electrical stimulations, MEA recordings unveil the complex patterns of electrical activities that arise from the signaling processes within the retinal network. Here, we present a stepwise approach for using microelectrode arrays (MEAs) for recording action potentials from the mouse retina in response to electrical and light stimuli. We provide detailed techniques accompanied by description of a custom optical system, example recordings, troubleshooting guidelines, and data processing methods including spike sorting and code resources for analyzing light and electrical responses. The comprehensive nature of this paper aims to guide researchers in utilizing MEAs effectively for investigating retinal functionality. In particular, it can be easy to have a MEA experiment fail, but hard to identify the source of the failure. This paper is meant to demystify that process. It includes:•A description of MEA setup, recording, and spike data validation.•A troubleshooting guide for common failure modes in MEA recordings from mouse retina.•Spike detection and sorting to precisely extract distinctive action potential.

Electronic approaches to restitute vision in patients with neurodegenerative diseases of the retina.

Degenerations of the outer retina are hereditary diseases leading to significant loss of vision. Several concepts of active electrical stimulation of the remaining retinal network resulted in the development of retinal visual implants and prosthetic vision. Subretinal and epiretinal visual implants are currently the leading approaches in restoring functional vision in blind humans with retinitis pigmentosa or other outer retinal degenerations. This review gives a short overview about the principles, advantages, limitations and vision outcome of the up-to-date published artificial vision by electronic visual implants, as well as their known biocompatibility and safety issues.

Total colourblindness is caused by mutations in the gene encoding the alpha-subunit of the cone photoreceptor cGMP-gated cation channel.

Total colourblindness (OMIM 216900), also referred to as rod monochromacy (RM) or complete achromatopsia, is a rare, autosomal recessive inherited and congenital disorder characterized by photophobia, reduced visual acuity, nystagmus and the complete inability to discriminate between colours. Electroretinographic recordings show that in RM, rod photoreceptor function is normal, whereas cone photoreceptor responses are absent. The locus for RM has been mapped to chromosome 2q11 (ref. 2), however the gene underlying RM has not yet been identified. Recently, a suitable candidate gene, CNGA3, encoding the alpha-subunit of the cone photoreceptor cGMP-gated cation channel, a key component of the phototransduction pathway, has been cloned and assigned to human chromosome 2q11 (refs 3,4). We report the identification of missense mutations in CNGA3 in five families with RM. Homozygous mutations are present in two families, whereas the remaining families show compound heterozygous mutations. In all cases, the segregation pattern of the mutations is consistent with the autosomal recessive inheritance of the disease and all mutations affect amino acids that are highly conserved among cyclic nucleotide gated channels (CNG) in various species. This is the first report of a colour vision disorder caused by defects other than mutations in the cone pigment genes, and implies at least in this instance a common genetic basis for phototransduction in the three different cone photoreceptors of the human retina.

New views on RPE65 deficiency: the rod system is the source of vision in a mouse model of Leber congenital amaurosis.

Leber congenital amaurosis (LCA) is the most serious form of the autosomal recessive childhood-onset retinal dystrophies. Mutations in the gene encoding RPE65, a protein vital for regeneration of the visual pigment rhodopsin in the retinal pigment epithelium, account for 10-15% of LCA cases. Whereas previous studies of RPE65 deficiency in both animal models and patients attributed remaining visual function to cones, we show here that light-evoked retinal responses in fact originate from rods. For this purpose, we selectively impaired either rod or cone function in Rpe65-/- mice by generating double- mutant mice with models of pure cone function (rhodopsin-deficient mice; Rho-/-) and pure rod function (cyclic nucleotide-gated channel alpha3-deficient mice; Cnga3-/-). The electroretinograms (ERGs) of Rpe65-/- and Rpe65-/-Cnga3-/- mice were almost identical, whereas there was no assessable response in Rpe65-/-Rho-/- mice. Thus, we conclude that the rod system is the source of vision in RPE65 deficiency. Furthermore, we found that lack of RPE65 enables rods to mimic cone function by responding under normally cone-isolating lighting conditions. We propose as a mechanism decreased rod sensitivity due to a reduction in rhodopsin content to less than 1%. In general, the dissection of pathophysiological processes in animal models through the introduction of additional, selective mutations is a promising concept in functional genetics.

An L-type calcium-channel gene mutated in incomplete X-linked congenital stationary night blindness.

The locus for the incomplete form of X-linked congenital stationary night blindness (CSNB2) maps to a 1.1-Mb region in Xp11.23 between markers DXS722 and DXS255. We identified a retina-specific calcium channel alpha1-subunit gene (CACNA1F) in this region, consisting of 48 exons encoding 1966 amino acids and showing high homology to L-type calcium channel alpha1-subunits. Mutation analysis in 13 families with CSNB2 revealed nine different mutations in 10 families, including three nonsense and one frameshift mutation. These data indicate that aberrations in a voltage-gated calcium channel, presumably causing a decrease in neurotransmitter release from photoreceptor presynaptic terminals, are a frequent cause of CSNB2.

A gene (RPGR) with homology to the RCC1 guanine nucleotide exchange factor is mutated in X-linked retinitis pigmentosa (RP3).

X-linked retinitis pigmentosa (xlRP) is a severe progressive retinal degeneration which affects about 1 in 25,000 of the population. The most common form of xlRP, RP3, has been localised to the interval between CYBB and OTC in Xp21.1 by linkage analysis and deletion mapping. Identification of microdeletions within this region has now led to the positional cloning of a gene, RPGR, that spans 60 kg of genomic DNA and is ubiquitously expressed. The predicted 90 kD protein contains in its N-terminal half a tandem repeat structure highly similar to RCC1 (regulator of chromosome condensation), suggesting an interaction with a small GTPase. The C-terminal half contains a domain, rich in acidic residues, and ends in a potential isoprenylation anchorage site. The two intragenic deletions, two nonsense and three missense mutations within conserved domains provide evidence that RPGR (retinitis pigmentosa GTPase regulator) is the RP3 gene.

GPR98 mutations cause Usher syndrome type 2 in males.

Mutations in the large GPR98 gene underlie Usher syndrome type 2C (USH2C), and all patients described to date have been female. It was speculated that GPR98 mutations cause a more severe, and eventually lethal, phenotype in males. We describe for the first time two male patients with USH2 with novel GPR98 mutations. Clinical characterization of a male patient and his affected sister revealed a typical USH2 phenotype in both. GPR98 may have been excluded from systematic investigation in previous studies, and the proportion of patients with USH2C probably underestimated. GPR98 should be considered in patients with USH2 of both sexes.

[Subretinal visual implants]. / Subretinale visuelle Implantate.

Visual implants are medical technologies that replace parts of the visual neuronal pathway. The subretinal implant developed by our group is being used in a human trials since 2005 and replaces the function of degenerated photoreceptors by an electronic device in blind patients. The subretinal implant consists of a 70-µm thin microchip with 1500 microphotodiodes each with an amplifier and an electrode with area of 3 mm × 3 mm. The power supply is provided by a subdermal power supply cable. The microchip is implanted under the macula and transforms the light signal into an electrical one, which is referred directly to the bipolar cells. Requirements for a good function of the implant are a preserved function of the inner retina, as well as clear optic media and a good visual acuity in the earlier life. The current technology can mediate a visual field of 10 - 12° and a computed resolution of up to 0.25° visual angle (corresponding to a visual acuity of 63 / 1000 - 80 / 1000) in blind patients. The so far best results from our studies reached a visual acuity of 21 / 1000 in blind retinitis pigmentosa patients. This overview is intended to inform the ophthalmologist about the current state of the technology and help him/her to advise interested patients.

Enchancement of luminance flicker by color-opponent mechanisms.

Color-opponent ganglion cells in the monkey retina respond to luminance flicker at high temporal frequencies. Color opponency, which makes these cells so selective of wavelength at low temporal frequencies, is progressively lost at high frequencies. This loss is due to a frequency-dependent phase shift between the responses of spectrally different center and surround mechanisms in the receptive field of each of these cells. Center and surround responses, which are antagonistic at low temporal frequencies, become synergistic at high ones, making these cells most responsive at high frequencies to those wavelengths to which they are least responsive at low frequencies. This phenomenon can explain the differences between chromatic and luminance flicker in human vision.

Spike-triggered average electrical stimuli as input filters for bionic vision-a perspective.

Bionic retinal implants are gaining acceptance in the treatment of blindness from degenerative diseases including retinitis pigmentosa and macular degeneration. OBJECTIVE: A current obstacle to the improved performance of such implants is the difficulty of comparing the results of disparate experiments. Another obstacle is the current difficulty in selectively activating the many different retinal ganglion cell types that are used as separate pathways for visual information to the brain. APPROACH: To address these obstacles, we propose a modelling framework based on white noise stimulation and reverse correlation. In this perspective, we first outline early developments in visual retinal physiology leading up to the implementation of white noise stimuli and spike-triggered averaging. We then review recent efforts to adapt the white noise method for electrical stimulation of the retina and some of the nuances of this approach. MAIN RESULTS: Based on such white noise methods, we describe a modelling framework whereby the effect of any arbitrary electrical stimulus on a ganglion cell's neural code can be better understood. SIGNIFICANCE: This framework should additionally disentangle the effects of stimulation on photoreceptor, bipolar cell and retinal ganglion cell-ultimately supporting selective stimulation of specific ganglion cell types for a more nuanced bionic retinal implant. Finally, we point to upcoming considerations in this rapidly developing domain of research.