Programmable retinal dynamics in a CMOS mixed-signal array processor chip

The low-level image processing that takes place in the retina is intended to compress the relevant visual information to a manageable size. The behavior of the external layers of the biological retina has been successfully modelled by a Cellular Neural Network, whose evolution can be described by a set of coupled nonlinear differential equations. A mixed-signal VLSI implementation of the focal-plane low-level image processing based upon this biological model constitutes a feasible and cost effective alternative to conventional digital processing in real-time applications. For these reasons, a programmable array processor prototype chip has been designed and fabricated in a standard 0.5μm CMOS technology. The integrated system consists of a network of two coupled layers, containing 32 × 32 elementary processors, running at different time constants. Involved image processing algorithms can be programmed on this chip by tuning the appropriate interconnections weights. Propagative, active wave phenomena and retina-like effects can be observed in this chip. Design challenges, trade-offs, the buildings blocks and some test results are presented in this paper.Office of Naval Research (USA) N00014-00-10429European Community IST-1999-19007Ministerio de Ciencia y Tecnología TIC1999-082

Disruption in murine Eml1 perturbs retinal lamination during early development.

During mammalian development, establishing functional neural networks in stratified tissues of the mammalian central nervous system depends upon the proper migration and positioning of neurons, a process known as lamination. In particular, the pseudostratified neuroepithelia of the retina and cerebrocortical ventricular zones provide a platform for progenitor cell proliferation and migration. Lamination defects in these tissues lead to mispositioned neurons, disrupted neuronal connections, and abnormal function. The molecular mechanisms necessary for proper lamination in these tissues are incompletely understood. Here, we identified a nonsense mutation in the Eml1 gene in a novel murine model, tvrm360, displaying subcortical heterotopia, hydrocephalus and disorganization of retinal architecture. In the retina, Eml1 disruption caused abnormal positioning of photoreceptor cell nuclei early in development. Upon maturation, these ectopic photoreceptors possessed cilia and formed synapses but failed to produce robust outer segments, implying a late defect in photoreceptor differentiation secondary to mislocalization. In addition, abnormal positioning of Müller cell bodies and bipolar cells was evident throughout the inner neuroblastic layer. Basal displacement of mitotic nuclei in the retinal neuroepithelium was observed in tvrm360 mice at postnatal day 0. The abnormal positioning of retinal progenitor cells at birth and ectopic presence of photoreceptors and secondary neurons upon maturation suggest that EML1 functions early in eye development and is crucial for proper retinal lamination during cellular proliferation and development

A Novel Rhodopsin Gene from Octopus vulgaris for Optobioelectronics Materials

The unique photochromic retinal protein from rhabdomeric octopus membranes \u2013 octopus rhodopsin (OctR) has emerged as promising material for biomolecular photonic applications due to its unique properties and advantages. Here we report isolation of the novel full length octR gene from retina cDNA of Octopus vulgaris eyes and its sequence comparison with rhodopsins of other cephalopods and vertebrates. The isolated gene can be used to develop various expression systems for production of recombinant OctR for structural studies and novel optobioelectronic applications. The alignment of amino acid (a.a.) sequence with different opsins revealed similarity to cephalopoda rhodopsins (Rho) and to human melanopsin from intrinsically photosensitive retinal ganglion cells. The alingment of OctR a.a. sequence with mammalian and cephalopoda Rho with known 3D structures revealed promising substitutions V2C and W292C for developing stable and functionally active recombinant OctR after heterologous expression

High accuracy decoding of dynamical motion from a large retinal population

Motion tracking is a challenge the visual system has to solve by reading out the retinal population. Here we recorded a large population of ganglion cells in a dense patch of salamander and guinea pig retinas while displaying a bar moving diffusively. We show that the bar position can be reconstructed from retinal activity with a precision in the hyperacuity regime using a linear decoder acting on 100+ cells. The classical view would have suggested that the firing rates of the cells form a moving hill of activity tracking the bar's position. Instead, we found that ganglion cells fired sparsely over an area much larger than predicted by their receptive fields, so that the neural image did not track the bar. This highly redundant organization allows for diverse collections of ganglion cells to represent high-accuracy motion information in a form easily read out by downstream neural circuits.Comment: 23 pages, 7 figure

Identification of deubiquitinating enzyme genes relevant for the regulation of retina-specific genes

[eng] Protein post-translational modifications are regulatory mechanisms that cells use in response to intra- and extracellular signals. These signals modulate a panoply of conjugating enzymes that modify proteins post-translationally by the conjugation of a small functional group or peptide. The consequences of these modifications are very diverse, but they all present a common feature: they shift protein fate, localization or function. Besides, in the case of covalent protein modifications, such as ubiquitination, these regulatory mechanisms are reversible and dynamic. In mammals, only two E1 and around thirty E2 ligases have been described to participate in the ubiquitin cycle, in contrast to the approximately 600 E3 ligases identified. Note that this is a highly dynamic process and thus, cells also contain a group of deubiquitinating enzymes (DUBs), responsible for detaching ubiquitin from its substrates. DUBs process the ubiquitin precursor that has been transcribed from several genes as fusion proteins; deubiquitinate substrates and rescue them from protein degradation; and finally, they recycle Ub molecules from proteins committed to be degraded20. DUBs play important roles in disease and cellular processes; however and despite its evident importance in the organism, data on their mode of regulation and substrate specificity is still scarce21 The retina is the part of the eye responsible for capturing light stimuli from our surroundings. It is a neuronal tissue located at the posterior part of the eye, which captures light photons, converts the luminic energy into electrochemical stimuli and finally sends visual information to the brain where it is integrated. Vertebrate retina is formed by seven neuronal cell types organized in six precise functional and structural layers. The main function of the retina, the phototransduction, is carried out by two types of photoreceptor cells (PhR), cones and rods (see below). Photoreceptors are highly specialized cellular types, which differ both structurally and functionally, but that develop from the same photoreceptor progenitor precursor. Therefore, there must be a fine and tight genetic control to successfully achieve a properly structured and functional tissue. The regulation by photoreceptor-specific transcription factors has been amply described1,35,36; however, it is not their bare action that determines photoreceptor fate, since SUMO conjugation –and possibly other post-tranlational modifications, such ubiquitination– of TFs play a key role in this process37 In mammals, several comprehensive surveys of DUBs have been reported resulting in: in silico inventories of the DUBs in the human genome20,21; identification of protein interactors by cell-based proteomics analysis45; studies of subcellular localization46; functional involvement in maintaining genome integrity47. However, detailed expression and functional analysis for most DUBs on particular tissues or organs, such as the retina, is still missing. For these reasons, this work is intended as a study of deubiquitinating enzymes in the retina and their possible role in photoreceptor development and homeostasis. To that end, several objectives have been set and reached: 1. Analysis of the involvement of deubiquitinating enzymes (DUBs) in the mouse retina: I. Analysis of mRNA expression and pattern of expression via Real Time qPCR and in situ hybridization. II. Determination of the protein localization via fluorescent immunohistochemistry. III. Sequence and functional conservation analysis through phylogenetic and phenotypic studies. IV. Transcriptomic analysis of DUBs’ mRNA expression in the retina during developmentPreliminary analysis of the functional role of selected DUBs in mouse retinal development, via in vivo gene knockdown. 2. Devising an in-vivo cell system to study the role of DUB enzymes on the regulation of retinal promoters: I. Establishing a cell culture system of study II. Knockdown of DUB genes via shRNA and siRNA silencing techniques. 3. Identification of CRX post-translational modifications, particularly ubiquitynation.[spa] Las modificaciones post-traduccionales de proteínas son mecanismos de regulación que las células utilizan en respuestas a intra y extracelulares. En el caso de las modificaciones covalentes con proteínas, como lo es la ubiquitinación, estos mecanismos de regulación son reversibles y dinámicos. La ubiquitinación se hace reversible por la acción de las enzimas deubiquitinantes (DUBs), encargados de hidrolizar el enlace entre la ubiquitina y sus proteínas sustrato. Las DUBs juegan importantes roles en enfermedades y procesos celulares, no obstante, todavía hay poca información sobre su implicación y regulación de tejidos específicos, como la retina. La retina órgano de la visión responsable de capturar estímulos lumínicos, convertirlos en estímulos electroquímicos y finalmente mandar esta información hacia el córtex visual. Existen dos tipos de fotoreceptores, los conos y los bastones, que difieren funcional y estructuralmente, pero que provienen de una misma célula precursora. Se ha descrito que esta estricta diferenciación se lleva a cabo gracias a la acción combinada de factores de transcripción específicos junto a las modificaiones post-traduccionales, como lo és la molecula ubiquitin-like, SUMO. El presente trabajo pretende analizar la acción de los enzimas deubiquitinantes en el desarrollo de la retina y los fotoreceptores. Para ello, se ha llevado a cabo una primera descripción de los niveles y el patrón de expresión de estos enzimas en la retina; así como un anàlisis transcriptómico en diferentes estadíos de desarrollo retinal murino y humano. Además, se ha realizado un estudio de su conservación evolutiva y su implicación en fenotipos neuronales y de retina. Asimismo, se ha puesto a punto un sistema celular que replica condiciones fisiológicas similares a las retinales y se ha realizado un análisis de silenciamiento de los genes que codifican para las DUBs utilizando shRNA, siRNA y Gapmers. Finalmente, se ha realizado un análisis de las posibles modificaciones post-traduccionales en el factor de transcripción retinal CRX

Functional Properties of Visual Pigments using A1 and A2 Chromophore : From Molecules to Ecology

The first event in vision is the absorption of a photon by a visual pigment molecule in a retinal photoreceptor cell. Activation of the molecule triggers a chemical amplification cascade, which finally leads to a change in the membrane potential of the cell. However, a visual pigment molecule may also be spontaneously activated by thermal energy. The resulting electrical response is identical to that caused by a photon. Such false light signals form a background noise limiting the detection of dim light. The absorption spectrum of a visual pigment (its ability to use different wavelengths of light) and its propensity for thermal activation both depend on the minimum amount of energy required for activation (the activation energy Ea). These properties of the pigment can be tuned on an evolutionary time scale by changes in the amino acid sequence of the protein part (the opsin) or on a physiological time scale by changing the light-sensitive cofactor bound to the opsin, the chromophore. The latter option is accessible only to poikilothermic vertebrates having two alternative chromophores (retinal A1 and A2). In this thesis, functional consequences of the A1-A2 exchange were investigated. In the first part, the relation between the changes of the absorption spectrum and the activation energy was quantitatively measured in several species of amphibians and fishes using both chromophores. The A2-induced shift of the absorption spectrum towards longer wavelengths was found always to correlate with a decrease in Ea. Later investigations have confirmed that decreasing Ea increases the rate of thermal activations. Thus the switch from A1 to A2 in the same opsin gives a more red-sensitive but noisier pigment. Against this background, the second part of the thesis investigates chromophore usage in eight populations of nine-spined sticklebacks (Pungitius pungitius) from different light environments. The amino acid sequence of the rods was found to be identical in all populations, implying that variations in spectral sensitivity depended only on the A1:A2 ratios. The cone absorption spectra also suggested that the variation within each cone class was due to varying chromophore proportions alone. The differences between populations could not be consistently explained as adaptations to the different light environments. However, an important and quite unexpected result was that the same individual could have quite different chromophore proportions in rods and cones (more A2 in cones). This shows that there are mechanisms by which chromophore proportions in different photoreceptors can be regulated much more selectively than previously thought. Since pigment noise is sensitivity-limiting mainly in dim light, it may be suggested that cones (working mainly in brighter light) can better afford using the noisy A2 chromophore to shift their spectral sensitivities for a better match to a long-wavelength photic environment.Näkötapahtuma alkaa, kun verkkokalvon fotoreseptorisoluissa sijaitseva näköpigmenttimolekyyli absorboi fotonin. Molekyylin aktivoituminen käynnistää kemiallisen vahvistusketjun, jonka lopputuloksena solun kalvojännite muuttuu. Näköpigmenttimolekyyli voi kuitenkin aktivoitua myös spontaanisti lämpöenergian vaikutuksesta (termisesti), synnyttäen sähköisen vasteen joka on täysin samanlainen kuin fotonin aiheuttama. Tällaiset väärät valosignaalit muodostavat taustakohinan, joka rajoittaa heikkojen valojen havaitsemista. Näköpigmentin absorptiospektri (sen kyky käyttää valon eri aallonpituuksia) ja sen taipumus aktivoitua termisesti riippuvat molemmat aktivaation vaatimasta minimienergiamäärästä (ns. aktivaatioenergiasta Ea). Pigmentin ominaisuuksia voidaan säätää joko evolutiivisella aikaskaalalla proteiiniosan (opsiinin) aminohapposekvenssiä muuttamalla tai fysiologisella aikaskaalalla opsiiniin sidotun valoherkän kofaktorin, ns. kromoforin, vaihdolla. Jälkimmäinen optio on vain vaihtolämpöisillä selkärankaisilla, joilla on käytössään kaksi vaihtoehtoista kromoforia (retinaali A1 ja A2). Tässä väitöskirjassa tutkittiin A1-A2-vaihdon funktionaalisia seurauksia. Ensimmäisessä osassa mitattiin kvantitatiivisesti absorptiospektrin ja aktivaatioenergian muutosten suhdetta useilla sammakko- ja kalalajeilla. Todettiin että A2:een liittyvä absorptiospektrin siirtyminen pitempiin aallonpituuksiin korreloi aina Ea:n laskun kanssa. Myöhemmät tutkimukset ovat vahvistaneet, että Ea:n alentaminen lisää termisten aktivaatioiden määrää. A1-kromoforin vaihtaminen A2:een samassa opsiinissa antaa siis punaherkemmän mutta kohinaisemman pigmentin. Tätä taustaa vasten väitöskirjan toisessa osassa tutkittiin kromoforin käyttöä kahdeksassa, eri valoympäristöissä elävässä kymmenpiikkipopulaatiossa (Pungitius pungitius). Sauvasolujen opsiinien aminohapposekvenssi todettiin identtiseksi kaikissa populaatioissa, joten spektraaliherkkyyden vaihtelu johtui yksinomaan vaihtelevista A1:A2 suhteista. Myös tappisolujen absorptiospektrit viittasivat siihen, että kunkin tappiluokan sisäinen vaihtelu johtui vain kromoforisuhteista. Populaatioiden välisiä eroja ei pystytty johdonmukaisesti selittämään adaptaatioina eri valoympäristöihin. Sen sijaan tärkeä ja täysin odottamaton tulos oli, että saman yksilön sauvoissa ja tapeissa saattoi olla aivan eri kromoforisuhteet (tapeissa enemmän A2). Tämä osoittaa, että on mekanismeja joilla eri reseptoreiden kromoforisuhteita voidaan säätää paljon yksilöidymmin kuin on tiedetty. Koska pigmenttikohina rajoittaa näön herkkyyttä lähinnä heikossa valossa, voidaan ajatella, että nimenomaan tappien spektraaliherkkyyksiä on varaa siirtää A2:lla paremmin vastaamaan keltaisen järven valospektriä, ilman että kohinasta johtuva hinta on liian korkea

Translational Retinal Research and Therapies.

The following review summarizes the state of the art in representative aspects of gene therapy/translational medicine and evolves from a symposium held at the School of Veterinary Medicine, University of Pennsylvania on November 16, 2017 honoring Dr. Gustavo Aguirre, recipient of ARVO's 2017 Proctor Medal. Focusing on the retina, speakers highlighted current work on moving therapies for inherited retinal degenerative diseases from the laboratory bench to the clinic