Phototransduction in retinal rods and cones: effects of temperature and background light, and an application for testing drug delivery

The photoreceptor cells of the vertebrate retina share a common morphological design and molecular scheme for phototransduction. Within this framework, there are great functional differences with respect to response amplification, kinetics, and adaptability to different mean light levels, first, between the two main classes of photoreceptors, rods and cones, and second, between different taxonomic groups. The present thesis analyses functional differences and similarities between i) mammalian and amphibian photoreceptors, and ii) rods and cones by studying effects of temperature on electrophysiological response properties. The research is based on characterisation of sensitivity and photoresponse kinetics in rods and cones of two mammals (rat and mouse), and two amphibians (frog and toad). Photoresponses to light pulses of incremental strength were recorded by the electroretinogram (ERG) technique across isolated aspartate-treated retinas at different temperatures in the range 2 - 37 °C, and at different levels of mean illumination. One objective was to investigate how the major functional differences between different vertebrate photoreceptors can be explained without assuming large differences in the properties of the phototransduction molecules. A general conclusion is that at the same temperature photoreceptors of mammals and amphibians exhibit similar functional properties. In rods, the remaining differences in the electrophysiological properties can largely be explained by differences in outer-segment size and morphology. In cones the picture is more complex due to the highly folded structure of the outer segment as well as the presence of thermal isomerizations of visual pigment, which may occur at a rate possibly high enough to 'light-adapt' cones in darkness. Another objective was to relate the capacity for temporal integration of dark-adapted rod photoreceptors to the integration time of vision and absolute visual sensitivity. A strong correlation was found between temporal integration in rods and in a visually guided behaviour of toads at different temperatures. The results allow the conclusion that temporal integration is mainly set by the rods and explains a considerable part of differences in absolute visual sensitivity between amphibians and mammals. The thesis also includes a project in which the aim was to develop a method where the vertebrate retina could be used as a biosensor for monitoring controlled drug release from temperature-sensitive polymeric carriers. The developed method enabled accurate concentration determinations of the model drug 3-isobutyl-1-methylxanthine (IBMX) based on the square root dependence of photoresponse kinetics on [IBMX] discovered in the work. Moreover, the biocompatibility of drug carriers can be assessed by the degree to which rods retain stable function in the presence of the carrier molecule, or its monomers

The retinal pigment epithelium displays electrical excitability and lateral signal spreading

Background: The non-neuronal retinal pigment epithelium (RPE) functions in intimate association with retinal photoreceptors, performing a multitude of tasks critical for maintaining retinal homeostasis and collaborating with retinal glial cells to provide metabolic support and ionic buffering. Accordingly, the RPE has recently been shown to display dynamic properties mediated by an array of ion channels usually more characteristic of astrocytes and excitable cells. The recent discovery of canonical voltage-activated Na+ channels in the RPE and their importance for phagocytosis of photoreceptor outer segments raises a question about their electrogenic function. Here, we performed a detailed electrophysiological analysis related to the functioning of these channels in human embryonic stem cell (hESC)-derived RPE. Results: Our studies examining the electrical properties of the hESC-RPE revealed that its membrane mainly displays passive properties in a broad voltage range, with the exception of depolarization-induced spikes caused by voltage-activated Na+ current (INa). Spike amplitude depended on the availability of INa and spike kinetics on the membrane time constant, and the spikes could be largely suppressed by TTX. Membrane resistance fluctuated rapidly and strongly, repeatedly changing over the course of recordings and causing closely correlated fluctuations in resting membrane potential. In a minority of cells, we found delayed secondary INa-like inward currents characterized by comparatively small amplitudes and slow kinetics, which produced secondary depolarizing spikes. Up to three consecutive delayed inward current waves were detected. These currents could be rapidly and reversibly augmented by applying L-type Ca2+ channel blocker nifedipine to diminish influx of calcium and thus increase gap junctional conductance. Conclusions: This work shows, for the first time, that INa and INa-mediated voltage spikes can spread laterally through gap junctions in the monolayer of cells that are traditionally considered non-excitable. Our findings support a potential role of the RPE that goes beyond giving homeostatic support to the retina.publishedVersionPeer reviewe

Light-Responsive Bilayer Cell Culture Platform for Reversible Cell Guidance

publishedVersionPeer reviewe

Chromophore supply rate-limits mammalian photoreceptor dark adaptation

Efficient regeneration of visual pigment following its destruction by light is critical for the function of mammalian photoreceptors. Here, we show that misexpression of a subset of cone genes in the rd7 mouse hybrid rods enables them to access the normally cone-specific retina visual cycle. The rapid supply of chromophore by the retina visual cycle dramatically accelerated the mouse rod dark adaptation. At the same time, the competition between rods and cones for retina-derived chromophore slowed cone dark adaptation, indicating that the cone specificity of the retina visual cycle is key for rapid cone dark adaptation. Our findings demonstrate that mammalian photoreceptor dark adaptation is dominated by the supply of chromophore. Misexpression of cone genes in rods may represent a novel approach to treating visual disorders associated with mutations of visual cycle proteins or with reduced retinal pigment epithelium function due to aging

Computational Model of Ca2+ Wave Propagation in Human Retinal Pigment Epithelial ARPE-19 Cells

Abstract Objective Computational models of calcium (Ca2+) signaling have been constructed for several cell types. There are, however, no such models for retinal pigment epithelium (RPE). Our aim was to construct a Ca2+ signaling model for RPE based on our experimental data of mechanically induced Ca2+ wave in the in vitro model of RPE, the ARPE-19 monolayer. Methods We combined six essential Ca2+ signaling components into a model: stretch-sensitive Ca2+ channels (SSCCs), P2Y2 receptors, IP3 receptors, ryanodine receptors, Ca2+ pumps, and gap junctions. The cells in our epithelial model are connected to each other to enable transport of signaling molecules. Parameterization was done by tuning the above model components so that the simulated Ca2+ waves reproduced our control experimental data and data where gap junctions were blocked. Results Our model was able to explain Ca2+ signaling in ARPE-19 cells, and the basic mechanism was found to be as follows: 1) Cells near the stimulus site are likely to conduct Ca2+ through plasma membrane SSCCs and gap junctions conduct the Ca2+ and IP3 between cells further away. 2) Most likely the stimulated cell secretes ligand to the extracellular space where the ligand diffusion mediates the Ca2+ signal so that the ligand concentration decreases with distance. 3) The phosphorylation of the IP3 receptor defines the cell’s sensitivity to the extracellular ligand attenuating the Ca2+ signal in the distance. Conclusions The developed model was able to simulate an array of experimental data including drug effects. Furthermore, our simulations predict that suramin may interfere ligand binding on P2Y2 receptors or accelerate P2Y2 receptor phosphorylation, which may partially be the reason for Ca2+ wave attenuation by suramin. Being the first RPE Ca2+ signaling model created based on experimental data on ARPE-19 cell line, the model offers a platform for further modeling of native RPE functions.Public Library of Science open acces

Light-Induced Nanoscale Deformation in Azobenzene Thin Film Triggers Rapid Intracellular Ca2+ Increase via Mechanosensitive Cation Channels

Epithelial cells are in continuous dynamic biochemical and physical interaction with their extracellular environment. Ultimately, this interplay guides fundamental physiological processes. In these interactions, cells generate fast local and global transients of Ca2+ ions, which act as key intracellular messengers. However, the mechanical triggers initiating these responses have remained unclear. Light-responsive materials offer intriguing possibilities to dynamically modify the physical niche of the cells. Here, a light-sensitive azobenzene-based glassy material that can be micropatterned with visible light to undergo spatiotemporally controlled deformations is used. Real-time monitoring of consequential rapid intracellular Ca2+ signals reveals that the mechanosensitive cation channel Piezo1 has a major role in generating the Ca2+ transients after nanoscale mechanical deformation of the cell culture substrate. Furthermore, the studies indicate that Piezo1 preferably responds to shear deformation at the cell-material interphase rather than to absolute topographical change of the substrate. Finally, the experimentally verified computational model suggests that Na+ entering alongside Ca2+ through the mechanosensitive cation channels modulates the duration of Ca2+ transients, influencing differently the directly stimulated cells and their neighbors. This highlights the complexity of mechanical signaling in multicellular systems. These results give mechanistic understanding on how cells respond to rapid nanoscale material dynamics and deformations.Peer reviewe

Rhodopsin kinase and arrestin binding control the decay of photoactivated rhodopsin and dark adaptation of mouse rods

Photoactivation of vertebrate rhodopsin converts it to the physiologically active Meta II (R*) state, which triggers the rod light response. Meta II is rapidly inactivated by the phosphorylation of C-terminal serine and threonine residues by G-protein receptor kinase (Grk1) and subsequent binding of arrestin 1 (Arr1). Meta II exists in equilibrium with the more stable inactive form of rhodopsin, Meta III. Dark adaptation of rods requires the complete thermal decay of Meta II/Meta III into opsin and all-trans retinal and the subsequent regeneration of rhodopsin with 11-cis retinal chromophore. In this study, we examine the regulation of Meta III decay by Grk1 and Arr1 in intact mouse rods and their effect on rod dark adaptation. We measure the rates of Meta III decay in isolated retinas of wild-type (WT), Grk1-deficient (Grk1(−/−)), Arr1-deficient (Arr1(−/−)), and Arr1-overexpressing (Arr1(ox)) mice. We find that in WT mouse rods, Meta III peaks ∼6 min after rhodopsin activation and decays with a time constant (τ) of 17 min. Meta III decay slows in Arr1(−/−) rods (τ of ∼27 min), whereas it accelerates in Arr1(ox) rods (τ of ∼8 min) and Grk1(−/−) rods (τ of ∼13 min). In all cases, regeneration of rhodopsin with exogenous 11-cis retinal is rate limited by the decay of Meta III. Notably, the kinetics of rod dark adaptation in vivo is also modulated by the levels of Arr1 and Grk1. We conclude that, in addition to their well-established roles in Meta II inactivation, Grk1 and Arr1 can modulate the kinetics of Meta III decay and rod dark adaptation in vivo

Ultrathin Polyimide Membrane as Cell Carrier for Subretinal Transplantation of Human Embryonic Stem Cell Derived Retinal Pigment Epithelium

In this study, we investigated the suitability of ultrathin and porous polyimide (PI) membrane as a carrier for subretinal transplantation of human embryonic stem cell (hESC) -derived retinal pigment epithelial (RPE) cells in rabbits. The in vivo effects of hESC-RPE cells were analyzed by subretinal suspension injection into Royal College of Surgeons (RCS) rats. Rat eyes were analyzed with electroretinography (ERG) and histology. After analyzing the surface and permeability properties of PI, subretinal PI membrane transplantations with and without hESC-RPE were performed in rabbits. The rabbits were followed for three months and eyes analyzed with fundus photography, ERG, optical coherence tomography (OCT), and histology. Animals were immunosuppressed with cyclosporine the entire follow-up time. In dystrophic RCS rats, ERG and outer nuclear layer (ONL) thickness showed some rescue after hESC-RPE injection. Cells positive for human antigen were found in clusters under the retina 41 days post-injection but not anymore after 105 days. In rabbits, OCT showed good placement of the PI. However, there was loss of pigmentation on the hESC-RPE-PI over time. In the eyes with PI alone, no obvious signs of inflammation or retinal atrophy were observed. In the presence of hESC-RPE, mononuclear cell infiltration and retinal atrophy were observed around the membranes. The porous ultrathin PI membrane was well-tolerated in the subretinal space and is a promising scaffold for RPE transplantation. However, the rejection of the transplanted cells seems to be a major problem and the given immunosuppression was insufficient for reduction of xenograft induced inflammation.Public Library of Science open acces

Menetelmä mallilääkeaineen vapaan pitoisuuden mittaamiseksi nisäkkään verkkokalvossa

Funktionaalisten polymeerien avulla pyritään annostelemaan lääkeainetta elimistöön kontrolloidusti tietyllä hetkellä ja tietyllä aikaskaalalla. Polymeerirakenteisiin sidottu lääkeaine vapautetaan polymeeriä ympäröivään liuokseen esimerkiksi lämpötilan avulla. Lääkeainevapautusmenetelmiä kehitettäessä on tärkeää pystyä määrittämään lääkeaineen vapaa pitoisuus liuoksessa hyvin tarkasti. Erityisen tärkeää on lääkeaineen vapaan pitoisuuden määrittäminen elävässä kudoksessa. Tämän diplomityön tavoitteena oli kehittää menetelmä mallilääkeaineen vapaan pitoisuuden mittaamiseksi nisäkkään hermokudoksessa eri lämpötiloissa. Lisäksi pyrkimyksenä oli selvittää, miten erilaisten mono- ja polymeerien bioyhteensopivuutta voitaisiin testata luotettavasti. Työssä kehitetty menetelmä perustuu nisäkkään verkkokalvon näköaistinsolujen valovastekinetiikan monotoniseen riippuvuuteen mallilääkeaineen (3-isobutyyli-1-metyyliksantiini, IBMX) pitoisuudesta. Valovastekinetiikassa tapahtuvia muutoksia seurattiin elektroretinogrammitekniikalla (ERG). Preparaattina käytettiin rotan (Rattus norvegicus) eristettyä verkkokalvoa. Työssä havaittiin, että rotan sauvasolujen lineaarisen alueen valovasteiden kinetiikka on suoraan verrannollinen IBMX-konsentraation neliöjuureen. Kalibraatiokäyrän (IBMX:n pitoisuus valovastekinetiikan funktiona) avulla pystytään määrittämään polymeerirakenteista vapautuneen IBMX:n pitoisuus rotan verkkokalvossa pitoisuusalueella 3-300 µM. Työssä todettiin myös, että rotan verkkokalvoa voidaan käyttää erilaisten molekyylien bioyhteensopivuuden testaamiseen, sillä molekyylien mahdolliset vähäisetkin haittavaikutukset näkyvät muutoksina sauvasolujen valovasteiden amplitudissa, herkkyydessä ja/tai kinetiikassa. Tämä diplomityö osoittaa, kuinka nisäkkään verkkokalvoa voidaan käyttää hyvin herkkänä biosensorina. Työssä kehitetty menetelmä tullaan esittämään kansainvälisessä sarjajulkaisussa