Physiological Features of the S- and M-cone Photoreceptors of Wild-type Mice from Single-cell Recordings

Cone cells constitute only 3% of the photoreceptors of the wild-type (WT) mouse. While mouse rods have been thoroughly investigated with suction pipette recordings of their outer segment membrane currents, to date no recordings from WT cones have been published, likely because of the rarity of cones and the fragility of their outer segments. Recently, we characterized the photoreceptors of Nrl−/− mice, using suction pipette recordings from their “inner segments” (perinuclear region), and found them to be cones. Here we report the use of this same method to record for the first time the responses of single cones of WT mice, and of mice lacking the α-subunit of the G-protein transducin (Gtα−/−), a loss that renders them functionally rodless. Most cones were found to functionally co-express both S- (λmax = 360 nm) and M- (λmax = 508 nm) cone opsins and to be maximally sensitive at 360 nm (“S-cones”); nonetheless, all cones from the dorsal retina were found to be maximally sensitive at 508 nm (“M-cones”). The dim-flash response kinetics and absolute sensitivity of S- and M-cones were very similar and not dependent on which of the coexpressed cone opsins drove transduction; the time to peak of the dim-flash response was ∼70 ms, and ∼0.2% of the circulating current was suppressed per photoisomerization. Amplification in WT cones (A ∼4 s−2) was found to be about twofold lower than in rods (A ∼8 s−2). Mouse M-cones maintained their circulating current at very nearly the dark adapted level even when >90% of their M-opsin was bleached. S-cones were less tolerant to bleached S-opsin than M-cones to bleached M-opsin, but still far more tolerant than mouse rods to bleached rhodopsin, which exhibit persistent suppression of nearly 50% of their circulating current following a 20% bleach. Thus, the three types of mouse opsin appear distinctive in the degree to which their bleached, unregenerated opsins generate “dark light.

Photoreceptors of Nrl −/− Mice Coexpress Functional S- and M-cone Opsins Having Distinct Inactivation Mechanisms

The retinas of mice null for the neural retina leucine zipper transcription factor (Nrl −/−) contain no rods but are populated instead with photoreceptors that on ultrastructural, histochemical, and molecular criteria appear cone like. To characterize these photoreceptors functionally, responses of single photoreceptors of Nrl −/− mice were recorded with suction pipettes at 35–37°C and compared with the responses of rods of WT mice. Recordings were made either in the conventional manner, with the outer segment (OS) drawn into the pipette (“OS in”), or in a novel configuration with a portion of the inner segment drawn in (“OS out”). Nrl −/− photoreceptor responses recorded in the OS-out configuration were much faster than those of WT rods: for dim-flash responses tpeak = 91 ms vs. 215 ms; for saturating flashes, dominant recovery time constants, τD = 110 ms vs. 240 ms, respectively. Nrl −/− photoreceptors in the OS-in configuration had reduced amplification, sensitivity, and slowed recovery kinetics, but the recording configuration had no effect on rod response properties, suggesting Nrl −/− outer segments to be more susceptible to damage. Functional coexpression of two cone pigments in a single mammalian photoreceptor was established for the first time; the responses of every Nrl −/− cell were driven by both the short-wave (S, λmax ≈ 360 nm) and the mid-wave (M, λmax ≈ 510 nm) mouse cone pigment; the apparent ratio of coexpressed M-pigment varied from 1:1 to 1:3,000 in a manner reflecting a dorso-ventral retinal position gradient. The role of the G-protein receptor kinase Grk1 in cone pigment inactivation was investigated in recordings from Nrl −/−/Grk1−/− photoreceptors. Dim-flash responses of cells driven by either the S- or the M-cone pigment were slowed 2.8-fold and 7.5-fold, respectively, in the absence of Grk1; the inactivation of the M-pigment response was much more seriously retarded. Thus, Grk1 is essential to normal inactivation of both S- and M-mouse cone opsins, but S-opsin has access to a relatively effective, Grk1-independent inactivation pathway

Restoration of Visual Function by Expression of a Light-Gated Mammalian Ion Channel in Retinal Ganglion Cells or ON-Bipolar Cells

Most inherited forms of blindness are caused by mutations that lead to photoreceptor cell death but spare second- and third-order retinal neurons. Expression of the light-gated excitatory mammalian ion channel light-gated ionotropic glutamate receptor (LiGluR) in retinal ganglion cells (RGCs) of the retina degeneration (rd1) mouse model of blindness was previously shown to restore some visual functions when stimulated by UV light. Here, we report restored retinal function in visible light in rodent and canine models of blindness through the use of a second-generation photoswitch for LiGluR, maleimide-azobenzene-glutamate 0 with peak efficiency at 460 nm (MAG0460). In the blind rd1 mouse, multielectrode array recordings of retinal explants revealed robust and uniform light-evoked firing when LiGluR-MAG0460 was targeted to RGCs and robust but diverse activity patterns in RGCs when LiGluR-MAG0460 was targeted to ON-bipolar cells (ON-BCs). LiGluR-MAG0460 in either RGCs or ON-BCs of the rd1 mouse reinstated innate light-avoidance behavior and enabled mice to distinguish between different temporal patterns of light in an associative learning task. In the rod-cone dystrophy dog model of blindness, LiGluR-MAG0460 in RGCs restored robust light responses to retinal explants and intravitreal delivery of LiGluR and MAG0460 was well tolerated in vivo. The results in both large and small animal models of photoreceptor degeneration provide a path to clinical translation

ОЦЕНКА ВЕЛИЧИН СЕЙСМИЧЕСКИХ ВОЗДЕЙСТВИЙ ПО НАРУШЕНИЯМ И СМЕЩЕНИЯМ В СКАЛЬНЫХ МАССИВАХ

Local rock dislocations and shifts, which occur due to strong seismic impacts, are studied. Natural cases under review include displacements that occurred after strong earthquakes in the 20th century and paleoseismic dislocations revealed by the authors in the crustal rocks in the south-eastern (Russian) part of the Fennoscandia shield. The major goal of the study was to determine parameters of the paleoseismic events in terms of intensity, I, and magnitude, M. Two independent options were used: (1) estimation with reference to similar cases in the instrumental measurements period and to the currently applied scaling systems, and (2) introduction of physical characteristics of disturbances of particular types and habitus and fixed values of displacements of the rock blocks. Numerous local disturbances of the rock massifs with significant rock shifts were systematically reviewed as a set of standard models. Values of mass velocities of seismic impacts (peak ground velocities, PGV), which were needed for initiation of the revealed dislocations, were estimated. In many cases, PGV values were above 1 m/sec, i.e. considerably higher than values conventionally accepted (for ground conditions). For clarifications, data on strong movements and explosions were used, and the whole set of data was found reasonably consistent. It was concluded that the cases with PGV>1 m/sec corresponded to focal areas of earthquakes with M>6 (mainly in the deglaciation period). A graphical chart is proposed for estimation of magnitudes and hypocentral distances of initiating earthquakes in case of maximum PGV within a range from 0.01 to 5.00 m/sec. Based on the graphical chart, parameters are estimated for earthquakes that might have caused the dislocations observed on the main sites of the region under study.В работе рассматриваются локальные нарушения и смещения в скальных породах, возникающие при сильных сейсмических воздействиях. Натурный материал представлен примерами такого рода нарушений при известных сильных землетрясениях XX века, а также древними нарушениями и смещениями на ключевых участках юго-восточной части Фенноскандинавского кристаллического щита (по наблюдениям авторов). Конечная цель состояла в параметризации (в терминах интенсивности, I, и магнитуды, M) палеоземлетрясений по оставленным ими специфическим сейсмонарушениям. Это осуществлялось двумя независимыми путями, а именно, c использованием аналогий других инструментальных наблюдений, а также существующих шкал (1) и путем выведения физических характеристик нарушений определенного типа, габитуса и фиксированных величин смещений скальных блоков (2). Систематизация многочисленных локальных нарушений скальных массивов со значительными смещениями блоков породы представлена в виде ряда типовых моделей. Оценены значения массовых скоростей импульсных воздействий, необходимых для инициирования наблюденных смещений. Во многих случаях они превысили 1 м/с, т.е. оказались существенно больше обычно принимаемых для грунтовых условий. Для объяснения привлечены данные по сильным движениям и по взрывам и показано согласие всего этого комплекса данных. Делается вывод о том, что случаи массовых скоростей >1 м/с соответствуют эпицентральным областям землетрясений с магнитудой M>6 (в основном в период дегляциации). Предложена номограмма для оценки магнитуды и гипоцентрального расстояния инициирующего землетрясения при значениях максимальных массовых скоростей в диапазоне от 0.01 до 5.00 м/с. На основании номограммы оценены параметры землетрясений, которые могли вызвать наблюденные перемещения скальных блоков на ключевых участках региона

Cone-like morphological, molecular, and electrophysiological features of the photoreceptors of the Nrl knockout mouse

PURPOSE. To test the hypothesis that Nrl Ϫ/Ϫ photoreceptors are cones, by comparing them with WT rods and cones using morphological, molecular, histochemical, and electrophysiological criteria. METHODS. The photoreceptor layer of fixed retinal tissue of 4-to 6-week-old mice was examined in plastic sections by electron microscopy, and by confocal microscopy in frozen sections immunolabeled for the mouse UV-cone pigment and colabeled with PNA. Quantitative immunoblot analysis was used to determine the levels of expression of key cone-specific proteins. Single-and paired-flash methods were used to extract the spectral sensitivity, kinetics, and amplification of the awave of the ERG. RESULTS. Outer segments of Nrl Ϫ/Ϫ photoreceptors (ϳ7 m) are shorter than those of wild-type (WT) rods (ϳ25 m) and cones (ϳ15 m); but, like WT cones, they have 25 or more basal discs open to the extracellular space, extracellular matrix sheaths stained by PNA, chromatin "clumping" in their nuclei, and mitochondria two times shorter than rods. Nrl Ϫ/Ϫ photoreceptors express the mouse UV cone pigment, cone transducin, and cone arrestin in amounts expected, given the relative size and density of cones in the two retinas. The ERG a-wave was used to assay the properties of the photocurrent response. The sensitivity of the Nrl -/-a-wave is at its maximum at 360 nm, with a secondary mode at 510 nm having approximately one-tenth the maximum sensitivity. These wavelengths are the max of the two mouse cone pigments. The time to peak of the dim-flash photocurrent response was ϳ50 ms, more than two times faster than that of rods. CONCLUSIONS. Many morphological, molecular, and electrophysiological features of the Nrl Ϫ/Ϫ photoreceptors are cone-like, and strongly distinguish these cells from rods. This retina provides a model for the investigation of cone function and cone-specific genetic disease. (Invest Ophthalmol Vis Sci

Photochemical Restoration of Light Sensitivity in the Degenerated Canine Retina

Photopharmacological compounds such as azobenzene-based photoswitches have been shown to control the conductivity of ionic channels in a light-dependent manner and are considered a potential strategy to restore vision in patients with end-stage photoreceptor degeneration. Here, we report the effects of DENAQ, a second-generation azobenzene-based photoswitch on retinal ganglion cells (RGC) in canine retinas using multi-electrode array (MEA) recordings (from nine degenerated and six WT retinas). DENAQ treatment conferred increased light sensitivity to RGCs in degenerated canine retinas. RGC light responses were observed in degenerated retinas following ex vivo application of 1 mM DENAQ (n = 6) or after in vivo DENAQ injection (n = 3, 150 μL, 3–10 mM) using 455 nm light at intensities as low as 0.2 mW/cm2. The number of light-sensitive cells and the per cell response amplitude increased with light intensity up to the maximum tested intensity of 85 mW/cm2. Application of DENAQ to degenerated retinas with partially preserved cone function caused appearance of DENAQ-driven responses both in cone-driven and previously non-responsive RGCs, and disappearance of cone-driven responses. Repeated stimulation slowed activation and accelerated recovery of the DENAQ-driven responses. The latter is likely responsible for the delayed appearance of a response to 4 Hz flicker stimulation. Limited aqueous solubility of DENAQ results in focal drug aggregates associated with ocular toxicity. While this limits the therapeutic potential of DENAQ, more potent third-generation photoswitches may be more promising, especially when delivered in a slow-release formulation that prevents drug aggregation

Theory of Formation of Intensive Quasi-Stationary Electronic Beams in Gas-Filled Systems

Available from VNTIC / VNTIC - Scientific & Technical Information Centre of RussiaSIGLERURussian Federatio

SEISMIC IMPACT ESTIMATION FROM DATA ON DEFORMATIONS AND DISPLACEMENTS IN ROCK MASSIFS

Local rock dislocations and shifts, which occur due to strong seismic impacts, are studied. Natural cases under review include displacements that occurred after strong earthquakes in the 20th century and paleoseismic dislocations revealed by the authors in the crustal rocks in the south-eastern (Russian) part of the Fennoscandia shield. The major goal of the study was to determine parameters of the paleoseismic events in terms of intensity, I, and magnitude, M. Two independent options were used: (1) estimation with reference to similar cases in the instrumental measurements period and to the currently applied scaling systems, and (2) introduction of physical characteristics of disturbances of particular types and habitus and fixed values of displacements of the rock blocks. Numerous local disturbances of the rock massifs with significant rock shifts were systematically reviewed as a set of standard models. Values of mass velocities of seismic impacts (peak ground velocities, PGV), which were needed for initiation of the revealed dislocations, were estimated. In many cases, PGV values were above 1 m/sec, i.e. considerably higher than values conventionally accepted (for ground conditions). For clarifications, data on strong movements and explosions were used, and the whole set of data was found reasonably consistent. It was concluded that the cases with PGV>1 m/sec corresponded to focal areas of earthquakes with M>6 (mainly in the deglaciation period). A graphical chart is proposed for estimation of magnitudes and hypocentral distances of initiating earthquakes in case of maximum PGV within a range from 0.01 to 5.00 m/sec. Based on the graphical chart, parameters are estimated for earthquakes that might have caused the dislocations observed on the main sites of the region under study

3-D COUPLED SIMULATION OF A VVER 1000 WITH PARCS/ATHLET

A new OECD/NEA benchmark entitled “Reactivity compensation with diluted boron by stepwise insertion of control rod cluster” is starting. This benchmark, based on high quality measurements performed at the NPP Rostov Unit 2, aims to validate and assess high fidelity multi-physics simulation code capabilities. The Benchmark is divided in two phases: assembly wise and pin-by-pin resolution of steady-state and transient multi-physics problems. Multi-physics simulation requires the generation of parametrized few-group cross-sections. This task used to be done with deterministic (2-D) lattice codes, but in the past few years the Monte-Carlo code SERPENT has demonstrate its ability to generate accurate few-group homogenized cross-section without approximations, neither on the geometry nor in the nuclear data. Since the whole core SERPENT models for production of such cross-section libraries would be computationally costly (and the standard 2-D approach may introduce unnecessary large approximations), 3-D models of each assembly type in infinite radial lattice configurations have been created. These cross-sections are then used to evaluate effective multiplication factors for different core configurations with the diffusion code PARCS. The results are compared with the reference SERPENT calculations. In the next step, a thermal-hydraulic model with the system code ATHLET applying an assembly-wise description of the core (i.e. one channel per fuel assembly) has been developed for coupled PARCS/ATHLET transient test calculations. This paper describes in detail the models and techniques used for the generation of the few-group parameterized cross section libraries, the PARCS model and the ATHLET model. Additionally, a simple exercise with coupled code system PARCS/ATHLET is presented and analysed