Dark Current and Photocurrent in Retinal Rods

The interstitial voltages, currents, and resistances of the receptor layer of the isolated rat retina have been investigated with arrays of micropipette electrodes inserted under direct visual observation by infrared microscopy. In darkness a steady current flows inward through the plasma membrane of the rod outer segments. It is balanced by equal outward current distributed along the remainder of each rod. Flashes of light produce a photocurrent which transiently reduces the dark current with a waveform resembling the PII and a-wave components of the electroretinogram. The photocurrent is produced by a local action of light within 12 μm of its point of absorption in the outer segments. The quantum current gain of the photocurrent is greater than 10⁶. The electrical space constant of rat rods is greater than 25 μm, so that the electrical effects of the photocurrent are large enough at the rod synapses to permit single absorbed photons to be detected by the visual system. The photocurrent is apparently the primary sensory consequence of light absorption by rhodopsin.     

Kinetics of Synaptic Transmission at Ribbon Synapses of Rods and Cones

The ribbon synapse is a specialized structure that allows photoreceptors to sustain the continuous release of vesicles for hours upon hours and years upon years but also respond rapidly to momentary changes in illumination. Light responses of cones are faster than those of rods and, mirroring this difference, synaptic transmission from cones is also faster than transmission from rods. This review evaluates the various factors that regulate synaptic kinetics and contribute to kinetic differences between rod and cone synapses. Presynaptically, the release of glutamate-laden synaptic vesicles is regulated by properties of the synaptic proteins involved in exocytosis, influx of calcium through calcium channels, calcium release from intracellular stores, diffusion of calcium to the release site, calcium buffering, and extrusion of calcium from the cytoplasm. The rate of vesicle replenishment also limits the ability of the synapse to follow changes in release. Post-synaptic factors include properties of glutamate receptors, dynamics of glutamate diffusion through the cleft, and glutamate uptake by glutamate transporters. Thus, multiple synaptic mechanisms help to shape the responses of second-order horizontal and bipolar cells.     

Kinetics of the Hydrolysis of 8-Bromo-Cyclic GMP by the Light-Activated Phosphodiesterase of Toad Rods

The hypothesis that cyclic GMP is the internal transmitter of retinal rod phototransduction, when combined with the observations that 8-bromo-cyclic GMP opens the cyclic GMP-dependent outer segment conductance and that rods into which 8-bromo-cyclic GMP has been injected still respond to light, predicts that the light-activated phosphodiesterase (EC 3.1.4.17) must catalyze the hydrolysis of 8-bromo-cyclic GMP. This hypothesis was tested by measuring light-activated toad rod disk membrane phosphodiesterase with a pH assay technique. Phosphodiesterase-catalyzed hydrolysis of 8-bromo-cyclic GMP was confirmed: at pH 8.0, total proton production after flash activation was identical to total amount of 8-bromo-cyclic GMP added as substrate. Photoactivated phosphodiesterase was remarkably less efficient in catalyzing the hydrolysis of 8-bromo-cyclic GMP than of cyclic GMP: Vmax for 8-bromo-cyclic GMP was 0.063 M/M rhodopsin/s, whereas that for cyclic GMP was 11 M/M rhodopsin/s--170 times greater. The Km for 8-bromo-cyclic GMP was 160 microM, and for cyclic GMP, 590 microM. 8-bromo-cyclic GMP competitively inhibited phosphodiesterase-catalyzed hydrolysis of cyclic GMP with a Ki of 1.2 mM. Complete reaction progress curves were analyzed for obedience to Michaelis-Menten kinetics: cyclic GMP hydrolysis, 8-bromo-cyclic GMP hydrolysis, and cyclic GMP hydrolysis in the presence of 8-bromo-cyclic GMP as competitive inhibitor were found to follow the integrated form of the Michaelis-Menten equation over the time course of the reactions, assuming phosphodiesterase was activated as a step. The kinetic parameters extracted from reaction progress curves were consistent with those derived from analysis of the initial velocity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Control of Retinal Sensitivity : I. Light and Dark Adaptation of Vertebrate Rods and Cones

Rods and cones in Necturus respond with graded hyperpolarization to test flashes spanning about 3.5 log units of intensity. Steady background levels hyperpolarize the rods, and the rod responses become progressively smaller as background level is increased. In cones, higher background levels reduce the effectiveness of test flashes, so higher ranges of test intensities are required to elicit the full range of graded responses. When backgrounds are terminated, cones return rapidly, but rods return slowly to the dark potential level. The effects of backgrounds on both rods and cones can be observed at intensities that cause negligible bleaching as determined by retinal densitometry. During dark adaptation, changes are observed in the rods and cones that are similar to those produced by backgrounds. Receptor sensitivities, derived from these results, show that rods saturate, cones obey Weber's law, and sensitization during dark adaptation follows a two-phase time-course.     

The Fine Structure of the Retina Studied with the Electron Microscope : IV. Morphogenesis of Outer Segments of Retinal Rods

The morphogenesis of the outer segments of retinal rods was studied mainly in the kitten before the opening of the eye, and the probable sequence of the morphogenetic stages is deduced. Since the development of retinal rods is not synchronous, the deductions were based on observations of many single and serial sections. One centriole extends ciliary tubules of about 0.5 µ long, in the growing primitive cilium. Beyond this length, each ciliary tubule becomes a row of small vesicles (called "ciliary vesicles" in this paper), which penetrate into the distal region of the cilium. Where the ciliary vesicles establish contact with the plasma membrane of the distal region of the cilium, more or less deep infoldings of the plasma membrane are observed. In the distal region can be seen rows of tubular or vesicular structures. A few of these membranous structures are continuous with the bottoms of the infoldings. At the following stage, the infoldings disappear and the ciliary vesicles lose contact with the distal plasma membrane. Nonetheless, the formation of the tubular structures continues in the distal region of the primitive outer segment. The tubular structures appear to be transformed into the primitive rod sacs by sidewise enlargement. At a subsequent time, presumably, these primitive rod sacs flatten and are rearranged into a position perpendicular to the long axis of the outer segment. The detailed structure of the basal body of the connecting cilium was also studied by means of serial sections.     

Kinetics of Efficient Recombinant Adeno-Associated Virus Transduction in Retinal Pigment Epithelial Cells

The aim of this study was to investigate the premise that retinal pigment epithelial (RPE) cells are more permissive to recombinant adeno-associated virus (rAAV) transduction than other cells. We investigated the kinetics and mechanisms of rAAV transduction in RPE cells and found that the transduction efficiencies of cultured RPE cells HRPE51 and ARPE19 were significantly higher than those of 293 (P < 0.008) and HeLa (P < 0.025) cells. In addition, RPE cells reached maximum transduction efficiency at a much lower m.o.i. (m.o.i. 10) than 293 cells (m.o.i. 25). Competition experiments using 1 microg/ml heparin inhibited the high level of transduction in RPE cells by 30%, but additional heparin failed to reduce rAAV transduction further. Southern hybridization of low-molecular-weight DNA from transduced RPE cells indicated that 42% of single-stranded rAAV DNA was translocated into the nucleus by 2 h postinfection. By 6 h postinfection, double-stranded rAAV DNA was observed, which coincided with the onset of transgene expression. Southern and fluorescence in situ hybridization of total genomic DNA indicated that long-term transgene expression in RPE cells was maintained by the integration of rAAV into the cellular chromosome. Together, these results suggest that the high permissiveness of RPE cells is not related to the presence of heparan sulfate receptors or nuclear trafficking but may be due to an enhanced rate of second-strand synthesis and that integration in RPE cells is responsible for long-term transgene expression.     

Photocurrent measurements of the purple membrane oriented in a polyacrylamide gel.

When illuminated, oriented purple membranes isolated from Halobacterium halobium give a photoelectric effect. The frequency response of a photocurrent measuring system for purple membranes oriented and immobilized in a polyacrylamide gel is analyzed from DC to 100 MHz. The waveform of the photocurrent can depend on both the sample conditions (including bathing solution) and the measuring system (electrode and ammeter) at both the low and high frequency ends. In the DC-1 kHz range (millisecond signals), the apparent lifetime of the photocurrent component is distorted if the electrode is not platinized and if the conductivity of the bathing solution is not low. In the 1 kHz to 1 MHz range (microsecond signals), the frequency response is flat under most conditions. In the MHz range (nanosecond signals), the apparent lifetime of the photocurrent component will be distorted if the conductivity of the bathing solution is not high and if the input impedance of the ammeter is not low and constant throughout the frequency range. With our optimized apparatus, we could measure the photocurrent components from oriented purple membrane with lifetimes from 70 ms to 32 ns without distortion by the measuring system.     

Photocurrent response of bacteriorhodopsin adsorbed on bimolecular lipid membranes

The photo response of bacteriorhodopsin adsorbed on a bimolecular lipid membrane has been investigated using short-circuit current measurements. The results revealed a biphasic current vs. time curve for the photocurrent at pH values of approx. 7. This phenomenon could be modified by altering either the value of the external applied electrical field or the proton concentration differences.The observed effects of the external applied voltage, pH gradient and lipophilic proton carriers enabled us to conclude that the bacteriorhodopsin can be adsorbed in two different states, which give rise to a pumping effect and a flux of protons in opposite directions.A theoretical analysis of the photocycle in relation to the electrical field which acts on the proton uptake and release is proposed. The main effect of this field is to diminish the pumping rate due to the proton motive force resulting from the creation of space-charge in the vicinity of purple membrane fragments.     

Fundamental Understanding of Photocurrent Hysteresis in Perovskite Solar Cells

Organic–inorganic hybrid perovskite solar cells (PSCs) have become a promising candidate in the photovoltaic field due to their high power conversion efficiency and low material cost. However, the development of PSCs is limited by their poor stability under practical conditions in the presence of oxygen, moisture, sunlight, heat, and the current–voltage (I–V) hysteresis. In particular, the hysteretic I–V issue casts doubt on the validity of the photovoltaic performance results that are achieved, making it difficult to evaluate the authentic performance of PSCs. This review article focuses on understanding the I–V hysteresis behavior in PSCs and on exploring the possible reasons leading to this hysteresis phenomenon. The various strategies attempted to suppress the I–V hysteresis in PSCs are summarized, and a brief future recommendation is provided.     

Agar Pitting Gram-negative Rods in the Subgingival Flora of Dogs

The phenotypic characteristics as well as the prevalence and quantity (colony forming units/mL/sample) of 39 bacterial isolates of agar pitting Gram-negative rods, from two diseased and one healthy subgingival site from each of 16 pet dogs with naturally occurring periodontitis, were investigated. Phenotypic features were determined with use of standard biochemical methods, by enzymatic profiling with the API ZYM system, and by cellular fatty acid profiling. The organisms detected were motile, catalase-negative Campylobacter sp., present in 69% of the dogs and in 29% of the subgingival samples (69/29), motile, catalase-positive Campylobacter sp. (63/29), Eikenella corrodens (25/10), organisms closely resembling E. corrodens but nitrate-negative and unable to grow in air, designated E. corrodens -like (19/8), Bilophila wadsworthia (6/2), and non-motile Campylobacter sp. (6/2). The most frequent organisms were the motile Campylobacter isolates constituting 72% of all isolates. No statistically significant differences were detected between the diseased and healthy subgingival sites, with regard to the prevalence of any of these groups of organisms. Furthermore, the bacterial isolates were detected in almost equal numbers in the diseased and healthy sample sites. Hence, no association between dog periodontitis and the agar pitting Gram-negative rods was established. The phenotypic data also suggest, that the organisms that were only presumptively identified in the present study (Campylobacter sp., E. corrodens -like), may not have been previously described.     

Isolation and Characterization of Cytoplasmic Cofilin-Actin Rods

Cofilin-actin bundles (rods), which form in axons and dendrites of stressed neurons, lead to synaptic dysfunction and may mediate cognitive deficits in dementias. Rods form abundantly in the cytoplasm of non-neuronal cells in response to many treatments that induce rods in neurons. Rods in cell lysates are not stable in detergents or with added calcium. Rods induced by ATP-depletion and released from cells by mechanical lysis were first isolated from two cell lines expressing chimeric actin-depolymerizing factor (ADF)/cofilin fluorescent proteins by differential and equilibrium sedimentation on OptiPrep gradients and then from neuronal and non-neuronal cells expressing only endogenous proteins. Rods contain ADF/cofilin and actin in a 1:1 ratio. Isolated rods are stable in dithiothreitol, EGTA, Ca²⁺, and ATP. Cofilin-GFP-containing rods are stable in 500 mm NaCl, whereas rods formed from endogenous proteins are significantly less stable in high salt. Proteomic analysis of rods formed from endogenous proteins identified other potential components whose presence in rods was examined by immunofluorescence staining of cells. Only actin and ADF/cofilin are in rods during all phases of their formation; furthermore, the rapid assembly of rods in vitro from these purified proteins at physiological concentration shows that they are the only proteins necessary for rod formation. Cytoplasmic rod formation is inhibited by cytochalasin D and jasplakinolide. Time lapse imaging of rod formation shows abundant small needle-shaped rods that coalesce over time. Rod filament lengths measured by ultrastructural tomography ranged from 22 to 1480 nm. These results suggest rods form by assembly of cofilin-actin subunits, followed by self-association of ADF/cofilin-saturated F-actin.     

Photocurrent Enhancement in Si-Ge Photodetectors by Utilizing Surface Plasmons

A circular slit-groove surface plasmon polaritons (SPPs) launcher surrounding a photodetector is employed theoretically to enhance the photocurrent of atypical Si-Ge photodetectors. The slit and grooves are designed such that the SPPs are focused at the center of the absorption layer of the photodetector to result in additional electric current. Fabry–Perot resonance condition accurately calculates the period of the groove, slit-groove distance, photodetector radius, and slit-photodetector distance. The manipulation leads to constructive interference between the incident light impinging from the top and the SPPs propagating toward the photodetector. Simulation result shows that photocurrent increases by approximately 13-fold when the SPPs are introduced.     

New Diagnostic System for the Identification of Lactose-fermenting Gram-negative Rods

The identification of prompt lactose-fermenting gram-negative rods has generally relied heavily upon colonial morphology coupled with one or more indole, methyl red, Voges-Proskauer, citrate (IMViC) parameters, hydrogen sulfide, and motility. Studies were undertaken to compare diagnoses dependent solely upon the more orthodox criteria to a system for identification based upon hydrogen sulfide, ornithine decarboxylase, and citrate utilization (HOC). The results suggest that the IMViC scheme of identification is neither consistent nor applicable when applied to the current nomenclature of the above group of organisms and should be discarded, whereas the HOC system may prove to be of significant value to clinical microbiologists.