Polypyrrole-coated electrodes show thickness-dependent stability in different conditions during 42-day follow-up in vitro.

Electroconductive polypyrrole/dodecylbenzenesulphonate (PPy/DBS) has been proposed as novel electrode coating for biomedical applications. However, as yet, little is known about its long-term stability in moist conditions. This study compares the stability of PPy/DBS-coated platinum electrodes that are either dry-stored, incubated, or both incubated and electrically stimulated. The electrical and material properties of three different coating thicknesses were monitored for 42 days. Initially, the PPy/DBS-coating decreased the low frequency impedance of the platinum electrodes by 52% to 79%. The dry-stored electrodes remained stable during the follow-up, whereas the properties of all the incubated electrodes were altered in three stages with thickness-dependent duration: stabilization, stable, and degradation. The coated electrodes would be applicable for short-term, low-frequency in vitro measurements of up to 14 days without electrical stimulation, and up to 7 days with stimulation. The coating thickness is bound to other coating properties, and should therefore be selected according to the specific target application. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2202-2213, 2018.

Bleaching of mouse rods: microspectrophotometry and suction-electrode recording.

When a substantial fraction of rhodopsin in a rod photoreceptor is exposed to bright light, the rod is desensitized by a process known as bleaching adaptation. Experiments on isolated photoreceptors in amphibians have revealed many of the features of bleaching adaptation, but such experiments have not so far been possible in mammals. We now describe a method for making microspectrophotometric measurements of pigment concentration and suction-electrode recording of electrical responses over a wide range of bleaching exposures from isolated mouse rods or pieces of mouse retina. We show that if pigment is bleached at a low rate in the presence of bovine serum albumin (BSA), and intermediate photoproducts are allowed to decay, mouse rods are stably desensitized; subsequent treatment with exogenous 11-cis retinal results in pigment regeneration and substantial recovery of sensitivity to the dark-adapted value. Stably bleached wild-type (WT) rods show a decrease in circulating current and acceleration of the time course of decay, much as in steady background light; similar effects are seen in guanylyl cyclase-activating protein knockout (GCAPs(-/-)) rods, indicating that regulation of guanylyl cyclase is not necessary for at least a part of the adaptation produced by bleaching. Our experiments demonstrate that in mammalian rods, as in amphibian rods, steady-state desensitization after bleaching is produced by two components: (1) a reduction in the probability of photon absorption produced by a decrease in rhodopsin concentration; and (2) an equivalent background light whose intensity is proportional to the fraction of bleached pigment, and which adapts the rod like real background light. These two mechanisms together fully account for the 'log-linear' relationship in mammalian retina between sensitivity and per cent bleach, which can be measured in the steady state following exposure to bright light. Our methods will now make possible an examination of bleaching adaptation and pigment regeneration in mouse animal lines with mutations or other alterations in the proteins of transduction.

Mesopic background lights enhance dark-adapted cone ERG flash responses in the intact mouse retina: a possible role for gap junctional decoupling.

The cone-driven flash responses of mouse electroretinogram (ERG) increase as much as twofold over the course of several minutes during adaptation to a rod-compressing background light. The origins of this phenomenon were investigated in the present work by recording preflash-isolated (M-)cone flash responses ex vivo in darkness and during application of various steady background lights. In this protocol, the cone stimulating flash was preceded by a preflash that maintains rods under saturation (hyperpolarized) to allow selective stimulation of the cones at varying background light levels. The light-induced growth was found to represent true enhancement of cone flash responses with respect to their dark-adapted state. It developed within minutes, and its overall magnitude was a graded function of the background light intensity. The threshold intensity of cone response growth was observed with lights in the low mesopic luminance region, at which rod responses are partly compressed. Maximal effect was reached at intensities sufficient to suppress ∼ 90% of the rod responses. Light-induced enhancement of the cone photoresponses was not sensitive to antagonists and agonists of glutamatergic transmission. However, applying gap junction blockers to the dark-adapted retina produced qualitatively similar changes in the cone flash responses as did background light and prevented further growth during subsequent light-adaptation. These results are consistent with the idea that cone ERG photoresponses are suppressed in the dark-adapted mouse retina by gap junctional coupling between rods and cones. This coupling would then be gradually and reversibly removed by mesopic background lights, allowing larger functional range for the cone light responses.

Temperature dependence of dark-adapted sensitivity and light-adaptation in photoreceptors with A1 visual pigments: a comparison of frog L-cones and rods.

Flash responses of L-cones and rods were recorded as ERG mass potentials in the frog retina at different temperatures (2-25 degrees C). The purpose was to elucidate factors that make cones faster and less sensitive than rods, particularly the possible role of thermal activation of L-cone visual pigment in maintaining a "light-adapted" state even in darkness. Up to ca. 15 degrees C, cones and rods were desensitized roughly equally by warming (Q(10) approximately 2.2-2.7), retaining a 5-fold sensitivity difference. In this range, the cone/rod difference must depend on factors other than thermal activation of the visual pigment. Above 15 degrees C, cones showed an additional component of desensitization compared with rods, coupled to accelerated response shut-off. This behavior is consistent with light-adaptation from temperature-dependent intrinsic activity (dark light). The apparent dark light as measured by the minimum background intensities needed to affect sensitivity and/or kinetics increased by ca. 10-fold between 15 and 25 degrees C, whereas reported increases in visual-pigment activation rates over this range are less than 5-fold. We conclude that the dark state of frog L-cones above 15 degrees C may be largely set by thermal activation of the phototransduction machinery, but only part of the experimentally determined dark light can be ascribed to the visual pigment.

Mouse cone photoresponses obtained with electroretinogram from the isolated retina.

We characterize the dark-adapted photoresponses from mouse cones intact in the isolated retina, their virtually natural environment, by isolating pharmacologically the photoreceptor light responses from the electroretinogram (ERG). Due to the different photoresponse kinetics and sensitivity of rods and cones, the cone responses were readily attained by using a rod-saturating preflash. The stimulus wavelength (544 nm) was chosen to selectively stimulate the green sensitive ("M"-)pigment. Obtained responses were monophasic, showing fast kinetics (mean t(p)=51 ms) and low sensitivity (fractional single-photon response ca. 0.23%). The amplification coefficient of cones (4.6 s(-2)) was very close to that of rods (5.6 s(-2)), while the dominant time constant of recovery was clearly smaller for cones (33 ms) than for rods (160 ms).

Light responses and light adaptation in rat retinal rods at different temperatures.

Rod responses to brief pulses of light were recorded as electroretinogram (ERG) mass potentials across isolated, aspartate-superfused rat retinas at different temperatures and intensities of steady background light. The objective was to clarify to what extent differences in sensitivity, response kinetics and light adaptation between mammalian and amphibian rods can be explained by temperature and outer-segment size without assuming functional differences in the phototransduction molecules. Corresponding information for amphibian rods from the literature was supplemented by new recordings from toad retina. All light intensities were expressed as photoisomerizations per rod (Rh*). In the rat retina, an estimated 34% of incident photons at the wavelength of peak sensitivity caused isomerizations in rods, as the (hexagonally packed) outer segments measured 1.7 microm x 22 microm and had specific absorbance of 0.016 microm(-1) on average. Fractional sensitivity (S) in darkness increased with cooling in a similar manner in rat and toad rods, but the rat function as a whole was displaced to a ca 0.7 log unit higher sensitivity level. This difference can be fully explained by the smaller dimensions of rat rod outer segments, since the same rate of phosphodiesterase (PDE) activation by activated rhodopsin will produce a faster drop in cGMP concentration, hence a larger response in rat than in toad. In the range 15-25 degrees C, the waveform and absolute time scale of dark-adapted dim-flash photoresponses at any given temperature were similar in rat and toad, although the overall temperature dependence of the time to peak (t(p)) was somewhat steeper in rat (Q(10) approximately 4 versus 2-3). Light adaptation was similar in rat and amphibian rods when measured at the same temperature. The mean background intensity that depressed S by 1 log unit at 12 degrees C was in the range 20-50 Rh* s(-1) in both, compared with ca 4500 Rh* s(-1) in rat rods at 36 degrees C. We conclude that it is not necessary to assume major differences in the functional properties of the phototransduction molecules to account for the differences in response properties of mammalian and amphibian rods.