Hormetic Heat Shock Enhances Autophagy through HSF1 in Retinal Pigment Epithelium Cells.

To maintain homeostasis, cells have evolved stress-response pathways to cope with exogenous and endogenous stress factors. Diverse stresses at high doses may be detrimental, albeit low doses of stress, known as hormesis, can be beneficial. Upon exposure to stress, such as temperature rise, the conventional heat shock response (HSR) regulated by the heat shock transcription factor 1 (HSF1) facilitates refolding of misfolded proteins with the help of heat shock proteins (HSPs). However, the role and molecular mechanisms underlying the beneficial effects of HSR with other clearance processes, such as autophagy, remain poorly understood. In this study, human ARPE-19 cells, an in vitro model of retinal pigment epithelium, were treated with hormetic heat shock (HHS) and the autophagy expression profile was examined using quantitative PCR (qPCR), immunoblotting, immunoprecipitation, and immunofluorescence. We demonstrate that HHS enhances the expression of fundamental autophagy-associated genes in ARPE-19 cells through the activation of HSF1. HHS transiently increases the level of SQSTM1 and LC3B-II and activates autophagy. These findings reveal a role for autophagic HSF1-regulated functions and demonstrate the contribution of autophagy to hormesis in the HSR by improving proteostasis.

Retinal Temperature Determination Based on Photopic Porcine Electroretinogram.

OBJECTIVE: Subthreshold retinal laser therapy (SLT) is a treatment modality where the temperature of the retinal pigment epithelium (RPE) is briefly elevated to trigger the therapeutic benefits of sublethal heat shock. However, the temperature elevation induced by a laser exposure varies between patients due to individual differences in RPE pigmentation and choroidal perfusion. This study describes an electroretinography (ERG)-based method for controlling the temperature elevation during SLT. METHODS: The temperature dependence of the photopic ERG response kinetics were investigated both ex vivo with isolated pig retinas and in vivo with anesthetized pigs by altering the temperature of the subject and recording ERG in different temperatures. A model was created for ERG-based temperature estimation and the feasibility of the model for controlling SLT was assessed through computational simulations. RESULTS: The kinetics of the photopic in vivo flash ERG signaling accelerated between 3.6 and 4.7%/°C, depending on the strength of the stimulus. The temperature dependence was 5.0%/°C in the entire investigated range of 33 to 44°C in ex vivo ERG. The simulations showed that the method is suitable for determining the steady-state temperature elevation in SLT treatments with a sufficiently long laser exposure and large spot size, e.g., during > 30 s laser exposures with > 3 mm stimulus spot diameter. CONCLUSIONS: The described ERG-based temperature estimation model could be used to control SLT treatments such as transpupillary thermotherapy. SIGNIFICANCE: The introduced ERG-based method for controlling SLT could improve the repeatability, safety, and efficacy of the treatment of various retinal disorders.

Pinosylvin Extract Retinari™ Sustains Electrophysiological Function, Prevents Thinning of Retina, and Enhances Cellular Response to Oxidative Stress in NFE2L2 Knockout Mice.

Chronic oxidative stress eventually leads to protein aggregation in combination with impaired autophagy, which has been observed in age-related macular degeneration. We have previously shown an effective age-related macular degeneration disease model in mice with nuclear factor-erythroid 2-related factor-2 (NFE2L2) knockout. We have also shown pinosylvin, a polyphenol abundant in bark waste, to increase human retinal pigment epithelium cell viability in vitro. In this work, the effects of commercial natural pinosylvin extract, Retinari™, were studied on the electroretinogram, optical coherence tomogram, autophagic activity, antioxidant capacity, and inflammation markers. Wild-type and NFE2L2 knockout mice were raised until the age of 14.8 ± 3.8 months. They were fed with either regular or Retinari™ chow (141 ± 17.0 mg/kg/day of pinosylvin) for 10 weeks before the assays. Retinari™ treatment preserved significant retinal function with significantly preserved a- and b-wave amplitudes in the electroretinogram responses. Additionally, the treatment prevented thinning of the retina in the NFE2L2 knockout mice. The NFE2L2 knockout mice showed reduced ubiquitin-tagged protein accumulation in addition to local upregulation of complement factor H and antioxidant enzymes superoxide dismutase 1 and catalase. Therefore, the treatment in the NFE2L2 KO disease model led to reduced chronic oxidative stress and sustained retinal function and morphology. Our results demonstrate that pinosylvin supplementation could potentially lower the risk of age-related macular degeneration onset and slow down its progression.

A p97/Valosin-Containing Protein Inhibitor Drug CB-5083 Has a Potent but Reversible Off-Target Effect on Phosphodiesterase-6.

CB-5083 is an inhibitor of p97/valosin-containing protein (VCP), for which phase I trials for cancer were terminated because of adverse effects on vision, such as photophobia and dyschromatopsia. Lower dose CB-5083 could combat inclusion body myopathy with early-onset Paget disease and frontotemporal dementia or multisystem proteinopathy caused by gain-of-function mutations in VCP. We hypothesized that the visual impairment in the cancer trial was due to CB-5083's inhibition of phosphodiesterase (PDE)-6, which mediates signal transduction in photoreceptors. To test our hypothesis, we used in vivo and ex vivo electroretinography (ERG) in mice and a PDE6 activity assay of bovine rod outer segment (ROS) extracts. Additionally, histology and optical coherence tomography were used to assess CB-5083's long-term ocular toxicity. A single administration of CB-5083 led to robust ERG signal deterioration, specifically in photoresponse kinetics. Similar recordings with known PDE inhibitors sildenafil, tadalafil, vardenafil, and zaprinast showed that only vardenafil had as strong an effect on the ERG signal in vivo as did CB-5083. In the biochemical assay, CB-5083 inhibited PDE6 activity with a potency higher than sildenafil but lower than that of vardenafil. Ex vivo ERG revealed a PDE6 inhibition constant of 80 nM for CB-5083, which is 7-fold smaller than that for sildenafil. Finally, we showed that the inhibitory effect of CB-5083 on visual function is reversible, and its chronic administration does not cause permanent retinal anomalies in aged VCP-disease model mice. Our results warrant re-evaluation of CB-5083 as a clinical therapeutic agent. We recommend preclinical ERG recordings as a routine drug safety screen. SIGNIFICANCE STATEMENT: This report supports the use of a valosin-containing protein (VCP) inhibitor drug, CB-5083, for the treatment of neuromuscular VCP disease despite CB-5083's initial clinical failure for cancer treatment due to side effects on vision. The data show that CB-5083 displays a dose-dependent but reversible inhibitory action on phosphodiesterase-6, an essential enzyme in retinal photoreceptor function, but no long-term consequences on retinal function or structure.

Functional modulation of phosphodiesterase-6 by calcium in mouse rod photoreceptors.

Phosphodiesterase-6 (PDE6) is a key protein in the G-protein cascade converting photon information to bioelectrical signals in vertebrate photoreceptor cells. Here, we demonstrate that PDE6 is regulated by calcium, contrary to the common view that PDE1 is the unique PDE class whose activity is modulated by intracellular Ca2+. To broaden the operating range of photoreceptors, mammalian rod photoresponse recovery is accelerated mainly by two calcium sensor proteins: recoverin, modulating the lifetime of activated rhodopsin, and guanylate cyclase-activating proteins (GCAPs), regulating the cGMP synthesis. We found that decreasing rod intracellular Ca2+ concentration accelerates the flash response recovery and increases the basal PDE6 activity (ßdark) maximally by ~ 30% when recording local electroretinography across the rod outer segment layer from GCAPs-/- recoverin-/- mice. Our modeling shows that a similar elevation in ßdark can fully explain the observed acceleration of flash response recovery in low Ca2+. Additionally, a reduction of the free Ca2+ in GCAPs-/- recoverin-/- rods shifted the inhibition constants of competitive PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) against the thermally activated and light-activated forms of PDE6 to opposite directions, indicating a complex interaction between IBMX, PDE6, and calcium. The discovered regulation of PDE6 is a previously unknown mechanism in the Ca2+-mediated modulation of rod light sensitivity.

Induction of Heat Shock Protein 70 in Mouse RPE as an In Vivo Model of Transpupillary Thermal Stimulation.

The induction of heat shock response in the macula has been proposed as a useful therapeutic strategy for retinal neurodegenerative diseases by promoting proteostasis and enhancing protective chaperone mechanisms. We applied transpupillary 1064 nm long-duration laser heating to the mouse (C57Bl/6J) fundus to examine the heat shock response in vivo. The intensity and spatial distribution of heat shock protein (HSP) 70 expression along with the concomitant probability for damage were measured 24 h after laser irradiation in the mouse retinal pigment epithelium (RPE) as a function of laser power. Our results show that the range of heating powers for producing heat shock response while avoiding damage in the mouse RPE is narrow. At powers of 64 and 70 mW, HSP70 immunostaining indicates 90 and 100% probability for clearly elevated HSP expression while the corresponding probability for damage is 20 and 33%, respectively. Tunel staining identified the apoptotic regions, and the estimated 50% damaging threshold probability for the heating (ED50) was ~72 mW. The staining with Bestrophin1 (BEST1) demonstrated RPE cell atrophy with the most intense powers. Consequently, fundus heating with a long-duration laser provides an approachable method to develop heat shock-based therapies for the RPE of retinal disease model mice.

In vivo monitoring of mouse retinal temperature by ERG photoresponses.

Non-damaging heating of the retina and RPE provides a promising treatment for retinal diseases. However, the lack of proper control over the temperature hinders the development of safe and repeatable procedures. Here, we demonstrate with mice a non-invasive method for estimating the temperature changes in the retina and the RPE during a heating procedure. The method is based on monitoring the temperature dependent properties of retinal photoresponses recorded by electroretinography (ERG). In this study, our aim was to investigate the feasibility of ERG signal for retinal temperature estimation, utilizing a-wave and b-wave kinetics as the source of temperature information. We quantified the temperature dependencies of photoresponse kinetics and developed two linear regression models between the temperature and the photoresponse features, enabling temperature estimation. With the first model, based on the a-wave of a single photoresponse, the RMS error obtained for retinal temperature estimation was <0.9 °C. The second model, applying the b-waves of five dim flash responses, an RMS error of <0.7 °C was achieved. In addition, we tested the sensitivity of the method to small changes in light stimulus strength and investigated suitable stimulus intervals for continuous retinal temperature monitoring. The proposed method provides a convenient technique for monitoring mouse retinal and RPE temperature with ERG recording when studying controlled retinal heating. Similar temperature dependencies exist in human ERG suggesting that this approach could also be applicable in clinical heating treatments.

Determination of basal phosphodiesterase activity in mouse rod photoreceptors with cGMP clamp.

Light regulates cGMP concentration in the photoreceptor cytoplasm by activating phosphodiesterase (PDE) molecules through a G-protein signalling cascade. Spontaneous PDE activity is present in rod outer segments even in darkness. This basal PDE activity (ßdark) has not been determined in wild type mammalian photoreceptor cells although it plays a key role in setting the sensitivity and recovery kinetics of rod responses. We present a novel method for determination of ßdark using local electroretinography (LERG) from isolated mouse retinas. The method is based on the ability of PDE inhibitors to decrease ßdark, which can be counterbalanced by increasing PDE activity with light. This procedure clamps cytoplasmic cGMP to its dark value. ßdark can be calculated based on the amount of light needed for the "cGMP clamp" and information extracted from the registered rod photoresponses. Here we apply this method to determine ßdark values for the first time in the mammalian rods and obtain the following estimates for different mouse models: 3.9 s-1 for wild type, 4.5 s-1 for guanylate cyclase activating proteins (GCAPs) knockout, and 4.4 s-1 for GCAPs and recoverin double knockout mice. Our results suggest that depletion of GCAPs or recoverin do not affect ßdark.

Loss of NRF-2 and PGC-1α genes leads to retinal pigment epithelium damage resembling dry age-related macular degeneration.

Age-related macular degeneration (AMD) is a multi-factorial disease that is the leading cause of irreversible and severe vision loss in the developed countries. It has been suggested that the pathogenesis of dry AMD involves impaired protein degradation in retinal pigment epithelial cells (RPE). RPE cells are constantly exposed to oxidative stress that may lead to the accumulation of damaged cellular proteins, DNA and lipids and evoke tissue deterioration during the aging process. The ubiquitin-proteasome pathway and the lysosomal/autophagosomal pathway are the two major proteolytic systems in eukaryotic cells. NRF-2 (nuclear factor-erythroid 2-related factor-2) and PGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1 alpha) are master transcription factors in the regulation of cellular detoxification. We investigated the role of NRF-2 and PGC-1α in the regulation of RPE cell structure and function by using global double knockout (dKO) mice. The NRF-2/PGC-1α dKO mice exhibited significant age-dependent RPE degeneration, accumulation of the oxidative stress marker, 4-HNE (4-hydroxynonenal), the endoplasmic reticulum stress markers GRP78 (glucose-regulated protein 78) and ATF4 (activating transcription factor 4), and damaged mitochondria. Moreover, levels of protein ubiquitination and autophagy markers p62/SQSTM1 (sequestosome 1), Beclin-1 and LC3B (microtubule associated protein 1 light chain 3 beta) were significantly increased together with the Iba-1 (ionized calcium binding adaptor molecule 1) mononuclear phagocyte marker and an enlargement of RPE size. These histopathological changes of RPE were accompanied by photoreceptor dysmorphology and vision loss as revealed by electroretinography. Consequently, these novel findings suggest that the NRF-2/PGC-1α dKO mouse is a valuable model for investigating the role of proteasomal and autophagy clearance in the RPE and in the development of dry AMD.

Electrophysiological determination of phosphodiesterase-6 inhibitor inhibition constants in intact mouse retina.

Cyclic nucleotide phosphodiesterases (PDEs) hydrolyze the second messengers cAMP and cGMP. PDEs control numerous cellular processes making them promising targets for the development of therapeutic agents. Unfortunately, many PDE inhibitor molecules are non-selective among PDE classes and efficient methods for quantitative studies on the isoform-specificity of PDE inhibitors in the natural environments of PDEs are unavailable. The PDE in photoreceptors, PDE6, mediates the conversion of photon information into electrical signals making the retina an exceptional model system for examinations of the pharmacological effects of PDE inhibitors on PDE6. Here we introduce electroretinography-based methods for determining the inhibition constants of PDE inhibitors towards the naturally occurring light-activated and spontaneously activated forms of PDE6. We compare our results to earlier biochemical determinations with trypsin-activated PDE6 with disintegrated PDE6 γ-subunit. The potencies of PDE inhibitors were determined by stimulating the photoreceptors of isolated mouse retinas with light and tracking the inhibitor-induced changes in their electrical responses. The methods were tested with three PDE inhibitors, 3-isobutyl-1-methylxanthine (IBMX), sildenafil, and zaprinast. The inhibition constants towards light-activated, spontaneously activated, and trypsin-activated PDE6 differed significantly from each other for sildenafil and zaprinast but were closely similar for IBMX. We hypothesize that this is due to the ability of the PDE6 γ-subunit to compete with sildenafil and zaprinast from the same binding sites near the catalytic domain of PDE6. The introduced methods are beneficial both for selecting potent molecules for PDE6 inhibition and for testing the drugs targeted at other PDE isoforms against adverse effects on visual function.

Transretinal ERG in Studying Mouse Rod Phototransduction: Comparison With Local ERG Across the Rod Outer Segments.

Purpose: Electroretinography (ERG) is the gold standard in clinical examinations of retinal function. Corneal ERG is widely used for diagnostics, but ERG components from the inner retina complicate quantitative investigations of the phototransduction cascade. Transretinal ERG (TERG) recorded ex vivo enables pharmacologic isolation of signals generated by photoreceptor cells, establishing an appealing electrophysiologic method for diverse studies of phototransduction. Pharmacologically isolated TERG, however, contains components arising in the photoreceptor inner segments. Here, we compared simultaneously recorded TERG and local ERG across the outer segment layer (LERG-OS) to determine how consistently TERG reflects changes in the rod outer segment current signaling. Methods: Recordings were made from dark-adapted, isolated C57BL/6J mouse retinas superfused with HEPES or bicarbonate buffered solution containing 2-mM aspartate or 20-µM DL-2-amino-4-phosphonobutyric acid to block synaptic transmission, and 50-µM BaCl2 to block the Müller cell component. TERG responses were recorded with macroelectrodes on both sides of the retina while responses across different retinal layers were recorded with microelectrodes. Results: The time-to-peak and the dominant time constant values were slightly smaller and the half-saturating stimulus was somewhat stronger in TERG compared with LERG-OS. No differences in light adaptation data were observed between the methods. LERG responses recorded across the whole photoreceptor layer were similar to those by TERG. Conclusions: TERG photoreceptor responses correspond well with the LERG-OS responses. The main differences are the nose component and slightly faster response kinetics observed by TERG. We conclude that TERG can be used for reliable quantitative investigation of phototransduction.

A Novel Method for Mouse Retinal Temperature Determination Based on ERG Photoresponses.

This study introduces a novel retinal temperature determination method based on the temperature dependent properties of photoresponses recorded by electroretinography (ERG). The kinetics and amplitudes of ERG photoresponses depend on retinal temperature. Additionally, raising retinal temperature increases the probability of long-wavelength photon absorption, which manifests as temperature dependence of photoreceptor sensitivity. In this study we extract a number of features that represent these properties from the a- and b-waves of mouse ex vivo ERG flash responses and construct three multivariable regression models between temperature and the selected features. The performance of these models was evaluated against a separate test dataset and for two of the models, an RMS temperature determination error of less than 0.50 °C could be reached. Our results demonstrate that the method can be successfully used for reliable retinal temperature determination ex vivo. The method, reflecting the temperature of distal retina, can be applied also in the estimation of retinal pigment epithelium temperature.

Lack of P4H-TM in mice results in age-related retinal and renal alterations.

Age-related macular degeneration (AMD), affecting the retinal pigment epithelium (RPE), is the leading cause of blindness in middle-aged and older people in developed countries. Genetic and environmental risk factors have been identified, but no effective cure exists. Using a mouse model we show that a transmembrane prolyl 4-hydroxylase (P4H-TM), which participates in the oxygen-dependent regulation of the hypoxia-inducible factor (HIF), is a potential novel candidate gene for AMD. We show that P4h-tm had its highest expression levels in the mouse RPE and brain, heart, lung, skeletal muscle and kidney. P4h-tm-/- mice were fertile and had a normal life span. Lack of P4h-tm stabilized HIF-1α in cortical neurons under normoxia, while in hypoxia it increased the expression of certain HIF target genes in tissues with high endogenous P4h-tm expression levels more than in wild-type mice. Renal erythropoietin levels increased in P4h-tm-/- mice with aging, but the resulting ∼2-fold increase in erythropoietin serum levels did not lead to erythrocytosis. Instead, accumulation of lipid-containing lamellar bodies in renal tubuli was detected in P4h-tm-/- mice with aging, resulting in inflammation and fibrosis, and later glomerular sclerosis and albuminuria. Lack of P4h-tm was associated with retinal thinning, rosette-like infoldings and drusen-like structure accumulation in RPE with aging, as is characteristic of AMD. Photoreceptor recycling was compromised, and electroretinograms revealed functional impairment of the cone pathway in adult P4h-tm-/- mice and cone and rod deficiency in middle-aged mice. P4H-TM is therefore imperative for normal vision, and potentially a novel candidate for age-induced diseases, such as AMD.

A novel Ca2+-feedback mechanism extends the operating range of mammalian rods to brighter light.

Sensory cells adjust their sensitivity to incoming signals, such as odor or light, in response to changes in background stimulation, thereby extending the range over which they operate. For instance, rod photoreceptors are extremely sensitive in darkness, so that they are able to detect individual photons, but remain responsive to visual stimuli under conditions of bright ambient light, which would be expected to saturate their response given the high gain of the rod transduction cascade in darkness. These photoreceptors regulate their sensitivity to light rapidly and reversibly in response to changes in ambient illumination, thereby avoiding saturation. Calcium ions (Ca2+) play a major role in mediating the rapid, subsecond adaptation to light, and the Ca2+-binding proteins GCAP1 and GCAP2 (or guanylyl cyclase-activating proteins [GCAPs]) have been identified as important mediators of the photoreceptor response to changes in intracellular Ca2+. However, mouse rods lacking both GCAP1 and GCAP2 (GCAP-/-) still show substantial light adaptation. Here, we determined the Ca2+ dependency of this residual light adaptation and, by combining pharmacological, genetic, and electrophysiological tools, showed that an unknown Ca2+-dependent mechanism contributes to light adaptation in GCAP-/- mouse rods. We found that mimicking the light-induced decrease in intracellular [Ca2+] accelerated recovery of the response to visual stimuli and caused a fourfold decrease of sensitivity in GCAP-/- rods. About half of this Ca2+-dependent regulation of sensitivity could be attributed to the recoverin-mediated pathway, whereas half of it was caused by the unknown mechanism. Furthermore, our data demonstrate that the feedback mechanisms regulating the sensitivity of mammalian rods on the second and subsecond time scales are all Ca2+ dependent and that, unlike salamander rods, Ca2+-independent background-induced acceleration of flash response kinetics is rather weak in mouse rods.

Flash responses of mouse rod photoreceptors in the isolated retina and corneal electroretinogram: comparison of gain and kinetics.

PURPOSE: To examine the amplification and kinetics of murine rod photoresponses by recording ERG flash responses in vivo and ex vivo from the same retina. We also aimed to evaluate the two available methods for isolating the rod signal from the ERG flash response, that is, pharmacology and paired flash method on the isolated retina. METHODS: Dark-adapted ERG responses to full-field flashes of green light were recorded from anesthetized (ketamine/xylazine) C57BL/6N mice. ERG flash responses to homogenous light stimuli arriving from the photoreceptor side were then recorded transretinally from the same retinas, isolated and perfused with Ringer's or Ames' solution at 37°C. The responses were analyzed to determine the a-wave kinetics as well as the estimated flash sensitivity and kinetics of the full rod responses derived with the paired flash protocol. The analysis was complemented with pharmacologic blockade of glutamatergic transmission in the isolated retina. RESULTS: The a-waves were of comparable size, sensitivity and kinetics in vivo and in the isolated retina, but the onset of the b-wave was delayed in the isolated retina. The Lamb-Pugh activation constants determined for the a-waves were similar in both preparations. The kinetics of the derived photoreceptor responses were similar in both conditions, although the responses were consistently slightly slower ex vivo. This was not explicable as a direct effect of ketamine or xylazine on the photoreceptors or as their indirect effect through hyperglycemia, as tested on the isolated retina. CONCLUSIONS: Through comparison to the corneal ERG, the transretinal ERG is a valuable tool for assaying the physiologic state of isolated retinal tissue. The rod photoreceptor responses of the intact isolated retina correspond well to those recorded in vivo. The origin of their faster kinetics compared to single cell recordings remains to be determined.

Calcium sets the physiological value of the dominant time constant of saturated mouse rod photoresponse recovery.

BACKGROUND: The rate-limiting step that determines the dominant time constant (τ(D)) of mammalian rod photoresponse recovery is the deactivation of the active phosphodiesterase (PDE6). Physiologically relevant Ca(2+)-dependent mechanisms that would affect the PDE inactivation have not been identified. However, recently it has been shown that τ(D) is modulated by background light in mouse rods. METHODOLOGY/PRINCIPAL FINDINGS: We used ex vivo ERG technique to record pharmacologically isolated photoreceptor responses (fast PIII component). We show a novel static effect of calcium on mouse rod phototransduction: Ca(2+) shortens the dominant time constant (τ(D)) of saturated photoresponse recovery, i.e., when extracellular free Ca(2+) is decreased from 1 mM to ∼25 nM, the τ(D) is reversibly increased ∼1.5-2-fold. CONCLUSIONS: We conclude that the increase in τ(D) during low Ca(2+) treatment is not due to increased [cGMP], increased [Na(+)] or decreased [ATP] in rod outer segment (ROS). Also it cannot be due to protein translocation mechanisms. We suggest that a Ca(2+)-dependent mechanism controls the life time of active PDE.

Rod phototransduction determines the trade-off of temporal integration and speed of vision in dark-adapted toads.

Human vision is approximately 10 times less sensitive than toad vision on a cool night. Here, we investigate (1) how far differences in the capacity for temporal integration underlie such differences in sensitivity and (2) whether the response kinetics of the rod photoreceptors can explain temporal integration at the behavioral level. The toad was studied as a model that allows experimentation at different body temperatures. Sensitivity, integration time, and temporal accuracy of vision were measured psychophysically by recording snapping at worm dummies moving at different velocities. Rod photoresponses were studied by ERG recording across the isolated retina. In both types of experiments, the general timescale of vision was varied by using two temperatures, 15 and 25 degrees C. Behavioral integration times were 4.3 s at 15 degrees C and 0.9 s at 25 degrees C, and rod integration times were 4.2-4.3 s at 15 degrees C and 1.0-1.3 s at 25 degrees C. Maximal behavioral sensitivity was fivefold lower at 25 degrees C than at 15 degrees C, which can be accounted for by inability of the "warm" toads to integrate light over longer times than the rods. However, the long integration time at 15 degrees C, allowing high sensitivity, degraded the accuracy of snapping toward quickly moving worms. We conclude that temporal integration explains a considerable part of all variation in absolute visual sensitivity. The strong correlation between rods and behavior suggests that the integration time of dark-adapted vision is set by rod phototransduction at the input to the visual system. This implies that there is an inexorable trade-off between temporal integration and resolution.

Origin of the fast negative ERG component from isolated aspartate-treated mouse retina.

The leading edge of the a-wave of the ERG is generally believed to accurately reflect the changes in the circulating current through the cGMP-gated channels in the outer segment plasma membrane of rods and cones. The aspartate-isolated mammalian electroretinogram (ERG) to a rod-saturating flash contains a fast "nose"-like wave temporally overlapping with the a-wave. We characterize the nature of this nose, investigate the membrane current mechanisms involved in the nose mechanism, and propose a model that can explain the generation of the nose component in the rod inner segment. On the basis of pharmacological treatments and perfusate ion composition alterations we rule out the possible role of most of the known rod membrane current mechanisms that might participate in the generation of the ERG nose component and we propose that the nose is generated by the interplay of voltage-dependent K(x) and h channels together with the Na(+)/K(+) ATPase. Our results strengthen the view that the kinetics of the leading edge of the ERG photoresponses should correspond to that of the outer segment light-sensitive current.

A new method for measuring free drug concentration: retinal tissue as a biosensor.

PURPOSE: To develop a method of using isolated rat retina as a biosensor in experiments on controlled drug release for measuring the resultant concentration of free model drug in living tissue and for testing the biocompatibility of the polymers and polymeric nanostructures used as drug carriers. METHODS: The method is based on the monotonic dependence of the photoresponse kinetics of retinal rods on the concentration of the membrane-permeable phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Changes in the time to peak (tp) of linear-range rod photoresponses were followed by transretinal ERG mass potential recordings in the aspartate-treated, dark-adapted rat retina. The dependence of tp on [IBMX] was measured, and the calibration curve thus obtained was used to determine the amount of IBMX released from polymeric structures. The biocompatibility of the carrier was first assessed by the degree to which rods retained stable function in the presence of the polymer or monomers alone. RESULTS: The dependence of tp on [IBMX] was well-described by a second-order polynomial. After each change of [IBMX], a new equilibrium state was reached within 6 to 9 minutes, depending on temperature. The amounts of IBMX released from biocompatible polymeric structures were measurable with good accuracy in the range 10 to 300 microM. CONCLUSIONS: This method enables accurate concentration determinations of the model drug IBMX in retinal tissue in drug-release experiments. The concentration dependence of the photoresponse kinetics has to be calibrated for each retina and temperature. The same preparation can be used for rapid testing of possible bioincompatibility of various molecules.

The photoactivation energy of the visual pigment in two spectrally different populations of Mysis relicta (Crustacea, Mysida).

We report the first study of the relation between the wavelength of maximum absorbance (lambdamax) and the photoactivation energy (Ea) in invertebrate visual pigments. Two populations of the opossum shrimp Mysis relicta were compared. The two have been separated for 9,000 years and have adapted to different spectral environments ("Sea" and "Lake") with porphyropsins peaking at lambdamax=529 nm and 554 nm, respectively. The estimation of Ea was based on measurement of temperature effects on the spectral sensitivity of the eye. In accordance with theory (Stiles in Transactions of the optical convention of the worshipful company of spectacle makers. Spectacle Makers' Co., London, 1948), relative sensitivity to long wavelengths increased with rising temperature. The estimates calculated from this effect are Ea,529=47.8+/-1.8 kcal/mol and Ea,554=41.5+/-0.7 kcal/mol (different at P<0.01). Thus the red-shift of lambdamax in the "Lake" population, correlating with the long-wavelength dominated light environment, is achieved by changes in the opsin that decrease the energy gap between the ground state and the first excited state of the chromophore. We propose that this will carry a cost in terms of increased thermal noise, and that evolutionary adaptation of the visual pigment to the light environment is directed towards maximizing the signal-to-noise ratio rather than the quantum catch.