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.

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.

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.

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 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.