Rod Transducin Research Articles

Transducin-Deficient Rod Photoreceptors Evaluated With Optical Coherence Tomography and Oxygen Consumption Rate Energy Biomarkers.

To test the hypothesis that rod energy biomarkers in light and dark are similar in mice without functional rod transducin (Gnat1rd17). Gnat1rd17 and wildtype (WT) mice were studied in canonically low energy demand (light) and high energy demand (dark) conditions. We measured rod inner segment ellipsoid zone (ISez) profile shape, external limiting membrane-retinal pigment epithelium (ELM-RPE) thickness, and magnitude of a hyporeflective band (HB) intensity dip located between photoreceptor tips and apical RPE; antioxidants were given in a subset of mice. Oxygen consumption rate (OCR) and visual performance indexes were also measured. The lower energy demand expected in light-adapted wildtype retinas was associated with an elongated ISez, thicker ELM-RPE, and higher HB magnitude, and lower OCR compared to high energy demand conditions in the dark. Gnat1rd17 mice showed a wildtype-like ISez profile shape at 20 minutes of light that became rounder at 60 minutes; at both times, ELM-RPE was smaller than wildtype values, and the HB magnitude was unmeasurable. OCR was higher than in the dark. Light-adapted Gnat1rd17 mice biomarkers were unaffected by anti-oxidants. Gnat1rd17 mice showed modest outer nuclear layer thinning and no reduction in visual performance indexes. Light-stimulated changes in all biomarkers in WT mice are consistent with the established light-induced decrease in net energy demand. In contrast, biomarker changes in Gnat1rd17 mice raise the possibility that light increases net energy demand in the absence of rod phototransduction.

Transducin Partners Outside the Phototransduction Pathway.

Transducin mediates signal transduction in a classical G protein-coupled receptor (GPCR) phototransduction cascade. Interactions of transducin with the receptor and the effector molecules had been extensively investigated and are currently defined at the atomic level. However, partners and functions of rod transducin α (Gαt1) and βγ (Gβ1γ1) outside the visual pathway are not well-understood. In particular, light-induced redistribution of rod transducin from the outer segment to the inner segment and synaptic terminal (IS/ST) allows Gαt1 and/or Gβ1γ1 to modulate synaptic transmission from rods to rod bipolar cells (RBCs). Protein-protein interactions underlying this modulation are largely unknown. We discuss known interactors of transducin in the rod IS/ST compartment and potential pathways leading to the synaptic effects of light-dispersed Gαt1 and Gβ1γ1. Furthermore, we show that a prominent non-GPCR guanine nucleotide exchange factor (GEF) and a chaperone of Gα subunits, resistance to inhibitors of cholinesterase 8A (Ric-8A) protein, is expressed throughout the retina including photoreceptor cells. Recent structures of Ric-8A alone and in complexes with Gα subunits have illuminated the structural underpinnings of the Ric-8A activities. We generated a mouse model with conditional knockout of Ric-8A in rods in order to begin defining the functional roles of the protein in rod photoreceptors and the retina. Our analysis suggests that Ric-8A is not an obligate chaperone of Gαt1. Further research is needed to investigate probable roles of Ric-8A as a GEF, trafficking chaperone, or a mediator of the synaptic effects of Gαt1.

Dark noise and retinal degeneration from D190N-rhodopsin

Numerous rhodopsin mutations have been implicated in night blindness and retinal degeneration, often with unclear etiology. D190N-rhodopsin (D190N-Rho) is a well-known inherited human mutation causing retinitis pigmentosa. Both higher-than-normal spontaneous-isomerization activity and misfolding/mistargeting of the mutant protein have been proposed as causes of the disease, but neither explanation has been thoroughly examined. We replaced wild-type rhodopsin (WT-Rho) in RhoD190N/WT mouse rods with a largely "functionally silenced" rhodopsin mutant to isolate electrical responses triggered by D190N-Rho activity, and found that D190N-Rho at the single-molecule level indeed isomerizes more frequently than WT-Rho by over an order of magnitude. Importantly, however, this higher molecular dark activity does not translate into an overall higher cellular dark noise, owing to diminished D190N-Rho content in the rod outer segment. Separately, we found that much of the degeneration and shortened outer-segment length of RhoD190N/WT mouse rods was not averted by ablating rod transducin in phototransduction-also consistent with D190N-Rho's higher isomerization activity not being the primary cause of disease. Instead, the low pigment content, shortened outer-segment length, and a moderate unfolded protein response implicate protein misfolding as the major pathogenic problem. Finally, D190N-Rho also provided some insight into the mechanism of spontaneous pigment excitation.

Stimulus-Driven Retinal Intrinsic Signal Optical Imaging in Mouse Demonstrates a Dominant Rod-Driven Component.

PurposeThe primary hypotheses tested are that (1) there exist stimulus-driven intrinsic optical signals in the mouse retina similar to those previously observed in other species, and (2) these optical signals require an intact rod photoreceptor phototransduction cascade.MethodsWe used 38 wild-type C57BL6J mice and 18 genetic knockout Gnat1−/− mice to study the light-evoked retinal intrinsic response. A custom mouse fundus camera delivered visual stimuli and collected mouse retinal imaging data of changes in retinal reflectance for further analysis. The retina was stimulated in the high-mesopic range with a 505-nm light-emitting diode while also being illuminated with 780-nm near-infrared light.ResultsWild-type C57BL6J mice yielded retinal imaging signals that typically showed a stimulus-driven decrease in retinal reflectance of ∼0.1%, with a time course of several seconds. The signals exhibit spatial specificity in the retina. Overall, the mouse imaging signals are similar in sign and time course to those reported in other mammalian species but are of lower amplitude. In contrast, functional retinal imaging of Gnat1−/− mice that lack a functional rod transducin yielded no such stimulus-driven signals.ConclusionsPrevious studies have not shown which pathway component is essential for the generation of these imaged signals. The absence of the intrinsic signal responses in Gnat1−/− knockout mice indicates that a functional rod transducin is likely to be necessary for generating the retinal intrinsic signals. These studies, to the best of our knowledge, demonstrate for the first time in vivo mouse retinal functional imaging signals similar to those previously shown in other mammalian species.

Diminished Cone Sensitivity in cpfl3 Mice Is Caused by Defective Transducin Signaling.

Purpose Cone photoreceptor function loss 3 (Gnat2cpfl3/cpfl3 or cpfl3) is a mouse model commonly used as a functional cones null from a naturally occurring mutation in the α-subunit of cone transducin (Gnat2). We nevertheless detected robust cone-mediated light responses from cpfl3 animals, which we now explore.MethodsRecordings were made from whole retina and from identified cones with whole-cell patch clamp in retinal slices. Relative levels of GNAT2 protein and numbers of cones in isolated retinas were compared between cpfl3, rod transducin knockout (Gnat1−/−), cpfl3/Gnat1−/− double mutants, and control C57Bl/6J age-matched mice at 4, 9, and 14 weeks of age.ResultsCones from cpfl3 and cpfl3/Gnat1−/− mice 2 to 3 months of age displayed normal dark currents but greatly reduced sensitivity and amplification constants. Responses decayed more slowly than in control (C57Bl/6J) mice, indicating an altered mechanism of inactivation. At dim light intensities rod responses could be recorded from cpfl3 cones, indicating intact rod/cone gap junctions. The cpfl3 and cpfl3/Gnat1−/− mice express two-fold less GNAT2 protein compared with C57 at 4 weeks, and a four-fold decrease by 14 weeks. This is accompanied by a small decrease in the number of cones.Conclusions Cplf3 cones can respond to light with currents of normal amplitude and cannot be assumed to be a Gnat2 null. The decreased sensitivity and amplification rate of cones is not explained by a reduction in GNAT2 protein level, but instead by abnormal interactions of the mutant transducin with rhodopsin and the effector molecule, cGMP phosphodiesterase.

Voltage-clamp recordings of light responses from wild-type and mutant mouse cone photoreceptors.

We describe the first extensive study of voltage-clamp current responses of cone photoreceptors in unlabeled, dark-adapted mouse retina using only the position and appearance of cone somata as a guide. Identification was confirmed from morphology after dye filling. Photocurrents recorded from wild-type mouse cones were biphasic with a fast cone component and a slower rod component. The rod component could be eliminated with dim background light and was not present in mouse lines lacking the rod transducin-α subunit (Gnat1-/- ) or connexin 36 (Cx36-/- ). Cones from Gnat1-/- or Cx36-/- mice had resting membrane potentials between -45 and -55 mV, peak photocurrents of 20-25 picoamps (pA) at a membrane potential Vm = -50 mV, sensitivities 60-70 times smaller than rods, and a total membrane capacitance two to four times greater than rods. The rate of activation (amplification constant) was largely independent of the brightness of the flash and was 1-2 s-2, less than half that of rods. The role of Ca2+-dependent transduction modulation was investigated by recording from cones in mice lacking rod transducin (Gnat1), recoverin, and/or the guanylyl-cyclase-activating proteins (GCAPs). In confirmation of previous results, responses of Gnat1-/- ;Gcaps-/- cones and triple-mutant Gnat1-/- ;Gcaps-/- ;Rv-/- cones recovered more slowly both to light flashes and steps and were more sensitive than cones expressing the GCAPs. Cones from all four mouse lines showed significant recovery and escaped saturation even in bright background light. This recovery occurred too rapidly to be caused by pigment bleaching or metaII decay and appears to reflect some modulation of response inactivation in addition to those produced by recoverin and the GCAPs. Our experiments now make possible a more detailed understanding of the cellular physiology of mammalian cone photoreceptors and the role of conductances in the inner and outer segment in producing cone light responses.

Evolutionary signatures of photoreceptor transmutation in geckos reveal potential adaptation and convergence with snakes.

Most vertebrates use a combination of rod and cone photoreceptors to enable vision in conditions ranging from starlight to direct sunlight. Nocturnal geckos, however, have simplex retinas that contain only rods in terms of morphology and physiology, but these rods are thought to be derived from cones through an evolutionary process known as photoreceptor transmutation. To investigate this, we generated eye transcriptomes and analyzed patterns of phototransduction gene evolution in geckos in comparison to other reptiles. We confirm that geckos have lost several major components of the rod phototransduction pathway, including rod opsin (RH1), which we identified as a pseudogene in multiple genomes. We also identified a partial rod transducin transcript, but found no evidence of the protein in retinal sections. However, we find that geckos express several complete rod phototransduction transcripts in the eye, which may contribute to the rod-like physiology of nocturnal gecko photoreceptors. Finally, we found surprising evidence that even though photoreceptor transmutation evolved independently in geckos and snakes, they have experienced parallel shifts in selective constraint on phototransduction genes. These results implicate adaptive change in the underlying molecular machinery of visual transduction, in addition to the convergent changes in cellular morphology, during photoreceptor transmutation.

A Mixture of U.S. Food and Drug Administration-Approved Monoaminergic Drugs Protects the Retina From Light Damage in Diverse Models of Night Blindness.

PurposeThe purpose of this study was to test the extent of light damage in different models of night blindness and apply these paradigms in testing the therapeutic efficacy of combination therapy by drugs acting on the Gi, Gs, and Gq protein-coupled receptors.MethodsAcute bright light exposure was used to test susceptibility to light damage in mice lacking the following crucial phototransduction proteins: rod transducin (GNAT1), cone transducin (GNAT2), visual arrestin 1 (ARR1), and rhodopsin kinase 1 (GRK1). Mice were intraperitoneally injected with either vehicle or drug combination consisting of metoprolol (β1-receptor antagonist), bromocriptine (dopamine family-2 receptor agonist) and tamsulosin (α1-receptor antagonist) before bright light exposure. Light damage was primarily assessed with optical coherence tomography and inspection of cone population in retinal whole mounts. Retinal inflammation was assessed in a subset of experiments using autofluorescence imaging by scanning laser ophthalmoscopy and by postmortem inspection of microglia and astrocyte activity.ResultsThe Gnat1−/− mice showed slightly increased susceptibility to rod light damage, whereas the Gnat2−/− mice were very resistant. The Arr1−/− and Grk1−/− mice were sensitive for both rod and cone light damage and showed robust retinal inflammation 7 days after bright light exposure. Pretreatment with metoprolol + bromocriptine + tamsulosin rescued the retina in all genetic backgrounds, starting at doses of 0.2 mg/kg metoprolol, 0.02 mg/kg bromocriptine, and 0.01 mg/kg tamsulosin in the Gnat1−/− mice. The therapeutic drug doses increased in parallel with light-damage severity.ConclusionsOur results suggest that congenital stationary night blindness and Oguchi disease patients can be at an elevated risk of the toxic effects of bright light. Furthermore, systems pharmacology drug regimens that stimulate Gi signaling and attenuate Gs and Gq signaling present a promising disease-modifying therapy for photoreceptor degenerative diseases.

Elementary response triggered by transducin in retinal rods

G protein-coupled receptor (GPCR) signaling is crucial for many physiological processes. A signature of such pathways is high amplification, a concept originating from retinal rod phototransduction, whereby one photoactivated rhodopsin molecule (Rho*) was long reported to activate several hundred transducins (GT*s), each then activating a cGMP-phosphodiesterase catalytic subunit (GT*·PDE*). This high gain at the Rho*-to-GT* step has been challenged more recently, but estimates remain dispersed and rely on some nonintact rod measurements. With two independent approaches, one with an extremely inefficient mutant rhodopsin and the other with WT bleached rhodopsin, which has exceedingly weak constitutive activity in darkness, we obtained an estimate for the electrical effect from a single GT*·PDE* molecular complex in intact mouse rods. Comparing the single-GT*·PDE* effect to the WT single-photon response, both in Gcaps-/- background, gives an effective gain of only ∼12-14 GT*·PDE*s produced per Rho*. Our findings have finally dispelled the entrenched concept of very high gain at the receptor-to-G protein/effector step in GPCR systems.

ARL3 regulates trafficking of prenylated phototransduction proteins to the rod outer segment.

The small GTPase, ADP-ribosylation factor-like 3 (ARL3), has been proposed to participate in the transport of proteins in photoreceptor cells. Moreover, it has been implicated in the pathogenesis associated with X-linked retinitis pigmentosa (XLRP) resulting from mutations in the ARL3 GTPase activating protein, retinitis pigmentosa 2 (RP2). To determine the importance of ARL3 in rod photoreceptor cells, we generated transgenic mice expressing a dominant active form of ARL3 (ARL3-Q71L) under a rod-specific promoter. ARL3-Q71L animals exhibited extensive rod cell death after post-natal day 30 (PN30) and degeneration was complete by PN70. Prior to the onset of cell death, rod photoresponse was significantly reduced along with a robust decrease in rod phosphodiesterase 6 (PDE6) and G-protein receptor kinase-1 (GRK1) levels. Furthermore, assembled phosphodiesterase-6 (PDE6) subunits, rod transducin and G-protein receptor kinase-1 (GRK1) accumulated on large punctate structures within the inner segment in ARL3-Q71L retina. Defective trafficking of prenylated proteins is likely due to sequestration of prenyl binding protein δ (PrBPδ) by ARL3-Q71L as we demonstrate a specific interaction between these proteins in the retina. Unexpectedly, our studies also revealed a novel role for ARL3 in the migration of photoreceptor nuclei. In conclusion, this study identifies ARL3 as a key player in prenylated protein trafficking in rod photoreceptor cells and establishes the potential role for ARL3 dysregulation in the pathogenesis of RP2-related forms of XLRP.

Evolutionary transformation of rod photoreceptors in the all-cone retina of a diurnal garter snake

Vertebrate retinas are generally composed of rod (dim-light) and cone (bright-light) photoreceptors with distinct morphologies that evolved as adaptations to nocturnal/crepuscular and diurnal light environments. Over 70 years ago, the "transmutation" theory was proposed to explain some of the rare exceptions in which a photoreceptor type is missing, suggesting that photoreceptors could evolutionarily transition between cell types. Although studies have shown support for this theory in nocturnal geckos, the origins of all-cone retinas, such as those found in diurnal colubrid snakes, remain a mystery. Here we investigate the evolutionary fate of the rods in a diurnal garter snake and test two competing hypotheses: (i) that the rods, and their corresponding molecular machinery, were lost or (ii) that the rods were evolutionarily modified to resemble, and function, as cones. Using multiple approaches, we find evidence for a functional and unusually blue-shifted rhodopsin that is expressed in small single "cones." Moreover, these cones express rod transducin and have rod ultrastructural features, providing strong support for the hypothesis that they are not true cones, as previously thought, but rather are modified rods. Several intriguing features of garter snake rhodopsin are suggestive of a more cone-like function. We propose that these cone-like rods may have evolved to regain spectral sensitivity and chromatic discrimination as a result of ancestral losses of middle-wavelength cone opsins in early snake evolution. This study illustrates how sensory evolution can be shaped not only by environmental constraints but also by historical contingency in forming new cell types with convergent functionality.

A novel homozygous truncating GNAT1 mutation implicated in retinal degeneration

BackgroundThe GNAT1 gene encodes the α subunit of the rod transducin protein, a key element in the rod phototransduction cascade. Variants in GNAT1 have been implicated in stationary night-blindness in...

Knockout ofRP2decreases GRK1 and rod transducin subunits and leads to photoreceptor degeneration in zebrafish

Retinitis pigmentosa (RP) affects about 1.8 million individuals worldwide. X-linked retinitis pigmentosa (XLRP) is one of the most severe forms of RP. Nearly 85% of XLRP cases are caused by mutations in the X-linked retinitis pigmentosa 2 (RP2) and RPGR. RP2 has been considered to be a GTPase activator protein for ARL3 and to play a role in the traffic of ciliary proteins. The mechanism of how RP2 mutations cause RP is still unclear. In this study, we generated an RP2 knockout zebrafish line using transcription activator-like effector nuclease technology. Progressive retinal degeneration could be observed in the mutant zebrafish. The degeneration of rods' outer segments (OSs) is predominant, followed by the degeneration of cones' OS. These phenotypes are similar to the characteristics of RP2 patients, and also partly consistent with the phenotypes of RP2 knockout mice and morpholino-mediated RP2 knockdown zebrafish. For the first time, we found RP2 deletion leads to decreased protein levels and abnormal retinal localizations of GRK1 and rod transducin subunits (GNAT1 and GNB1) in zebrafish. Furthermore, the distribution of the total farnesylated proteins in zebrafish retina is also affected by RP2 ablation. These molecular alterations observed in the RP2 knockout zebrafish might probably be responsible for the gradual loss of the photoreceptors' OSs. Our work identified the progression of retinal degeneration in RP2 knockout zebrafish, provided a foundation for revealing the pathogenesis of RP caused by RP2 mutations, and would help to develop potential therapeutics against RP in further studies.

Confirming a prediction of the calcium hypothesis of photoreceptor aging in mice

Prior work in healthy rats supported a calcium hypothesis of photoreceptor aging, wherein progressive age-related declines in photopic vision are explainable by the extent of earlier escalating d-cis-diltiazem–insensitive increases in photoreceptor L-type calcium channel (LTCC) activity in vivo. Unlike rats, healthy mice have relatively stable photopic vision until after 18 months of age. We therefore hypothesized that photoreceptor LTCC activity in mice would not progressively increase until after 18 months. In 2–5, 10, 18, and 26 months male C57Bl/6J mice, photoreceptor LTCC activity and retinal thickness were evaluated in vivo (manganese-enhanced magnetic resonance imaging) with some groups also treated with d-cis-diltiazem; visual performance was evaluated (optokinetic tracking). Data were calibrated for cone-only responses using mice without rod transducin (GNAT1−/−). Photopic vision was stable until after 18 months without retinal thinning or progressive increases in retinal manganese uptake. We measured an uptake spike at 10 months. This spike, unlike that in the rat, was diltiazem sensitive in the dark and diltiazem insensitive in the light. Between dark and light, uptake in inner retina of older mice was unequal (unlike that in 2–5 months mice); outer retinal uptake was similar to that in 2–5 months mice. Stable murine photopic visual performance and nonescalating photoreceptor LTCC activity before 18 months of age were consistent with a prediction of the calcium hypothesis. Stark differences in the temporal evolution of mouse and rat photoreceptor LTCC activity suggest the need for personalized identification of the retinal mechanisms contributing to declines in photopic vision to ensure success of future treatment efforts.

Adaptation to Background Light Enables Contrast Coding at Rod Bipolar Cell Synapses

Rod photoreceptors contribute to vision over an ∼ 6-log-unit range of light intensities. The wide dynamic range of rod vision is thought to depend upon light intensity-dependent switching between two parallel pathways linking rods to ganglion cells: a rod → rod bipolar (RB) cell pathway that operates at dim backgrounds and a rod → cone → cone bipolar cell pathway that operates at brighter backgrounds. We evaluated this conventional model of rod vision by recording rod-mediated light responses from ganglion and AII amacrine cells and by recording RB-mediated synaptic currents from AII amacrine cells in mouse retina. Contrary to the conventional model, we found that the RB pathway functioned at backgrounds sufficient to activate the rod → cone pathway. As background light intensity increased, the RB's role changed from encoding the absorption of single photons to encoding contrast modulations around mean luminance. This transition is explained by the intrinsic dynamics of transmission from RB synapses.

Photoreceptors in whirler mice show defective transducin translocation and are susceptible to short-term light/dark changes-induced degeneration

Usher syndrome combines congenital hearing loss and retinitis pigmentosa (RP). Mutations in the whirlin gene (DFNB31/WHRN) cause a subtype of Usher syndrome (USH2D). Whirler mice have a defective whirlin gene. They have inner ear defects but usually do not develop retinal degeneration. Here we report that, in whirler mouse photoreceptors, the light-activated rod transducin translocation is delayed and its activation threshold is shifted to a higher level. Rhodopsin mis-localization is observed in rod inner segments. Continuous moderate light exposure can induce significant rod photoreceptor degeneration. Whirler mice reared under a 1500 lux light/dark cycle also develop severe photoreceptor degeneration. Previously, we have reported that shaker1 mice, a USH1B model, show moderate light-induced photoreceptor degeneration with delayed transducin translocation. Here, we further show that, in both whirler and shaker1 mice, short-term moderate light/dark changes can induce rod degeneration as severe as that induced by continuous light exposure. The results from shaker1 and whirler mice suggest that defective transducin translocation may be functionally related to light-induced degeneration, and these two symptoms may be caused by defects in Usher protein function in rods. Furthermore, these results indicate that both Usher syndrome mouse models possess a light-induced retinal phenotype and may share a closely related pathobiological mechanism.

Functional Comparison of Rod and Cone Gαt on the Regulation of Light Sensitivity*

The signaling cascades mediated by G protein-coupled receptors (GPCRs) exhibit a wide spectrum of spatial and temporal response properties to fulfill diverse physiological demands. However, the mechanisms that shape the signaling response of the GPCR are not well understood. In this study, we replaced cone transducin α (cTα) for rod transducin α (rTα) in rod photoreceptors of transgenic mice, which also express S opsin, to evaluate the role of Gα subtype on signal amplification from different GPCRs in the same cell; such analysis may explain functional differences between retinal rod and cone photoreceptors. We showed that ectopically expressed cTα 1) forms a heterotrimeric complex with rod Gβ(1)γ(1), 2) substitutes equally for rTα in generating photoresponses initiated by either rhodopsin or S-cone opsin, and 3) exhibited similar light-activated translocation as endogenous rTα in rods and endogenous cTα in cones. Thus, rTα and cTα appear functionally interchangeable. Interestingly, light sensitivity appeared to correlate with the concentration of cTα when expression is reduced below 35% of normal. However, quantification of endogenous cTα concentration in cones showed a higher level to rTα in rods. Thus, reduced sensitivity in cones cannot be explained by reduced coupling efficiency between the GPCR and G protein or a lower concentration of G protein in cones versus rods.

Expression and subcellular distribution of UNC119a, a protein partner of transducin α subunit in rod photoreceptors

A recently discovered interaction of rod transducin α subunit (Gαt1) with UNC119a is thought to be important for transducin trafficking in photoreceptors. In this study, we analyzed the subcellular distribution of UNC119a under different conditions of illumination in vivo. Analyses by immunofluorescence and Western blotting of retina serial tangential sections demonstrated that UNC119a resides predominantly in the rod inner segment, with a small fraction of UNC119a also appearing to infiltrate the rod outer segment. Such a distribution is consistent with the proposed role of UNC119a in facilitating transducin transport from the rod inner segment to the outer segment in the dark. In addition, UNC119a was present in smaller amounts in the cell body and synaptic region of rods. The profile of UNC119a subcellular distribution remained largely unchanged under all tested conditions of illumination, and correlated with the profile of Gαt1 following its light-dependent translocation. Quantification by Western blotting suggested that mouse retina contains ~17pmol of UNC119a, giving a ~1 to 4 molar ratio of UNC119a to Gαt1. Hence, light-translocated Gαt1 can serve as a major partner of UNC119a. Supporting this role, the levels of UNC119a were downregulated by about 2-fold in mouse retina lacking Gαt1. As a dominant partner, Gαt1 may potentially modulate the function of other known UNC119a-interacting proteins involved in photoreceptor ciliary trafficking and synaptic regulation, in a light-dependent manner.

Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light whereas cones operate continuously under rapidly changing bright light conditions. Absorption of light by rod- and cone-specific visual pigments in the outer segments of photoreceptors triggers a phototransduction cascade that eventually leads to closure of cyclic nucleotide-gated channels on the plasma membrane and cell hyperpolarization. This light-induced change in membrane current and potential can be registered as a photoresponse, by either classical suction electrode recording technique or by transretinal electroretinogram recordings (ERG) from isolated retinas with pharmacologically blocked postsynaptic response components. The latter method allows drug-accessible long-lasting recordings from mouse photoreceptors and is particularly useful for obtaining stable photoresponses from the scarce and fragile mouse cones. In the case of cones, such experiments can be performed both in dark-adapted conditions and following intense illumination that bleaches essentially all visual pigment, to monitor the process of cone photosensitivity recovery during dark adaptation. In this video, we will show how to perform rod- and M/L-cone-driven transretinal recordings from dark-adapted mouse retina. Rod recordings will be carried out using retina of wild type (C57Bl/6) mice. For simplicity, cone recordings will be obtained from genetically modified rod transducin α-subunit knockout (Tα(-/-)) mice which lack rod signaling(8).

CNGA3 Deficiency Affects Cone Synaptic Terminal Structure and Function and Leads to Secondary Rod Dysfunction and Degeneration

To investigate rod function and survival after cone dysfunction and degeneration in a mouse model of cone cyclic nucleotide-gated (CNG) channel deficiency. Rod function and survival in mice with cone CNG channel subunit CNGA3 deficiency (CNGA3-/- mice) were evaluated by electroretinographic (ERG), morphometric, and Western blot analyses. The arrangement, integrity, and ultrastructure of photoreceptor terminals were investigated by immunohistochemistry and electron microscopy. The authors found loss of cone function and cone death accompanied by impairment of rods and rod-driven signaling in CNGA3-/- mice. Scotopic ERG b-wave amplitudes were reduced by 15% at 1 month, 30% at 6 months, and 40% at 9 months and older, while scotopic a-wave amplitudes were decreased by 20% at 9 months, compared with ERGs of age-matched wild-type mice. Outer nuclear layer thickness in CNGA3-/- retina was reduced by 15% at 12 months compared with age-matched wild-type controls. This was accompanied by a 30%-40% reduction in expression of rod-specific proteins, including rhodopsin, rod transducin α-subunit, and glutamic acid-rich protein (GARP). Cone terminals in the CNGA3-/- retina showed a progressive loss of neurochemical and ultrastructural integrity. Abnormalities were observed as early as 1 month. Disorganized rod terminal ultrastructure was noted by 12 months. These findings demonstrate secondary rod impairment and degeneration after cone degeneration in mice with cone CNG channel deficiency. Loss of cone phototransduction accompanies the compromised integrity of cone terminals. With time, rod synaptic structure, function, and viability also become compromised.