Cone-Driven Retinal Responses Are Shaped by Rod But Not Cone HCN1.

Signal integration of converging neural circuits is poorly understood. One example is in the retina where the integration of rod and cone signaling is responsible for the large dynamic range of vision. The relative contribution of rods versus cones is dictated by a complex function involving background light intensity and stimulus temporal frequency. One understudied mechanism involved in coordinating rod and cone signaling onto the shared retinal circuit is the hyperpolarization activated current (Ih) mediated by hyperpolarization-activated cyclic nucleotide-gated 1 (HCN1) channels expressed in rods and cones. Ih opposes membrane hyperpolarization driven by activation of the phototransduction cascade and modulates the strength and kinetics of the photoreceptor voltage response. We examined conditional knock-out (KO) of HCN1 from mouse rods using electroretinography (ERG). In the absence of HCN1, rod responses are prolonged in dim light which altered the response to slow modulation of light intensity both at the level of retinal signaling and behavior. Under brighter intensities, cone-driven signaling was suppressed. To our surprise, conditional KO of HCN1 from mouse cones had no effect on cone-mediated signaling. We propose that Ih is dispensable in cones because of the high level of temporal control of cone phototransduction. Thus, HCN1 is required for cone-driven retinal signaling only indirectly by modulating the voltage response of rods to limit their output.SIGNIFICANCE STATEMENT Hyperpolarization gated hyperpolarization-activated cyclic nucleotide-gated 1 (HCN1) channels carry a feedback current that helps to reset light-activated photoreceptors. Using conditional HCN1 knock-out (KO) mice we show that ablating HCN1 from rods allows rods to signal in bright light when they are normally shut down. Instead of enhancing vision this results in suppressing cone signaling. Conversely, ablating HCN1 from cones was of no consequence. This work provides novel insights into the integration of rod and cone signaling in the retina and challenges our assumptions about the role of HCN1 in cones.

Temporal Contrast Sensitivity Increases despite Photoreceptor Degeneration in a Mouse Model of Retinitis Pigmentosa.

The detection of temporal variations in amplitude of light intensity, or temporal contrast sensitivity (TCS), depends on the kinetics of rod photoresponse recovery. Uncharacteristically fast rod recovery kinetics are facets of both human patients and transgenic animal models with a P23H rhodopsin mutation, a prevalent cause of retinitis pigmentosa (RP). Here, we show that mice with this mutation (RhoP23H/+) exhibit an age-dependent and illumination-dependent enhancement in TCS compared with controls. At retinal illumination levels producing ≥1000 R*/rod/s or more, postnatal day 30 (P30) RhoP23H/+ mice exhibit a 1.2-fold to 2-fold increase in retinal and optomotor TCS relative to controls in response to flicker frequencies of 3, 6, and 12 Hz despite significant photoreceptor degeneration and loss of flash electroretinogram (ERG) b-wave amplitude. Surprisingly, the TCS of RhoP23H/+ mice further increases as degeneration advances. Enhanced TCS is also observed in a second model (rhodopsin heterozygous mice, Rho+/-) with fast rod recovery kinetics and no apparent retinal degeneration. In both mouse models, enhanced TCS is explained quantitatively by a comprehensive model that includes photoresponse recovery kinetics, density and collecting area of degenerating rods. Measurement of TCS may be a non-invasive early diagnostic tool indicative of rod dysfunction in some forms of retinal degenerative disease.

Molecular and functional architecture of the mouse photoreceptor network.

Mouse photoreceptors are electrically coupled via gap junctions, but the relative importance of rod/rod, cone/cone, or rod/cone coupling is unknown. Furthermore, while connexin36 (Cx36) is expressed by cones, the identity of the rod connexin has been controversial. We report that FACS-sorted rods and cones both express Cx36 but no other connexins. We created rod- and cone-specific Cx36 knockout mice to dissect the photoreceptor network. In the wild type, Cx36 plaques at rod/cone contacts accounted for more than 95% of photoreceptor labeling and paired recordings showed the transjunctional conductance between rods and cones was ~300 pS. When Cx36 was eliminated on one side of the gap junction, in either conditional knockout, Cx36 labeling and rod/cone coupling were almost abolished. We could not detect direct rod/rod coupling, and cone/cone coupling was minor. Rod/cone coupling is so prevalent that indirect rod/cone/rod coupling via the network may account for previous reports of rod coupling.

Rod Photoreceptors Signal Fast Changes in Daylight Levels Using a Cx36-Independent Retinal Pathway in Mouse.

Temporal contrast detected by rod photoreceptors is channeled into multiple retinal rod pathways that ultimately connect to cone photoreceptor pathways via Cx36 gap junctions or via chemical synapses. However, we do not yet understand how the different rod pathways contribute to the perception of temporal contrast (changes in luminance with time) at mesopic light levels, where both rods and cones actively respond to light. Here, we use a forced-choice, operant behavior assay to investigate rod-driven, temporal contrast sensitivity (TCS) in mice of either sex. Transgenic mice with desensitized cones (GNAT2 cpfl3 line) were used to identify rod contributions to TCS in mesopic lights. We found that at low mesopic lights (400 photons/s/µm2 at the retina), control and GNAT2 cpfl3 mice had similar TCS. Surprisingly, at upper mesopic lights (8000 photons/s/µm2), GNAT2 cpfl3 mice exhibited a relative reduction in TCS to low (<12 Hz) while maintaining normal TCS to high (12-36 Hz) temporal frequencies. The rod-driven responses to high temporal frequencies developed gradually over time (>30 min). Furthermore, the TCS of GNAT2 cpfl3 and GNAT2 cpfl3 ::Cx36-/- mice matched closely, indicating that transmission of high-frequency signals (1) does not require the rod-cone Cx36 gap junctions as has been proposed in the past; and (2) a Cx36-independent rod pathway(s) (e.g., direct rod to OFF cone bipolar cell synapses and/or glycinergic synapses from AII amacrine cells to OFF ganglion cells) is sufficient for fast, mesopic rod-driven vision. These findings extend our understanding of the link between visual circuits and perception in mouse.SIGNIFICANCE STATEMENT The contributions of specific retinal pathways to visual perception are not well understood. We found that the temporal processing properties of rod-driven vision in mice change significantly with light level. In dim lights, rods relay relatively slow temporal variations. However, in daylight conditions, rod pathways exhibit high sensitivity to fast but not to slow temporal variations, whereas cone-driven responses supplement the loss in rod-driven sensitivity to slow temporal variations. Our findings highlight the dynamic interplay of rod- and cone-driven vision as light levels rise from night to daytime levels. Furthermore, the fast, rod-driven signals do not require the rod-to-cone Cx36 gap junctions as proposed in the past, but rather, can be relayed by alternative Cx36-independent rod pathways.

Rod Photoresponse Kinetics Limit Temporal Contrast Sensitivity in Mesopic Vision.

The mammalian visual system operates over an extended range of ambient light levels by switching between rod and cone photoreceptors. Rod-driven vision is sluggish, highly sensitive, and operates in dim or scotopic lights, whereas cone-driven vision is brisk, less sensitive, and operates in bright or photopic lights. At intermediate or mesopic lights, vision transitions seamlessly from rod-driven to cone-driven, despite the profound differences in rod and cone response dynamics. The neural mechanisms underlying such a smooth handoff are not understood. Using an operant behavior assay, electrophysiological recordings, and mathematical modeling we examined the neural underpinnings of the mesopic visual transition in mice of either sex. We found that rods, but not cones, drive visual sensitivity to temporal light variations over much of the mesopic range. Surprisingly, speeding up rod photoresponse recovery kinetics in transgenic mice improved visual sensitivity to slow temporal variations, in the range where perceptual sensitivity is governed by Weber's law of sensation. In contrast, physiological processes acting downstream from phototransduction limit sensitivity to high frequencies and temporal resolution. We traced the paradoxical control of visual temporal sensitivity to rod photoresponses themselves. A scenario emerges where perceptual sensitivity is limited by: (1) the kinetics of neural processes acting downstream from phototransduction in scotopic lights, (2) rod response kinetics in mesopic lights, and (3) cone response kinetics as light levels rise into the photopic range.SIGNIFICANCE STATEMENT Our ability to detect flickering lights is constrained by the dynamics of the slowest step in the visual pathway. Cone photoresponse kinetics limit visual temporal sensitivity in bright (photopic) lights, whereas mechanisms in the inner retina limit sensitivity in dim (scotopic) lights. The neural mechanisms underlying the transition between scotopic and photopic vision in mesopic lights, when both rods are cones are active, are unknown. This study provides a missing link in this mechanism by establishing that rod photoresponse kinetics limit temporal sensitivity during the mesopic transition. Surprisingly, this range is where Weber's Law of Sensation governs temporal contrast sensitivity in mouse. Our results will help guide future studies of complex and dynamic interactions between rod-cone signals in the mesopic retina.

Visual Temporal Contrast Sensitivity in the Behaving Mouse Shares Fundamental Properties with Human Psychophysics.

The mammalian visual system has a remarkable capacity to detect differences in contrast across time, which is known as temporal contrast sensitivity (TCS). Details of the underlying neural mechanisms are rapidly emerging as a result of a series of elegant electrophysiological studies performed largely with the mouse as an experimental model. However, rigorous psychophysical methods are necessary to pair the electrophysiology with temporal visual behavior in mouse. The optomotor response is frequently used as a proxy for retinal temporal processing in rodents. However, subcortical reflexive pathways drive the optomotor response rather than cortical decision-making areas. To address this problem, we have developed an operant behavior assay that measures TCS in behaving mice. Mice were trained to perform a forced-choice visual task and were tested daily on their ability to distinguish flickering from nonflickering overhead lights. Correct responses (Hit and Correct Rejections) were rewarded. Contrast, temporal frequency, and mean illumination of the flicker were the independent variables. We validated and applied the theory of signal detection to estimate the discriminability factor (d´), a measure of performance that is independent of response bias and motivation. The empirical contrast threshold was defined as the contrast necessary to elicit d´ = 1 and TCS as the inverse of the contrast threshold. With this approach, we established in the mouse a model of human vision that shares fundamental properties of human temporal psychophysics such as Weber adaptation in response to low temporal frequency flicker and illumination-dependent increases in critical flicker frequency as predicted by the Ferry-Porter law.

A system to measure the pupil response to steady lights in freely behaving mice.

BACKGROUND: Transgenic mice are widely used for the study of basic visual function and retinal disease, including in psychophysical tests. Mice have a robust pupillary light reflex that controls the amount of light that enters the eye, and the attenuating effects of the pupil must be considered during such tests. Measurement of the size of pupils at various luminance levels requires that mice remain stable over prolonged periods of time; however, sedation of mice with anesthesia and/or manual restraint can influence the size of their pupils. NEW METHOD: We present a system to measure the pupillary light response to steady lights of freely behaving mice using a custom-built, portable device that automatically acquires close-up images of their eyes. The device takes advantage of the intrinsic nature of mice to inspect objects of interest and can be used to measure pupillary responses in optomotor or operant behavior testing chambers. RESULTS: The size of the pupils in freely behaving mice decreased gradually with luminance from a maximal area in the dark of 3.8mm2 down to a minimum 0.14mm2 at 80 scotopic cd/m2. The data was well fit with a Hill equation with Lo equal to 0.21cd/m2 and coefficient h=0.48. COMPARISON WITH EXISTING METHODS: These values agree with prior measurements of the pupillary response of unrestrained mice that use more laborious and time consuming approaches. CONCLUSIONS: Our new method facilitates practical, straightforward and accurate measurements of pupillary responses made under the same experimental conditions as those used during psychophysical testing.

Onecut1 is essential for horizontal cell genesis and retinal integrity.

Horizontal cells are interneurons that synapse with photoreceptors in the outer retina. Their genesis during development is subject to regulation by transcription factors in a hierarchical manner. Previously, we showed that Onecut 1 (Oc1), an atypical homeodomain transcription factor, is expressed in developing horizontal cells (HCs) and retinal ganglion cells (RGCs) in the mouse retina. Herein, by knocking out Oc1 specifically in the developing retina, we show that the majority (∼80%) of HCs fail to form during early retinal development, implying that Oc1 is essential for HC genesis. However, no other retinal cell types, including RGCs, were affected in the Oc1 knock-out. Analysis of the genetic relationship between Oc1 and other transcription factor genes required for HC development revealed that Oc1 functions downstream of FoxN4, in parallel with Ptf1a, but upstream of Lim1 and Prox1. By in utero electroporation, we found that Oc1 and Ptf1a together are not only essential, but also sufficient for determination of HC fate. In addition, the synaptic connections in the outer plexiform layer are defective in Oc1-null mice, and photoreceptors undergo age-dependent degeneration, indicating that HCs are not only an integral part of the retinal circuitry, but also are essential for the survival of photoreceptors. In sum, these results demonstrate that Oc1 is a critical determinant of HC fate, and reveal that HCs are essential for photoreceptor viability, retinal integrity, and normal visual function.

Loss of scotopic contrast sensitivity in the optomotor response of diabetic mice.

PURPOSE: Diabetes reduces retinal and visual sensitivity to dim light flashes. However, the impact of diabetes on contrast sensitivity in dim light is unknown. Based on the lowered visual sensitivity previously observed, we hypothesized that contrast sensitivity would similarly be reduced. We therefore examined scotopic contrast sensitivity of the optomotor response in the Ins2(Akita/+) mouse model of type 1 diabetes. METHODS: A longitudinal study of spatial and temporal contrast sensitivity in Ins2(Akita/+) mice and wild-type Ins2(+/+) littermates was conducted. Contrast sensitivity of the optomotor response to rotating gratings of various spatial and temporal frequencies was measured at a dim luminance level (2.6 · 10(-5) cd/m2) known to elicit rod- but not cone-driven responses. RESULTS: An early, progressive loss in scotopic contrast sensitivity was observed in Ins2(Akita/+) mice that was absent from Ins2(+/+) littermate controls. The loss in contrast sensitivity developed over a 3- to 4-month period after the onset of hyperglycemia. Ins2(Akita/+) mice exhibited a nonselective 40% loss in sensitivity to all spatial frequencies and a selective loss in sensitivity to fast but not to slow varying gratings (temporal frequencies > 0.1 Hz or, equivalently, speeds > 3 deg/s). Such losses in sensitivity were prevented by glycemic control with insulin treatment. CONCLUSIONS: An association between a model of type 1 diabetes and scotopic contrast sensitivity of the optomotor response is indicated. Ins2(Akita/+) mice exhibit a uniform loss in optomotor contrast sensitivity to all spatial frequencies that, unexpectedly, can be explained as being secondary to a retinal or central loss in sensitivity to high temporal frequencies.

AAV-mediated gene therapy in the guanylate cyclase (RetGC1/RetGC2) double knockout mouse model of Leber congenital amaurosis.

Mutations in GUCY2D are associated with recessive Leber congenital amaurosis-1 (LCA1). GUCY2D encodes photoreceptor-specific, retinal guanylate cyclase-1 (RetGC1). Reports of retinal degeneration in LCA1 are conflicting; some describe no obvious degeneration and others report loss of both rods and cones. Proof of concept studies in models representing the spectrum of phenotypes is warranted. We have previously demonstrated adeno-associated virus (AAV)-mediated RetGC1 is therapeutic in GC1ko mice, a model exhibiting loss of cones only. The purpose of this study was to characterize AAV-mediated gene therapy in the RetGC1/RetGC2 double knockout (GCdko) mouse, a model lacking rod and cone function and exhibiting progressive loss of both photoreceptor subclasses. Use of this model also allowed for the evaluation of the functional efficiency of transgenic RetGC1 isozyme. Subretinal delivery of AAV8(Y733F) vector containing the human rhodopsin kinase (hGRK1) promoter driving murine Gucy2e was performed in GCdko mice at various postnatal time points. Treatment resulted in restoration of rod and cone function at all treatment ages and preservation of retinal structure in GCdko mice treated as late as 7 weeks of age. Functional gains and structural preservation were stable for at least 1 year. Treatment also conferred cortical- and subcortical-based visually-guided behavior. Functional efficiency of transgenic RetGC1 was indistinguishable from that of endogenous isozyme in congenic wild-type (WT) mice. This study clearly demonstrates AAV-mediated RetGC1 expression restores function to and preserves structure of rod and cone photoreceptors in a degenerative model of retinal guanylate cyclase deficiency, further supporting development of an AAV-based vector for treatment of LCA1.

The relationship between slow photoresponse recovery rate and temporal resolution of vision.

The rate at which photoreceptors recover from excitation is thought to be critical for setting the temporal resolution of vision. Indeed, mutations in RGS9 (regulator of G-protein signaling 9) and R9AP (RGS9 anchor protein) proteins mediating rapid photoresponse recovery impair patients' ability to see moving objects. In this study, we analyzed temporal properties of retinal sensitivity and spatiotemporal aspects of visual behavior in R9AP knock-out mice. Surprisingly, we have found that this knock-out does not affect dim-light vision mediated by rods acting as single-photon counters. Under these conditions, vision was also unaffected in mice overexpressing R9AP in rods, which causes accelerated photoresponse recovery. However, in brighter light, slow photoresponse recovery in rods and cones impaired visual responses to high temporal frequency stimuli, as reported for the daylight vision of human patients. Therefore, the speed of photoresponse recovery can affect temporal resolution and motion detection when photoreceptors integrate signals from multiple photons but not when they act as single-photon counters.

AAV-mediated cone rescue in a naturally occurring mouse model of CNGA3-achromatopsia.

Achromatopsia is a rare autosomal recessive disorder which shows color blindness, severely impaired visual acuity, and extreme sensitivity to bright light. Mutations in the alpha subunits of the cone cyclic nucleotide-gated channels (CNGA3) are responsible for about 1/4 of achromatopsia in the U.S. and Europe. Here, we test whether gene replacement therapy using an AAV5 vector could restore cone-mediated function and arrest cone degeneration in the cpfl5 mouse, a naturally occurring mouse model of achromatopsia with a CNGA3 mutation. We show that gene therapy leads to significant rescue of cone-mediated ERGs, normal visual acuities and contrast sensitivities. Normal expression and outer segment localization of both M- and S-opsins were maintained in treated retinas. The therapeutic effect of treatment lasted for at least 5 months post-injection. This study is the first demonstration of substantial, relatively long-term restoration of cone-mediated light responsiveness and visual behavior in a naturally occurring mouse model of CNGA3 achromatopsia. The results provide the foundation for development of an AAV5-based gene therapy trial for human CNGA3 achromatopsia.

Partial rescue of retinal function in chronically hypoglycemic mice.

PURPOSE: Mice rendered hypoglycemic by a null mutation in the glucagon receptor gene Gcgr display late-onset retinal degeneration and loss of retinal sensitivity. Acute hyperglycemia induced by dextrose ingestion does not restore their retinal function, which is consistent with irreversible loss of vision. The goal of this study was to establish whether long-term administration of high dietary glucose rescues retinal function and circuit connectivity in aged Gcgr-/- mice. METHODS: Gcgr-/- mice were administered a carbohydrate-rich diet starting at 12 months of age. After 1 month of treatment, retinal function and structure were evaluated using electroretinographic (ERG) recordings and immunohistochemistry. RESULTS: Treatment with a carbohydrate-rich diet raised blood glucose levels and improved retinal function in Gcgr-/- mice. Blood glucose increased from moderate hypoglycemia to euglycemic levels, whereas ERG b-wave sensitivity improved approximately 10-fold. Because the b-wave reflects the electrical activity of second-order cells, we examined for changes in rod-to-bipolar cell synapses. Gcgr-/- retinas have 20% fewer synaptic pairings than Gcgr+/- retinas. Remarkably, most of the lost synapses were located farthest from the bipolar cell body, near the distal boundary of the outer plexiform layer (OPL), suggesting that apical synapses are most vulnerable to chronic hypoglycemia. Although treatment with the carbohydrate-rich diet restored retinal function, it did not restore these synaptic contacts. CONCLUSIONS: Prolonged exposure to diet-induced euglycemia improves retinal function but does not reestablish synaptic contacts lost by chronic hypoglycemia. These results suggest that retinal neurons have a homeostatic mechanism that integrates energetic status over prolonged periods of time and allows them to recover functionality despite synaptic loss.

Long-term retinal function and structure rescue using capsid mutant AAV8 vector in the rd10 mouse, a model of recessive retinitis pigmentosa.

The retinal degeneration 10 (rd10) mouse is a well-characterized model of autosomal recessive retinitis pigmentosa (RP), which carries a spontaneous mutation in the ß subunit of rod cGMP-phosphodiesterase (PDEß). Rd10 mouse exhibits photoreceptor dysfunction and rapid rod photoreceptor degeneration followed by cone degeneration and remodeling of the inner retina. Here, we evaluate whether gene replacement using the fast-acting tyrosine-capsid mutant AAV8 (Y733F) can provide long-term therapy in this model. AAV8 (Y733F)-smCBA-PDEß was subretinally delivered to postnatal day 14 (P14) rd10 mice in one eye only. Six months after injection, spectral domain optical coherence tomography (SD-OCT), electroretinogram (ERG), optomotor behavior tests, and immunohistochemistry showed that AAV8 (Y733F)-mediated PDEß expression restored retinal function and visual behavior and preserved retinal structure in treated rd10 eyes for at least 6 months. This is the first demonstration of long-term phenotypic rescue by gene therapy in an animal model of PDEß-RP. It is also the first example of tyrosine-capsid mutant AAV8 (Y733F)-mediated correction of a retinal phenotype. These results lay the groundwork for the development of PDEß-RP gene therapy trial and suggest that tyrosine-capsid mutant AAV vectors may be effective for treating other rapidly degenerating models of retinal degeneration.

Functional and behavioral restoration of vision by gene therapy in the guanylate cyclase-1 (GC1) knockout mouse.

BACKGROUND: Recessive mutations in guanylate cyclase-1 (Gucy2d) are associated with severe, early onset Leber congenital amaurosis-1(LCA1). Gucy2d encodes guanylate cyclase (GC1) is expressed in photoreceptor outer segment membranes and produces cGMP in these cells. LCA1 patients present in infancy with severely impaired vision and extinguished electroretinogram (ERG) but retain some photoreceptors in both their macular and peripheral retina for years. Like LCA1 patients, loss of cone function in the GC1 knockout (GC1KO) mouse precedes cone degeneration. The purpose of this study was to test whether delivery of functional GC1 to cone cells of the postnatal GC1KO mouse could restore function to these cells. METHODOLOGY/PRINCIPAL FINDINGS: Serotype 5 AAV vectors containing either a photoreceptor-specific, rhodopsin kinase (hGRK1) or ubiquitous (smCBA) promoter driving expression of wild type murine GC1 were subretinally delivered to one eye of P14 GC1KO mice. Visual function (ERG) was analyzed in treated and untreated eyes until 3 months post injection. AAV-treated, isogenic wild type and uninjected control mice were evaluated for restoration of visual behavior using optomotor testing. At 3 months post injection, all animals were sacrificed, and their treated and untreated retinas assayed for expression of GC1 and localization of cone arrestin. Cone-mediated function was restored to treated eyes of GC1KO mice (ERG amplitudes were approximately 45% of normal). Treatment effect was stable for at least 3 months. Robust improvements in cone-mediated visual behavior were also observed, with responses of treated mice being similar or identical to that of wild type mice. AAV-vectored GC1 expression was found in photoreceptors and cone cells were preserved in treated retinas. CONCLUSIONS/SIGNIFICANCE: This is the first demonstration of gene-based restoration of both visual function/vision-elicited behavior and cone preservation in a mammalian model of GC1 deficiency. Importantly, results were obtained using a well characterized, clinically relevant AAV vector. These results lay the ground work for the development of an AAV-based gene therapy vector for the treatment of LCA1.

Impaired cone function and cone degeneration resulting from CNGB3 deficiency: down-regulation of CNGA3 biosynthesis as a potential mechanism.

The cone cyclic nucleotide-gated (CNG) channel is essential for central and color vision and visual acuity. This channel is composed of two structurally related subunits, CNGA3 and CNGB3; CNGA3 is the ion-conducting subunit, whereas CNGB3 is a modulatory subunit. Mutations in both subunits are associated with achromatopsia and progressive cone dystrophy, with mutations in CNGB3 alone accounting for 50% of all known cases of achromatopsia. However, the molecular mechanisms underlying cone diseases that result from CNGB3 deficiency are unknown. This study investigated the role of CNGB3 in cones, using CNGB3(-/-) mice. Cone dysfunction was apparent at the earliest time point examined (post-natal day 30) in CNGB3(-/-) mice. When compared with wild-type (WT) controls: photopic electroretingraphic (ERG) responses were decreased by approximately 75%, whereas scotopic ERG responses were unchanged; visual acuity was decreased by approximately 20%, whereas contrast sensitivity was unchanged; cone density was reduced by approximately 40%; photoreceptor apoptosis was detected; and outer segment disorganization was observed in some cones. Notably, CNGA3 protein and mRNA levels were significantly decreased in CNGB3(-/-) mice; in contrast, mRNA levels of S-opsin, Gnat2 and Pde6c were unchanged, relative to WT mice. Hence, we show that loss of CNGB3 reduces biosynthesis of CNGA3 and impairs cone CNG channel function. We suggest that down-regulation of CNGA3 contributes to the pathogenic mechanism by which CNGB3 mutations lead to human cone disease.

Light responses in rods of vitamin A-deprived Xenopus.

PURPOSE: Accumulation of free opsin by mutations in rhodopsin or insufficiencies in the visual cycle can lead to retinal degeneration. Free opsin activates phototransduction; however, the link between constitutive activation and retinal degeneration is unclear. In this study, the photoresponses of Xenopus rods rendered constitutively active by vitamin A deprivation were examined. Unlike their mammalian counterparts, Xenopus rods do not degenerate. Contrasting phototransduction in vitamin A-deprived Xenopus rods with phototransduction in constitutively active mammalian rods may provide new understanding of the mechanisms that lead to retinal degeneration. METHODS: The photocurrents of Xenopus tadpole rods were measured with suction electrode recordings, and guanylate cyclase activity was measured with the IBMX (3-isobutyl-1-methylxanthine) jump technique. The amount of rhodopsin in rods was determined by microspectrophotometry. RESULTS: The vitamin A-deprived rod outer segments were 60% to 70% the length and diameter of the rods in age-matched animals. Approximately 90% of its opsin content was in the free or unbound form. Analogous to bleaching adaptation, the photoresponses were desensitized (10- to 20-fold) and faster. Unlike bleaching adaptation, the vitamin A-deprived rods maintained near normal saturating (dark) current densities by developing abnormally high rates of cGMP synthesis. Their rate of cGMP synthesis in the dark (15 seconds(-1)) was twofold greater than the maximum levels attainable by control rods ( approximately 7 seconds(-1)). CONCLUSIONS: Preserving circulating current density and response range appears to be an important goal for rod homeostasis. However, the compensatory changes associated with vitamin A deprivation in Xenopus rods come at the high metabolic cost of a 15-fold increase in basal ATP consumption.

Monitoring mouse retinal degeneration with high-resolution spectral-domain optical coherence tomography.

Progression of retinal degeneration in a mouse model was studied in vivo with high-resolution spectral-domain optical coherence tomography (SD-OCT). Imaging in 3D with high depth resolution (<3 mum), SD-OCT resolved all the major layers of the retina of control C57BL/6J mice. Images of transgenic mice having a null mutation of the rhodopsin gene revealed the anatomical consequences of retinal degeneration: thinning of the outer retina, including the outer plexiform layer (OPL), outer nuclear layer (ONL), and inner and outer segments (IS/OS). We monitored the progression of retinal degeneration in rd1 mice (C3H/HeJ) by periodically imaging the same mice from the time the pups opened their eyes on P13 to P34. SD-OCT images showed that the outer retina (OPL, ONL, IS/OS) had already thinned by 73% (100 to 27 mum) at eye opening. The retina continued to degenerate, and by P20 the outer retina was not resolvable. The thickness of entire retina decreased from 228 mum (control) to 152 mum on P13 and to 98 mum by P34, a 57% reduction with the complete loss in the outer retina. In summary, we show that SD-OCT can monitor the progression of retinal degeneration in transgenic mice.