Diurnal retinal and choroidal gene expression patterns support a role for circadian biology in myopia pathogenesis.

The prevalence of myopia (nearsightedness) is increasing to alarming levels, but its etiology remains poorly understood. Because both laboratory and clinical findings suggest an etiologic role for circadian rhythms in myopia development, we assayed gene expression by RNA-Seq in retina and choroid at the onset of unilateral experimental myopia in chick, isolating tissues every 4 h during a single 24-h period from myopic and contralateral control eyes. Occluded versus open eye gene expression differences varied considerably over the 24-h sampling period, with some occurring at multiple times of day but with others showing differences at only a single investigated timepoint. Some of the genes identified in retina or choroid of chick myopia were previously identified as candidate genes for common human myopia. Like differentially expressed genes, pathways identified by Gene Set Enrichment Analysis also varied dramatically by sampling time. Considered with other laboratory data, human genetic and epidemiology data, these findings further implicate circadian events in myopia pathogenesis. The present results emphasize a need to include time of day in mechanistic studies of myopia and to assess circadian biology directly in trying to understand better the origin of myopia and to develop more effective therapies.

Diabetic rats with high levels of endogenous dopamine do not show retinal vascular pathology.

Purpose: Limited research exists on the time course of long-term retinal and cerebral deficits in diabetic rodents. Previously, we examined short term (4-8 weeks) deficits in the Goto-Kakizaki (GK) rat model of Type II diabetes. Here, we investigated the long-term (1-8 months) temporal appearance of functional deficits (retinal, cognitive, and motor), retinal vascular pathology, and retinal dopamine levels in the GK rat. Methods: In GK rats and Wistar controls, retinal neuronal function (electroretinogram), cognitive function (Y-maze), and motor function (rotarod) were measured at 1, 2, 4, 6, and 8 months of age. In addition, we evaluated retinal vascular function (functional hyperemia) and glucose and insulin tolerance. Retinas from rats euthanized at ≥8 months were assessed for vascular pathology. Dopamine and DOPAC levels were measured via HPLC in retinas from rats euthanized at 1, 2, 8, and 12 months. Results: Goto-Kakizaki rats exhibited significant glucose intolerance beginning at 4 weeks and worsening over time (p < 0.001). GK rats also showed significant delays in flicker and oscillatory potential implicit times (p < 0.05 to p < 0.001) beginning at 1 month. Cognitive deficits were observed beginning at 6 months (p < 0.05), but no motor deficits. GK rats showed no deficits in functional hyperemia and no increase in acellular retinal capillaries. Dopamine levels were twice as high in GK vs. Wistar retinas at 1, 2, 8, and 12 months (p < 0.001). Conclusion: As shown previously, retinal deficits were detectable prior to cognitive deficits in GK rats. While retinal neuronal function was compromised, retinal vascular pathology was not observed, even at 12+ months. High endogenous levels of dopamine in the GK rat may be acting as an anti-angiogenic and providing protection against vascular pathology.

Deletion of histone demethylase Lsd1 (Kdm1a) during retinal development leads to defects in retinal function and structure.

Purpose: The purpose of this study was to investigate the role of Lysine specific demethylase 1 (Lsd1) in murine retinal development. LSD1 is a histone demethylase that can demethylate mono- and di-methyl groups on H3K4 and H3K9. Using Chx10-Cre and Rho-iCre75 driver lines, we generated novel transgenic mouse lines to delete Lsd1 in most retinal progenitor cells or specifically in rod photoreceptors. We hypothesize that Lsd1 deletion will cause global morphological and functional defects due to its importance in neuronal development. Methods: We tested the retinal function of young adult mice by electroretinogram (ERG) and assessed retinal morphology by in vivo imaging by fundus photography and SD-OCT. Afterward, eyes were enucleated, fixed, and sectioned for subsequent hematoxylin and eosin (H&E) or immunofluorescence staining. Other eyes were plastic fixed and sectioned for electron microscopy. Results: In adult Chx10-Cre Lsd1fl/fl mice, we observed a marked reduction in a-, b-, and c-wave amplitudes in scotopic conditions compared to age-matched control mice. Photopic and flicker ERG waveforms were even more sharply reduced. Modest reductions in total retinal thickness and outer nuclear layer (ONL) thickness were observed in SD-OCT and H&E images. Lastly, electron microscopy revealed significantly shorter inner and outer segments and immunofluorescence showed modest reductions in specific cell type populations. We did not observe any obvious functional or morphological defects in the adult Rho-iCre75 Lsd1fl/fl animals. Conclusion: Lsd1 is necessary for neuronal development in the retina. Adult Chx10-Cre Lsd1fl/fl mice show impaired retinal function and morphology. These effects were fully manifested in young adults (P30), suggesting that Lsd1 affects early retinal development in mice.

The Circadian Clock in the Retinal Pigment Epithelium Controls the Diurnal Rhythm of Phagocytic Activity.

The diurnal peak of phagocytosis by the retinal pigment epithelium (RPE) of photoreceptor outer segments (POS) is under circadian control and believed that this process involves interactions from the retina and RPE. Previous studies have demonstrated that a functional circadian clock exists within multiple retinal cell types and RPE. Thereby, the aim of this study was to determine whether the clock in the retina or RPE controls the diurnal phagocytic peak and whether disruption of the circadian clock in the RPE would affect cellular function and the viability during aging. To that, we generated and validated an RPE tissue-specific KO of the essential clock gene, Bmal1, and then determined the daily rhythm in phagocytic activity by the RPE in mice lacking a functional circadian clock in the retina or RPE. Then, using electroretinography, spectral domain-optical coherence tomography, and optomotor response of visual function we determined the effect of Bmal1 removal in young (6 months) and old (18 months) mice. RPE morphology and lipofuscin accumulation was determined in young and old mice. Our data shows that the clock in the RPE, rather than the retina clock, controls the diurnal phagocytic peak. Surprisingly, absence of a functional RPE clock and phagocytic peak does not result in any detectable age-related degenerative phenotype in the retina or RPE. Thus, our results demonstrate that the circadian clock in the RPE controls the daily peak of phagocytic activity. However, the absence of the clock in the RPE does not result in deterioration of photoreceptors or the RPE during aging.

ON than OFF pathway disruption leads to greater deficits in visual function and retinal dopamine signaling.

The visual system uses ON and OFF pathways to signal luminance increments and decrements. Increasing evidence suggests that ON and OFF pathways have different signaling properties and serve specialized visual functions. However, it is still unclear the contribution of ON and OFF pathways to visual behavior. Therefore, we examined the effects on optomotor response and the retinal dopamine system in nob mice with ON pathway dysfunction and Vsx1-/- mice with partial OFF pathway dysfunction. Spatial frequency and contrast sensitivity thresholds were determined, and values were compared to age-matched wild-type controls. Retinas were collected immediately after visual testing to measure levels of dopamine and its metabolite, DOPAC. At 4 weeks of age, we found that nob mice had significantly reduced spatial frequency (19%) and contrast sensitivity (60%) thresholds compared to wild-type mice. Vsx1-/- mice also exhibited reductions in optomotor responses (3% in spatial frequency; 18% in contrast sensitivity) at 4 weeks, although these changes were significantly smaller than those found in nob mice. Furthermore, nob mice had significantly lower DOPAC levels (53%) and dopamine turnover (41%) compared to controls while Vsx1-/- mice displayed a transient increase in DOPAC levels at 4 weeks of age (55%). Our results show that dysfunction of ON pathways leads to reductions in contrast sensitivity, spatial frequency threshold, and retinal dopamine turnover whereas partial loss of the OFF pathway has minimal effect. We conclude that ON pathways play a critical role in visual reflexes and retinal dopamine signaling, highlighting a potential association for future investigations.

Melanopsin modulates refractive development and myopia.

Myopia, or nearsightedness, is the most common form of refractive abnormality and is characterized by excessive ocular elongation in relation to ocular power. Retinal neurotransmitter signaling, including dopamine, is implicated in myopic ocular growth, but the visual pathways that initiate and sustain myopia remain unclear. Melanopsin-expressing retinal ganglion cells (mRGCs), which detect light, are important for visual function, and have connections with retinal dopamine cells. Here, we investigated how mRGCs influence normal and myopic refractive development using two mutant mouse models: Opn4-/- mice that lack functional melanopsin photopigments and intrinsic mRGC responses but still receive other photoreceptor-mediated input to these cells; and Opn4DTA/DTA mice that lack intrinsic and photoreceptor-mediated mRGC responses due to mRGC cell death. In mice with intact vision or form-deprivation, we measured refractive error, ocular properties including axial length and corneal curvature, and the levels of retinal dopamine and its primary metabolite, L-3,4-dihydroxyphenylalanine (DOPAC). Myopia was measured as a myopic shift, or the difference in refractive error between the form-deprived and contralateral eyes. We found that Opn4-/- mice had altered normal refractive development compared to Opn4+/+ wildtype mice, starting ∼4D more myopic but developing ∼2D greater hyperopia by 16 weeks of age. Consistent with hyperopia at older ages, 16 week-old Opn4-/- mice also had shorter eyes compared to Opn4+/+ mice (3.34 vs 3.42 mm). Opn4DTA/DTA mice, however, were more hyperopic than both Opn4+/+ and Opn4-/- mice across development ending with even shorter axial lengths. Despite these differences, both Opn4-/- and Opn4DTA/DTA mice had ∼2D greater myopic shifts in response to form-deprivation compared to Opn4+/+ mice. Furthermore, when vision was intact, dopamine and DOPAC levels were similar between Opn4-/- and Opn4+/+ mice, but higher in Opn4DTA/DTA mice, which differed with age. However, form-deprivation reduced retinal dopamine and DOAPC by ∼20% in Opn4-/- compared to Opn4+/+ mice but did not affect retinal dopamine and DOPAC in Opn4DTA/DTA mice. Lastly, systemically treating Opn4-/- mice with the dopamine precursor L-DOPA reduced their form-deprivation myopia by half compared to non-treated mice. Collectively our findings show that disruption of retinal melanopsin signaling alters the rate and magnitude of normal refractive development, yields greater susceptibility to form-deprivation myopia, and changes dopamine signaling. Our results suggest that mRGCs participate in the eye's response to myopigenic stimuli, acting partly through dopaminergic mechanisms, and provide a potential therapeutic target underling myopia progression. We conclude that proper mRGC function is necessary for correct refractive development and protection from myopia progression.

A Tropomycin-Related Kinase B Receptor Activator for the Management of Ocular Blast-Induced Vision Loss.

Pressure waves from explosions or other traumatic events can damage the neurons of the eye and visual centers of the brain, leading to functional loss of vision. There are currently few treatments for such injuries that can be deployed rapidly to mitigate damage. Brain-derived neurotrophic factor (BDNF) and activation of its receptor tropomycin-related kinase B (TrkB) have neuroprotective effects in a number of degeneration models. Small molecule activators of TrkB, such as N-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-2-oxopiperidine-3-carboxamide (HIOC), cross the blood-brain and blood-retina barriers after systemic administration. We characterize the effects of blast-induced ocular trauma on retinal and visual function. We show that systemic administration of HIOC, a potent small molecule activator of the BDNF/TrkB receptor, preserves visual function in mice exposed to ocular blast injury. The HIOC treatment for one week preserves visual function for at least four months. The HIOC treatment effectively protected vision when the initial dose was administered up to 3 h after blast, but not if the initial treatment was delayed for 24 h. We provide evidence that the therapeutic effect of HIOC is mediated by activation of BDNF/TrkB receptors. The results indicate that HIOC may be useful for managing ocular blast injury and other forms of traumatic optic neuropathy.

Ambient Light Regulates Retinal Dopamine Signaling and Myopia Susceptibility.

Purpose: Exposure to high-intensity or outdoor lighting has been shown to decrease the severity of myopia in both human epidemiological studies and animal models. Currently, it is not fully understood how light interacts with visual signaling to impact myopia. Previous work performed in the mouse retina has demonstrated that functional rod photoreceptors are needed to develop experimentally-induced myopia, alluding to an essential role for rod signaling in refractive development. Methods: To determine whether dim rod-dominated illuminance levels influence myopia susceptibility, we housed male C57BL/6J mice under 12:12 light/dark cycles with scotopic (1.6 × 10-3 candela/m2), mesopic (1.6 × 101 cd/m2), or photopic (4.7 × 103 cd/m2) lighting from post-natal day 23 (P23) to P38. Half the mice received monocular exposure to -10 diopter (D) lens defocus from P28-38. Molecular assays to measure expression and content of DA-related genes and protein were conducted to determine how illuminance and lens defocus alter dopamine (DA) synthesis, storage, uptake, and degradation and affect myopia susceptibility in mice. Results: We found that mice exposed to either scotopic or photopic lighting developed significantly less severe myopic refractive shifts (lens treated eye minus contralateral eye; -1.62 ± 0.37D and -1.74 ± 0.44D, respectively) than mice exposed to mesopic lighting (-3.61 ± 0.50D; P < 0.005). The 3,4-dihydroxyphenylacetic acid /DA ratio, indicating DA activity, was highest under photopic light regardless of lens defocus treatment (controls: 0.09 ± 0.011 pg/mg, lens defocus: 0.08 ± 0.008 pg/mg). Conclusions: Lens defocus interacted with ambient conditions to differentially alter myopia susceptibility and DA-related genes and proteins. Collectively, these results show that scotopic and photopic lighting protect against lens-induced myopia, potentially indicating that a broad range of light levels are important in refractive development.

Photoreceptor Degeneration in Homozygous Male Per2luc Mice During Aging.

The Per2luc mouse model developed by Takahashi laboratory is one of the most powerful models to study circadian rhythms in real time. In this study, we report that photoreceptors degenerate in male Per2luc mice during aging. Young (2.5- to 5-month-old) and aged (11- to 13.5-month-old) homozygous male Per2luc mice and C57BL/6J mice were used for this study. Retina structure and function were investigated via spectral domain optical coherence tomography (SD-OCT), fundus imaging, and electroretinography (ERG). Zonula occludens-1 (ZO-1) immunofluorescence was used to analyze the retinal pigment epithelium (RPE) morphology. Fundus examination revealed no difference between young Per2luc and wild-type (WT) mice. However, the fundus of aged Per2luc mice showed white deposits, suggestive of age-related drusen-like formation or microglia, which were absent in age-matched WT mice. No differences in retinal structure and function were observed between young Per2luc and WT mice. However, with age, Per2luc mice showed a significant reduction in total retinal thickness with respect to C57BL/6J mice. The reduction was mostly confined to the photoreceptor layer. Consistent with these results, we observed a significant decrease in the amplitude of a- and b-waves of the ERG in aged Per2luc mice. Analysis of the RPE morphology revealed that in aged Per2luc mice there was an increase in compactness and eccentricity with a decrease in solidity with respect to the values observed in WT, pointing toward signs of aging in the RPE of Per2luc mice. Our data demonstrate that homozygous Per2luc mice show photoreceptor degeneration during aging and a premature aging of the RPE.

Effects of Cone Connexin-36 Disruption on Light Adaptation and Circadian Regulation of the Photopic ERG.

Purpose: The present study tested the hypothesis that connexin-36 (Cx36) and gap junctions between photoreceptor cells contribute to the circadian rhythm of the photopic electroretinogram (ERG) b-wave amplitude. Methods: Cone-specific disruption of Cx36 was obtained in mice with a floxed Gjd2 gene and human red/green pigment promoter (HRGP)-driven Cre recombinase. Standard ERG, spectral-domain optical coherence tomography (SD-OCT) and histochemical methods were used. Results: HRGPcreGjd2fl/fl mice had a selective reduction in Cx36 protein in the outer plexiform layer; no reduction in Cx36 was observed in the inner plexiform layer. Cx36 disruption had no effect on the number of cones, the thickness of the photoreceptor layer, or the scotopic ERG responses. However, there was a reduction of the photopic ERG circadian rhythm, with b-wave amplitudes in the day and the night locked in the daytime, light-adapted state. In HRGPcreGjd2+/+and Gjd2fl/fl controls, the circadian rhythm of light-adapted ERG persisted, similar to that in wild type mice. Conclusions: Cx36 regulation contributes to the circadian rhythm of light-adapted ERG; in the absence of photoreceptor gap junctions, mice appear to be in a fully light-adapted state regardless of the time of day. The higher amplitudes and reduced circadian regulation of the b-wave of HRGPcreGjd2fl/fl mice may be due to increased synaptic strength at the cone to ON bipolar cell synapse due to electrotonic isolation of the terminals lacking gap junctions.

Dopamine 2 Receptor Signaling Controls the Daily Burst in Phagocytic Activity in the Mouse Retinal Pigment Epithelium.

Purpose: A burst in phagocytosis of spent photoreceptor outer fragments by RPE is a rhythmic process occurring 1 to 2 hours after the onset of light. This phenomenon is considered crucial for the health of the photoreceptors and RPE. We have recently reported that dopamine, via dopamine 2 receptor (D2R), shifts the circadian rhythm in the RPE. Methods: Here, we first investigated the impact of the removal of D2R on the daily peak of phagocytosis by RPE and then we analyzed the function and morphology of retina and RPE in the absence of D2R. Results: D2R knockout (KO) mice do not show a daily burst of phagocytic activity after the onset of light. RNA sequencing revealed a total of 394 differentially expressed genes (DEGs) between ZT 23 and ZT 1 in the control mice, whereas in D2R KO mice, we detected 1054 DEGs. Pathway analysis of the gene expression data implicated integrin signaling to be one of the upregulated pathways in control but not in D2R KO mice. Consistent with the gene expression data, phosphorylation of focal adhesion kinase (FAK) did not increase significantly in KO mice at ZT 1. No difference in retinal thickness, visual function, or morphology of RPE cells was observed between wild-type (WT) and D2R KO mice at the age of 3 and 12 months. Conclusions: Our data suggest that removal of D2R prevents the burst of phagocytosis and a related increase in the phosphorylation of FAK after light onset. The pathway analysis points toward a putative role of D2R in controlling integrin signaling, which is known to play an important role in the control of the daily burst of phagocytosis by the RPE. Our data also indicate that the absence of the burst of phagocytic activity in the early morning does not produce any apparent deleterious effect on the retina or RPE up to 1 year of age.

Visual Image Quality Impacts Circadian Rhythm-Related Gene Expression in Retina and in Choroid: A Potential Mechanism for Ametropias.

Purpose: Stimulated by evidence implicating diurnal/circadian rhythms and light in refractive development, we studied the expression over 24 hours of selected clock and circadian rhythm-related genes in retina/retinal pigment epithelium (RPE) and choroid of experimental ametropias in chicks. Methods: Newly hatched chicks, entrained to a 12-hour light/dark cycle for 12 to 14 days, either experienced nonrestricted vision OU (i.e., in both eyes) or received an image-blurring diffuser or a minus 10-diopter (D) or a plus 10-D defocusing lens over one eye. Starting 1 day later and at 4-hour intervals for 24 hours, the retina/RPE and choroid were separately dissected. Without pooling, total RNA was extracted, converted to cDNA, and assayed by quantitative PCR for the expression of the following genes: Opn4m, Clock, Npas2, Per3, Cry1, Arntl, and Mtnr1a. Results: The expression of each gene in retina/RPE and in choroid of eyes with nonrestricted vision OU varied over 24 hours, with equal levels OU for most genes and times. Altered visual input influenced gene expression in complex patterns that varied by gene, visual input, time, and eye, affecting experimental eyes with altered vision and also contralateral eyes with nonrestricted vision. Discussion: Altering visual input in ways known to induce ametropias alters the retinal/RPE and choroidal expression of circadian rhythm-related genes, further linking circadian biology with eye growth regulation. While further investigations are needed, studying circadian processes may help understand refractive mechanisms and the increasing myopia prevalence in contemporary societies where lighting patterns can desynchronize endogenous rhythms from the natural environmental light/dark cycle.

Dopamine metabolite levels in the vitreous of diabetic and non-diabetic humans.

Animal studies suggest that the retinal dysfunction in diabetic subjects that precedes overt clinical vasculopathy may be due to a retinal dopamine deficit. We analyzed levels of dopamine (DA) and its primary metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), in the vitreous of diabetic and non-diabetic human subjects. Adult patients undergoing pars plana vitrectomy for non-hemorrhagic indications were prospectively recruited from the Emory Eye Center in Atlanta, GA. Vitreous samples were analyzed using high performance liquid chromatography (HPLC) to measure levels of DOPAC and DA in the vitreous specimens. Vitreous samples from 9 diabetic patients and 20 from non-diabetic patients were analyzed. No eyes had apparent diabetic retinopathy. Mean normalized DA concentration in vitreous of diabetic subjects was 0.76 ± 0.12 pg/µL vs. 0.73 ± 0.08 pg/µL in non-diabetic vitreous (p = 0.849). DOPAC concentration was 8.84 ± 0.74 pg/µL in vitreous of diabetic subjects vs. 9.22 ± 0.56 pg/µL in vitreous of non-diabetic subjects (p = 0.691). No difference was observed in the concentrations of DA and DOPAC in the vitreous of people without diabetes compared to those with diabetes without retinopathy.

Novel Detection and Restorative Levodopa Treatment for Preclinical Diabetic Retinopathy.

Diabetic retinopathy (DR) is diagnosed clinically by directly viewing retinal vascular changes during ophthalmoscopy or through fundus photographs. However, electroretinography (ERG) studies in humans and rodents have revealed that retinal dysfunction is demonstrable prior to the development of visible vascular defects. Specifically, delays in dark-adapted ERG oscillatory potential (OP) implicit times in response to dim-flash stimuli (<-1.8 log cd · s/m2) occur prior to clinically recognized DR. Animal studies suggest that retinal dopamine deficiency underlies these early functional deficits. In this study, we randomized individuals with diabetes, without clinically detectable retinopathy, to treatment with either low- or high-dose Sinemet (levodopa plus carbidopa) for 2 weeks and compared their ERG findings with those of control subjects (no diabetes). We assessed dim-flash-stimulated OP delays using a novel handheld ERG system (RETeval) at baseline and 2 and 4 weeks. RETeval recordings identified significant OP implicit time delays in individuals with diabetes without retinopathy compared with age-matched control subjects (P < 0.001). After 2 weeks of Sinemet treatment, OP implicit times were restored to control values, and these improvements persisted even after a 2-week washout. We conclude that detection of dim-flash OP delays could provide early detection of DR and that Sinemet treatment may reverse retinal dysfunction.

Altered ocular parameters from circadian clock gene disruptions.

The pathophysiology of refractive errors is poorly understood. Myopia (nearsightedness) in particular both blurs vision and predisposes the eye to many blinding diseases during adulthood. Based on past findings of diurnal variations in the dimensions of the eyes of humans and other vertebrates, altered diurnal rhythms of these ocular dimensions with experimentally induced myopia, and evolving evidence that ambient light exposures influence refractive development, we assessed whether disturbances in circadian signals might alter the refractive development of the eye. In mice, retinal-specific knockout of the clock gene Bmal1 induces myopia and elongates the vitreous chamber, the optical compartment separating the lens and the retina. These alterations simulate common ocular findings in clinical myopia. In Drosophila melanogaster, knockouts of the clock genes cycle or period lengthen the pseudocone, the optical component of the ommatidium that separates the facet lens from the photoreceptors. Disrupting circadian signaling thus alters optical development of the eye in widely separated species. We propose that mechanisms of myopia include circadian dysregulation, a frequent occurrence in modern societies where myopia also is both highly prevalent and increasing at alarming rates. Addressing circadian dysregulation may improve understanding of the pathogenesis of refractive errors and introduce novel therapeutic approaches to ameliorate myopia development in children.

An opsin 5-dopamine pathway mediates light-dependent vascular development in the eye.

During mouse postnatal eye development, the embryonic hyaloid vascular network regresses from the vitreous as an adaption for high-acuity vision. This process occurs with precisely controlled timing. Here, we show that opsin 5 (OPN5; also known as neuropsin)-dependent retinal light responses regulate vascular development in the postnatal eye. In Opn5-null mice, hyaloid vessels regress precociously. We demonstrate that 380-nm light stimulation via OPN5 and VGAT (the vesicular GABA/glycine transporter) in retinal ganglion cells enhances the activity of inner retinal DAT (also known as SLC6A3; a dopamine reuptake transporter) and thus suppresses vitreal dopamine. In turn, dopamine acts directly on hyaloid vascular endothelial cells to suppress the activity of vascular endothelial growth factor receptor 2 (VEGFR2) and promote hyaloid vessel regression. With OPN5 loss of function, the vitreous dopamine level is elevated and results in premature hyaloid regression. These investigations identify violet light as a developmental timing cue that, via an OPN5-dopamine pathway, regulates optic axis clearance in preparation for visual function.

Lack of cone mediated retinal function increases susceptibility to form-deprivation myopia in mice.

Retinal photoreceptors are important in visual signaling for normal eye growth in animals. We used Gnat2cplf3/cplf3 (Gnat2-/-) mice, a genetic mouse model of cone dysfunction to investigate the influence of cone signaling in ocular refractive development and myopia susceptibility in mice. Refractive development under normal visual conditions was measured for Gnat2-/- and age-matched Gnat2+/+ mice, every 2 weeks from 4 to 14 weeks of age. Weekly measurements were performed on a separate cohort of mice that underwent monocular form-deprivation (FD) in the right eye from 4 weeks of age using head-mounted diffusers. Refraction, corneal curvature, and ocular biometrics were obtained using photorefraction, keratometry and optical coherence tomography, respectively. Retinas from FD mice were harvested, and analyzed for dopamine (DA) and 3,4-dihydroxyphenylacetate (DOPAC) using high-performance liquid chromatography. Under normal visual conditions, Gnat2+/+ and Gnat2-/- mice showed similar refractive error, axial length, and corneal radii across development (p > 0.05), indicating no significant effects of the Gnat2 mutation on normal ocular refractive development in mice. Three weeks of FD produced a significantly greater myopic shift in Gnat2-/- mice compared to Gnat2+/+ controls (-5.40 ±â€¯1.33 D vs -2.28 ±â€¯0.28 D, p = 0.042). Neither the Gnat2 mutation nor FD altered retinal levels of DA or DOPAC. Our results indicate that cone pathways needed for high acuity vision in primates are not as critical for normal refractive development in mice, and that both rods and cones contribute to visual signalling pathways needed to respond to FD in mammalian eyes.

Removal of clock gene Bmal1 from the retina affects retinal development and accelerates cone photoreceptor degeneration during aging.

The mammalian retina contains an autonomous circadian clock system that controls many physiological functions within this tissue. Previous studies on young mice have reported that removal of the key circadian clock gene Bmal1 from the retina affects the circadian regulation of visual function, but does not affect photoreceptor viability. Because dysfunction in the circadian system is known to affect cell viability during aging in other systems, we compared the effect of Bmal1 removal from the retina on visual function, inner retinal structure, and photoreceptor viability in young (1 to 3 months) and aged (24 to 26 months) mice. We found that removal of Bmal1 from the retina significantly affects visual information processing in both rod and cone pathways, reduces the thickness of inner retinal nuclear and plexiform layers, accelerates the decline of visual functions during aging, and reduces the viability of cone photoreceptors. Our results thus suggest that circadian clock dysfunction, caused by genetic or other means, may contribute to the decline of visual function during development and aging.

Dopaminergic amacrine cell number, plexus density, and dopamine content in the mouse retina: Strain differences and effects of Bax gene disruption.

Many types of retinal neuron modulate the distribution of their processes to ensure a uniform coverage of the retinal surface. Dendritic field area, for instance, is inversely related to the variation in cellular density for many cell types, observed either across retinal eccentricity or between different strains of mice that differ in cell number. Dopaminergic amacrine (DA) cells, by contrast, have dendritic arbors that bear no spatial relationship to the presence of their immediate homotypic neighbors, yet it remains to be determined whether their coverage upon the retina, as a population, is conserved across variation in their total number. The present study assessed the overall density of the dopaminergic plexus in the inner plexiform layer in the presence of large variation in the total number of DA cells, as well as their retinal dopamine content, to determine whether either of these features is conserved. We first compared these traits between two strains of mice (C57BL/6J and A/J) that exhibit a two-fold difference in DA cell number. We subsequently examined these same traits in littermate mice for which the pro-apoptotic Bax gene was either intact or knocked out, yielding a five-fold difference in DA cell number. In both comparisons, we found greater plexus density and DA content in the strain or condition with the greater number of DA cells. The population of DA cells, therefore, does not appear to self-regulate its process coverage to achieve a constant density as the DA mosaic is established during development, nor its functional dopamine content in maturity.

Rhythmic Regulation of Photoreceptor and RPE Genes Important for Vision and Genetically Associated With Severe Retinal Diseases.

Purpose: The aim of the present study was to identify candidate genes for mediating daily adjustment of vision. Methods: Genes important for vision and genetically associated with severe retinal diseases were tested for 24-hour rhythms in transcript levels in neuronal retina, microdissected photoreceptors, photoreceptor-related pinealocytes, and retinal pigment epithelium-choroid (RPE-choroid) complex by using quantitative PCR. Results: Photoreceptors of wildtype mice display circadian clock-dependent regulation of visual arrestins (Arr1, Arr4) and the visual cycle gene Rdh12, whereas cells of the RPE-choroid exhibit light-dependent regulation of the visual cycle key genes Lrat, Rpe65, and Rdh5. Clock-driven rhythmicity of Arr1, Arr4, and Rdh12 was observed also in rat pinealocytes, to persist in a mouse model of diabetic retinopathy (db/db) and, in the case of Arr1, to be abolished in retinae of mice deficient for dopamine D4 receptors. Therefore, the expression rhythms appear to be evolutionary conserved, to be unaffected in diabetic retinopathy, and, for Arr1, to require dopamine signaling via dopamine D4 receptors. Conclusions: The data of the present study suggest that daily adjustment of retinal function combines clock-dependent regulation of genes responsible for phototransduction termination (Arr1, Arr4) and detoxification (Rdh12) in photoreceptors with light-dependent regulation of genes responsible for retinoid recycling (Lrat, Rpe65, and Rdh5) in RPE. Furthermore, they indicate circadian and light-dependent regulation of genes genetically associated with severe retinal diseases.