Paul J. DeMarco

Generation of an Inbred Miniature Pig Model of Retinitis Pigmentosa

PURPOSE The Pro23His (P23H) rhodopsin (RHO) mutation underlies the most common form of human autosomal dominant retinitis pigmentosa (adRP). The objective of this investigation was to establish a transgenic miniature swine model of RP using the human P23H RHO gene. METHODS Somatic cell nuclear transfer (SCNT) was used to create transgenic miniature pigs that expressed the human P23H RHO mutation. From these experiments, six transgenic founders were identified whose retinal function was studied with full-field electroretinography (ffERG) from 3 months through 2 years. Progeny from one founder were generated and genotyped to determine transgene inheritance pattern. Retinal mRNA was isolated, and the ratio of P23H to wild-type pig RHO was measured. RESULTS A single transgene integration site was observed for five of the six founders. All founders had abnormal scotopic and photopic ffERGs after 3 months. The severity of the ffERG phenotype was grouped into moderately and severely affected groups. Offspring of one founder inherited the transgene as an autosomal dominant mutation. mRNA analyses demonstrated that approximately 80% of total RHO was mutant P23H. CONCLUSIONS Expression of the human RHO P23H transgene in the retina creates a miniature swine model with an inheritance pattern and retinal function that mimics adRP. This large-animal model can serve as a novel tool for the study of the pathogenesis and therapeutic intervention in the most common form of adRP.

Cone contributions to the photopic spectral sensitivity of the zebrafish ERG

Microspectrophotometry studies show that zebrafish (Danio rerio) possess four cone photopigments. The purpose of this study was to determine the cone contributions to the zebrafish photopic increment threshold spectral-sensitivity function. Electroretinogram (ERG) b-wave responses to monochromatic lights presented on a broadband or chromatic background were obtained. It was found that under the broadband background condition, the zebrafish spectral-sensitivity function showed several peaks that were narrower in sensitivity compared to the cone spectra. The spectral-sensitivity function was modeled with L - M and M - S opponent interactions and nonopponent S- and U-cone mechanisms. Using chromatic adaptation designed to suppress the contribution of the S-cones, a strong U-cone contribution to the spectral-sensitivity function was revealed, and the contributions of the S-cones to the M - S mechanism were reduced. These results show that the b-wave component of the ERG receives input from all four cone types and appears to reflect color opponent mechanisms. Thus, zebrafish may possess the fundamental properties necessary for color vision.

The transcription factor Nr2e3 functions in retinal progenitors to suppress cone cell generation.

The transcription factor Nr2e3 is an essential component for development and specification of rod and cone photoreceptors; however, the mechanism through which it acts is not well understood. In this study, we use Nr2e3(rd7/rd7) mice that harbor a mutation in Nr2e3, to serve as a model for the human retinal disease Enhanced S Cone Syndrome. Our studies reveal that NR2E3 is expressed in late retinal progenitors and differentiating photoreceptors of the developing retina and localized to the cell bodies of mature rods and cones. In particular, we demonstrate that the abnormal increase in cone photoreceptors observed in Nr2e3(rd7/rd7) mice arise from ectopic mitotic progenitor cells that are present in the outer nuclear layer of the mature Nr2e3(rd7/rd7) retina. A prolonged phase of proliferation is observed followed by abnormal retinal lamination with fragmented and disorganized photoreceptor synapses that result in a progressive loss of rod and cone function. An extended and pronounced wave of apoptosis is also detected at P30 and temporally correlates with the phase of prolonged proliferation. Approximately twice as many apoptotic cells were detected compared to proliferating cells. This wave of apoptosis appears to affect both rod and cone cells and thus may account for the concurrent loss of rod and cone function. We further show that Nr2e3(rd7/rd7) cones do not express rod specific genes and Nr2e3(rd7/rd7) rods do not express cone specific genes. Our studies suggest that, based on its temporal and spatial expression, NR2E3 acts simultaneously in different cell types: in late mitotic progenitors, newly differentiating post mitotic cells, and mature rods and cones. In particular, this study reveals the function of NR2E3 in mitotic progenitors is to repress the cone generation program. NR2E3 is thus one of the few genes known to influence the competency of retinal progenitors while simultaneously directing the rod and cone differentiation.

Anatomy and physiology of the visual system.

This article reviews the anatomy and physiology of the visual system. The physical stimulus is coded by several separate and parallel pathways at multiple sites in the nervous system. We outline the major parallel pathways of the system, from their beginning in the retina, to the multiple cortical areas that receive visual input. Emphasis has been placed on the functional properties of the neurons of these pathways and the various cortical processing areas as they are currently known. Clinical syndromes as a result of damage to specific visual cortical areas are also described.

Cone Photoreceptors Develop Normally in the Absence of Functional Rod Photoreceptors in a Transgenic Swine Model of Retinitis Pigmentosa

PURPOSE Human and swine retinas have morphological and functional similarities. In the absence of primate models, the swine is an attractive model to study retinal function and disease, with its cone-rich visual streak, our ability to manipulate their genome, and the differences in susceptibility of rod and cone photoreceptors to disease. We characterized the normal development of cone function and its subsequent decline in a P23H rhodopsin transgenic (TgP23H) miniswine model of autosomal dominant RP. METHODS Semen from TgP23H miniswine 53-1 inseminated domestic swine and produced TgP23H and Wt hybrid littermates. Retinal function was evaluated using ERGs between postnatal days (P) 14 and 120. Retinal ganglion cell (RGC) responses were recorded to full-field stimuli at several intensities. Retinal morphology was assessed using light and electron microscopy. RESULTS Scotopic retinal function matures in Wt pigs up to P60, but never develops in TgP23H pigs. Wt and TgP23H photopic vision matures similarly up to P30 and diverges at P60 where TgP23H cone vision declines. There are fewer TgP23H RGCs with visually evoked responses at all ages and their response to light is compromised. Photoreceptor morphological changes mirror these functional changes. CONCLUSIONS Lack of early scotopic function in TgP23H swine suggests it as a model of an aggressive form of RP. In this mammalian model of RP, normal cone function develops independent of rod function. Therefore, its retina represents a system in which therapies to rescue cones can be developed to prolong photopic visual function in RP patients.

Selective Rod Degeneration and Partial Cone Inactivation Characterize an Iodoacetic Acid Model of Swine Retinal Degeneration

PURPOSE. Transgenic pigs carrying a mutant human rhodopsin transgene have been developed as a large animal model of retinitis pigmentosa (RP). This model displays some key features of human RP, but the time course of disease progression makes this model costly, time consuming, and difficult to study because of the size of the animals at end-stage disease. Here, the authors evaluate an iodoacetic acid (IAA) model of photoreceptor degeneration in the pig as an alternative model that shares features of the transgenic pig and human RP. METHODS. IAA blocks glycolysis, thereby inhibiting photoreceptor function. The effect of the intravenous injection of IAA on swine rod and cone photoreceptor viability and morphology was followed by histologic evaluation of different regions of the retina using hematoxylin and eosin and immunostaining. Rod and cone function was analyzed by full-field electroretinography and multifocal electroretinography. RESULTS. IAA led to specific loss of rods in a central-to-peripheral retinal gradient. Although cones were resistant, they showed shortened outer segments, loss of bipolar cell synaptic connections, and a diminished flicker ERG, hallmarks of transition to cone dysfunction in RP patients. CONCLUSIONS. IAA provides an alternative rod-dominant model of retinal damage that shares a surprising number of features with the pig transgenic model of RP and with human RP. This IAA model is cost-effective and rapid, ensuring that the size of the animals does not become prohibitive for end-stage evaluation or therapeutic intervention.

APB selectively reduces visual responses in goldfish to high spatiotemporal frequencies

Visual responses of goldfish to rotating square-wave gratings were recorded before and after intraocular injection of 2-amino-4-phosphonobutyric acid (APB). High doses of APB reduced the rate of optokinetic nystagmus (OKN) to a relatively high spatial frequency grating moving at a high temporal frequency. Responses to a low spatial frequency grating were not altered, nor were responses to the higher spatial frequency when it rotated slowly. The effects of APB were transient and lasted no longer than 3 d. We conclude that APB reduces OKN to high spatiotemporal frequencies in goldfish.

Iodoacetic acid, but not sodium iodate, creates an inducible swine model of photoreceptor damage

Our purpose was to find a method to create a large animal model of inducible photoreceptor damage. To this end, we tested in domestic swine the efficacy of two chemical toxins, known to create photoreceptor damage in other species: Iodoacetic Acid (IAA) and Sodium Iodate (NaIO(3)). Intravenous (IV) administration of NaIO(3) up to 90 mg/kg had no effect on retinal function and 110 mg/kg was lethal. IV administration of IAA (5-20 mg/kg) produced concentration-dependent changes in visual function as measured by full-field and multi-focal electroretinograms (ffERG and mfERG), and 30 mg/kg IAA was lethal. The IAA-induced effects measured at two weeks were stable through eight weeks post-injection, the last time point investigated. IAA at 7.5, 10, and 12 mg/kg produce a concentration-dependent reduction in both ffERG b-wave and mfERG N1-P1 amplitudes compared to baseline at all post-injection times. Comparisons of dark- and light-adapted ffERG b-wave amplitudes show a more significant loss of rod relative to cone function. The fundus of swine treated with ≥10 mg/kg IAA was abnormal with thinner retinal vessels and pale optic discs, and we found no evidence of bone spicule formation. Histological evaluations show concentration-dependent outer retinal damage that correlates with functional changes. We conclude that NaIO(3,) is not an effective toxin in swine. In contrast, IAA can be used to create a rapidly inducible, selective, stable and concentration-dependent model of photoreceptor damage in swine retina. Because of these attributes this large animal model of controlled photoreceptor damage should be useful in the investigation of treatments to replace damaged photoreceptors.

Increment and decrement detection on temporally modulated fields.

Increment and decrement probe thresholds were measured during the presentation of two types of temporal masking stimuli. In Experiment 1, we measured thresholds for increment or decrement rectangular probes presented during the presentation of an increment or decrement Gaussian masking stimulus. We find that thresholds are higher when the probe and the Gaussian mask are of the same sign (e. g. both increments). However, both types of Gaussian mask raised increment and decrement probe thresholds above steady state conditions. In Experiment 2, we presented increment or decrement probes at one of eight possible phases of a 1 Hz luminance-modulated sine wave. For both increment and decrement probes, threshold variation with phase is non-sinusoidal in shape, but increment and decrement probe thresholds vary as a function of the sinusoid phase. These experiments show that increment and decrement thresholds vary as a function of the adaptation state of the visual system, and as a function of the direction of change in the adaptation state. Data from both experiments are discussed in terms of a recent neurophysiological model [Hood & Graham (1998). Threshold fluctuations on temporally modulated backgrounds: a possible physiological explanation based upon a recent computational model. Visual Neuroscience, 15 (5), 957-967]. We find that the predicted ON- and OFF-pathway responses do not correlate in a straightforward manner with the psychophysical thresholds, suggesting that detection of increment and decrement probes may not be performed exclusively by one pathway. Our data have implications for modeling visual performance under conditions where visual adaptation is dynamic, such as when scanning complex images or natural scenes.

Time course of adaptation to stimuli presented along cardinal lines in color space

Visual sensitivity is a process that allows the visual system to maintain optimal response over a wide range of ambient light levels and chromaticities. Several studies have used variants of the probe-flash paradigm to show that the time course of adaptation to abrupt changes in ambient luminance depends on both receptoral and postreceptoral mechanisms. Though a few studies have explored how these processes govern adaptation to color changes, most of this effort has targeted the L-M-cone pathway. The purpose of our work was to use the probe-flash paradigm to more fully explore light adaptation in both the L-M- and the S-cone pathways. We measured sensitivity to chromatic probes presented after the onset of a 2-s chromatic flash. Test and flash stimuli were spatially coextensive 2 degrees fields presented in Maxwellian view. Flash stimuli were presented as excursions from white and could extended in one of two directions along an equiluminant L-M-cone or S-cone line. Probes were presented as excursions from the adapting flash chromaticity and could extend either toward the spectrum locus or toward white. For both color lines, the data show a fast and slow adaptation component, although this was less evident in the S-cone data. The fast and slow components were modeled as first- and second-site adaptive processes, respectively. We find that the time course of adaptation is different for the two cardinal pathways. In addition, the time course for S-cone stimulation is polarity dependent. Our results characterize the rapid time course of adaptation in the chromatic pathways and reveal that the mechanics of adaptation within the S-cone pathway are distinct from those in the L-M-cone pathways.