Electroretinogram (ERG) studies identified a new mouse line with a normal a-wave but lacking the b-wave component. The ERG phenotype of this new allele, nob7, matched closely that of mouse mutants for Grm6, Lrit3, Trpm1, and Nyx, which encode for proteins expressed in depolarizing bipolar cells (DBCs). To identify the underlying mutation, we first crossed nob7 mice with Grm6nob3 mutants and measured the ERGs in offspring. All the offspring lacked the b-wave, indicating that nob7 is a new allele for Grm6: Grm6nob7. Sequence analyses of Grm6nob7 cDNAs identified a 28 base pair insertion between exons 8 and 9, which would result in a frameshift mutation in the open reading frame that encodes the metabotropic glutamate receptor 6 (Grm6). Sequencing both the cDNA and genomic DNA from exon 8 and intron 8, respectively, from the Grm6nob7 mouse revealed a G to A transition at the last position in exon 8. This mutation disrupts splicing and the normal exon 8 is extended by 28 base pairs, because splicing occurs 28 base pairs downstream at a cryptic splice donor. Consistent with the impact of the resulting frameshift mutation, there is a loss of mGluR6 protein (encoded by Grm6) from the dendritic tips of DBCs in the Grm6nob7 retina. These results indicate that Grm6nob7 is a new model of the complete form of congenital stationary night blindness, a human condition that has been linked to mutations of GRM6.

Streptozotocin (STZ)-induced diabetes is associated with reductions in the electrical response of the outer retina and retinal pigment epithelium (RPE) to light. Aldose reductase (AR) is the first enzyme required in the polyol-mediated metabolism of glucose, and AR inhibitors have been shown to improve diabetes-induced electroretinogram (ERG) defects. Here, we used control and AR−/− mice to determine if genetic inactivation of this enzyme likewise inhibits retinal electrophysiological defects observed in a mouse model of type 1 diabetes. STZ was used to induce hyperglycemia and type 1 diabetes. Diabetic and age-matched nondiabetic controls of each genotype were maintained for 22 weeks, after which ERGs were used to measure the light-evoked components of the RPE (dc-ERG) and the neural retina (a-wave, b-wave). In comparison to their nondiabetic controls, wildtype (WT) and AR−/− diabetic mice displayed significant decreases in the c-wave, fast oscillation, and off response components of the dc-ERG but not in the light peak response. Nondiabetic AR−/− mice displayed larger ERG component amplitudes than did nondiabetic WT mice; however, the amplitude of dc-ERG components in diabetic AR−/− animals were similar to WT diabetics. ERG a-wave amplitudes were not reduced in either diabetic group, but b-wave amplitudes were lower in WT and AR−/−diabetic mice. These findings demonstrate that the light-induced responses of the RPE and outer retina are disrupted in diabetic mice, but these defects are not due to photoreceptor dysfunction, nor are they ameliorated by deletion of AR. This latter finding suggests that benefits observed in other studies utilizing pharmacological inhibitors of AR might have been secondary to off-target effects of the drugs.

During the course of light adaptation, the amplitude and implicit time of the human cone ERG change systematically. In the present study, the effect of adapting field luminance on these ERG changes was assessed, and the hypothesis that light adaptation of the rod system is the primary determining factor was evaluated. Cone ERG responses, isolated through the use of 31.1-Hz flicker, were obtained from two visually normal subjects, initially under dark-adapted conditions and then repeatedly for 30 min following the onset of each of a series of ganzfeld adapting fields with luminances that ranged from –1.2 to 2.1 log cd/m. The increase in flicker ERG amplitude and decrease in implicit time during light adaptation were greatest at the highest adapting field luminances. Photopically equivalent achromatic and long-wavelength adapting fields induced comparable increases in flicker ERG amplitude, while scotopically equivalent adapting fields had considerably different effects. This latter finding demonstrates that the rod system is not a major determinant of the adaptation-induced increase in cone ERG amplitude.

Mutations in the human rod opsin gene have been shown to segregate with autosomal dominant retinitis pigmentosa (ADRP) and photoreceptor degeneration in transgenic mice. While these degenerations are characterized by the primary degeneration of rods, cones eventually die as well. To determine whether this subsequent cone degeneration is the result of expression of mutant rod opsin in the cones, the retinal cell-type specificity of a 221-bp fragment of the mouse rod opsin promoter was evaluated. Two transgenic mouse lines generated by injecting a fusion gene comprised of a 221-bp fragment of the mouse rod opsin promoter and the simian virus 40 large tumor antigen gene (Tag) were examined. The expression of Tag causes photoreceptor cell degeneration in members of both transgenic lines. However, the two lines differed with respect to the level of Tag expression and the rate and extent of photoreceptor cell degeneration. Immunocytochemical localization of opsin and Tag in surviving photoreceptor cells was determined and the results were confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). Rod- and cone-mediated function was evaluated by electroretinography (ERG). In the higher Tag-expressing transgenic line only one row of nuclei remained in the outer nuclear layer at postnatal day (P) 150. While these nuclei showed no antigenicity for rod opsin or Tag, they did stain with an antibody that reacts with both rod and cone S-antigens (arrestins), indicating that these cells were surviving photoreceptor nuclei. Positive staining with peanut agglutinin, which uniquely decorates matrix domains surrounding cones in the normal retina, confirmed that the surviving photoreceptor nuclei were of cone origin. RT-PCR substantiated the results from immunostaining; amplification product was obtained using blue cone opsin transcripts but not from either Tag or rod opsin transcripts. The second transgenic mouse line exhibited a much slower photoreceptor cell death that was associated with low levels of Tag transgene transcript. At P120, ~50% of photoreceptors remained and an ~45% reduction in the rod ERG a-wave was observed. Cone-mediated ERGs, however, were normal. The results demonstrate the rod-specific expression of Tag as directed by the 221-bp fragment of the mouse rod opsin promoter and suggest that the cone degeneration in ADRP or transgenic mice associated with mutations in the rod opsin gene is a secondary effect of rod degeneration.

Visual function was evaluated in transgenic mice expressing the simian virus 40 early region under the control of the promoter for phenylethanolamine-N-methyltransferase. These transgenic mice undergo a degeneration of the retinal horizontal cells and the outer plexiform layer. Electroretinograms (ERGs) were recorded under stimulus conditions chosen to elicit both receptoral and postreceptoral responses. The dark-adapted a-waves obtained from transgenic mice were not different from control recordings, indicating that the degenerative process does not interfere with function of the rod photoreceptors. In comparison, the ERG b-wave was markedly reduced in transgenic mice under both dark- and light-adapted conditions. Reproducible visual evoked potentials (VEPs) were recorded from transgenic mice in response to both low luminance stimuli that isolate rod function, and to higher luminance stimuli, indicating that retinal activity is transmitted centrally to the visual cortex. However, VEPs were delayed at all stimulus luminances compared to controls. Analysis of luminance-response functions suggests that the VEP delays could reflect the combination of a decrease in synaptic efficacy and an overall loss in visual sensitivity. These functional abnormalities correlate well with the anatomical abnormalities that have been previously observed in the transgenic retina (Hammang et al., 1993), namely a reduced number of synapses between photoreceptors and second-order neurons.

This study evaluated retinal function in mice following the expression of oncogenes under the control of photoreceptor-specific promoters in transgenic mice. Electroretinograms (ERGs) were recorded under stimulus conditions chosen to elicit rod- or cone-mediated components. In one transgenic line (MOT1), the simian virus 40 large tumor antigen was expressed under the control of the mouse opsin promoter. MOT1 mice exhibited an age-related decline in the amplitude of the rod-mediated ERG a-wave. In comparison, cone-mediated responses recorded from MOT1 mice remained normal up to four months of age, the oldest age tested. In the second transgenic line (CMYC), the rat c-myc gene was expressed under control of the human interphotoreceptor-retinoid binding protein promoter. CMYC mice exhibited a rapid reduction of cone-mediated responses and a gradual loss of the rod ERG a-wave. Analysis of rod ERG a-waves obtained from young MOT1 and CMYC mice indicated that the rod ERG abnormalities reflect a reduction in the number of rods contributing to the response with the retention of normal response properties in rods that remain. These results support the possibility that aberrant expression of oncogenes may underlie some forms of human rod and cone-rod dystrophy.

A 4 base pair deletion in a splice donor site of the Mfrp (membrane-type frizzled-related protein) gene, herein referred to as Mfrprd6/rd6, is predicted to lead to the skipping of exon 4 and photoreceptor degeneration in retinal degeneration 6 (rd6) mutant mice. Little, however, is known about the function of the protein or how the mutation causes the degenerative retinal phenotype. Here we examine ultrastructural changes in the retina of Mfrprd6/rd6 mice to determine the earliest effects of the mutation. We also extend the reported observations of the expression pattern of the dicistronic Mfrp/C1qtnf5 message and the localization of these and other retinal pigment epithelium (RPE) and retinal proteins during development and assess the ability of RPE cells to phagocytize outer segments (OSs) in mutant and wild-type (WT) mice. At the ultrastructural level, OSs do not develop normally in Mfrprd6/rd6 mutants. They are disorganized and become progressively shorter as mutant mice age. Additionally, there are focal areas in which there is a reduction of apical RPE microvilli. At P25, the rod electroretinogram (ERG) a-wave of Mfrprd6/rd6 mice is reduced in amplitude by ~50% as are ERG components generated by the RPE. Examination of β-catenin localization and Fos and Tcf-1 expression, intermediates of the canonical Wnt pathway, showed that they were not different between mutant and WT mice, suggesting that MFRP may operate through an alternative pathway. Finally, impaired OS phagocytosis was observed in Mfrprd6/rd6 mice both in standard ambient lighting conditions and with bright light exposure when compared to WT controls.

Glutamate release from photoreceptor terminals is controlled by voltage-dependent calcium channels (VDCCs). In humans, mutations in the Cacna1f gene, encoding the α1F subunit of VDCCs, underlie the incomplete form of X-linked congenital stationary night blindness (CSNB2). These mutations impair synaptic transmission from rod and cone photoreceptors to bipolar cells. Here, we report anatomical and functional characterizations of the retina in the nob2 (no b-wave 2) mouse, a naturally occurring mutant caused by a null mutation in Cacna1f. Not surprisingly, the b-waves of both the light- and dark-adapted electroretinogram are abnormal in nob2 mice. The outer plexiform layer (OPL) is disorganized, with extension of ectopic neurites through the outer nuclear layer that originate from rod bipolar and horizontal cells, but not from hyperpolarizing bipolar cells. These ectopic neurites continue to express mGluR6, which is frequently associated with profiles that label with the presynaptic marker Ribeye, indicating potential points of ectopic synapse formation. However, the morphology of the presynaptic Ribeye-positive profiles is abnormal. While cone pedicles are present their morphology also appears compromised. Characterizations of visual responses in retinal ganglion cells in vivo, under photopic conditions, demonstrate that ON-center cells have a reduced dynamic range, although their basic center-surround organization is retained; no alteration in the responses of OFF-center cells was evident. These results indicate that nob2 mice are a valuable model in which to explore the pathophysiological mechanisms associated with Cacna1f mutations causing CSNB2, and the subsequent effects on visual information processing. Further, the nob2 mouse represents a model system in which to define the signals that guide synapse formation and/or maintenance in the OPL.

Electroretinography provides a useful noninvasive approach to evaluate cone pathway activity. Despite wide application of the cone ERG to characterize retinal function in transgenic mice and mouse models of human hereditary retinal disease, the cellular origins of the mouse cone ERG have not been well defined. Here, we address this issue using a pharmacological approach that has been previously applied to other species. Agents that block receptor activation at well-defined retinal loci were dissolved in saline and injected into the vitreous of anesthetized adult BALBc/ByJ mice; cone ERGs were recorded 1–2 h later. Analysis of the resulting waveforms indicated that the mouse cone ERG includes a cornea-negative component that is derived from the activity of cone photoreceptors and retinal glial (Müller) cells. Similar to other species, activity of cone depolarizing bipolar cells contributes a large amplitude cornea-positive potential to the mouse cone ERG. In contrast to primate but similar to rat, the mouse cone ERG includes only a small contribution from hyperpolarizing bipolar cell activity. The inner retina appears to contribute to both the a- and b-waves of the mouse cone ERG. These results provide a foundation for interpreting changes in the waveform of the mouse cone ERG that may be observed following genetic alteration or other experimental treatment.

Primary open angle glaucoma (POAG) is a leading cause of late onset, progressive, irreversible blindness and, although its etiology is poorly understood, elevated intraocular pressure (IOP) often appears to be a contributory factor. Proteomic and Western analyses of trabecular meshwork (TM) from patients with POAG and age-matched controls originally implicated cochlin as possibly contributing to glaucoma pathogenesis. Cochlin deposits were subsequently detected in glaucomatous but not in control TM and older glaucomatous TM was found to contain higher levels of cochlin and significantly lower amounts of collagen type II. More recently, similar results were reported in DBA/2J mice, which at older ages develop elevated IOP, retinal ganglion cell degeneration, and optic nerve damage. Notably, cochlin was absent in TM from C57BL/6J, CD1, and BALBc/ByJ mice, which do not exhibit elevated IOP or glaucoma. Cochlin was found in the TM of very young DBA/2J mice, prior to elevated IOP, suggesting that over time the protein may contribute to the events leading to increased IOP and optic nerve damage. Here we review these findings and describe how future studies in DBA/2J mice can help resolve whether cochlin plays a causal role in mechanisms of POAG and elevated IOP.

We have used wild-type mice and mice possessing defects in specific retinal circuits in order to more clearly define functional circuits of the inner retina. The retina of the nob mouse lacks communication between photoreceptors and depolarizing bipolar cells (DBCs). Thus, all light driven activity in the nob mouse is mediated via remaining hyperpolarizing bipolar cell (HBC) circuits. Transducin null (Trα−/−) mice lack rod photoreceptor activity and thus remaining retinal circuits are solely generated via cone photoreceptor activity. Activation in inner retinal circuits in each of these mice was identified by monitoring light-induced expression of an immediate early gene, c-fos. The number of cells expressing c-fos in the inner retina was dependent upon stimulus intensity and was altered in a systematic fashion in mice with known retinal mutations. To determine whether c-fos is activated via circuits other than photoreceptors in the outer retina, we examined c-fos expression in tulp1−/− mice that lack photoreceptors in the outer retina; these mice showed virtually no c-fos activity following light exposure. Double-labeling immunohistochemical studies were carried out to more clearly define the population of c-fos expressing amacrine cells. Our results indicate that c-fos may be used to map functional circuits in the retina.

The electroretinogram (ERG) of the cone system provides a useful noninvasive measure of the activity of the cone pathway. Despite a wide application of the cone ERG in the study of rodent models of human hereditary retinal disease, the cellular origins of the rat cone ERG have not been well defined. Here, we address this issue using a pharmacological approach that has been used previously to derive ERG response components. Agents that impair synaptic transmission at well-defined retinal loci were dissolved in saline and injected into the vitreous of adult Sprague-Dawley rats anesthetized with ketamine/xylazine, and cone ERGs were recorded approximately 2 h later. Analysis of the resulting waveforms indicated that the rat cone ERG includes a relatively small-amplitude component of negative polarity that is derived from the activity of cone photoreceptors, and perhaps retinal glial (Müller) cells. The cone depolarizing bipolar cell pathway contributes a positive potential of large amplitude to the rat cone ERG. In comparison, the contribution of hyperpolarizing bipolar cells is of negative polarity and of much smaller amplitude. The inner retina contributes a negative wave upon which higher frequency oscillations are superimposed. These results provide a foundation for interpreting changes in the waveform of the rat cone ERG that may be observed following genetic alteration or other experimental treatment.

In the nob mouse, a mutation in nyctalopin results in a loss of signal transmission from photoreceptors to depolarizing bipolar cells (DBCs). We used immunohistochemical techniques to assess the expression pattern of proteins found at either the photoreceptor terminal or bipolar cell dendrites within the outer plexiform layer. We labeled normal and nob retinas with antibodies against mGluR6, PKC, G0α, bassoon, PSD-95, the α1F subunit of voltage-gated calcium channels, trkB, and dystrophin. All labeling patterns in nob and normal retinas were comparable to those previously reported in mouse retina. Our results indicate that the absence of nyctalopin does not disrupt the expression pattern of other proteins known to be required for synaptic transmission.

In response to light, the retinal pigment epithelium (RPE) generates a series of potentials that can be recorded using the dc-electroretinogram (dc-ERG). As these potentials can be related to specific cellular events, they provide information about RPE function and how that may be altered by disease or experimental manipulation. The purposes of the present study were to define a noninvasive means for recording the rat dc-ERG, to use this to define the stimulus–response properties of the major components, and to relate these results to measures of the rat electrooculogram (EOG). Parallel studies were conducted in two strains of rats (Long-Evans, LE; Sprague-Dawley, SD) that are commonly used in vision research. Rats were sedated with ketamine/xylazine and placed on a heating pad. Ag/AgCl wire electrodes were bridged with capillary tubes filled with Hanks balanced salt solution. The active electrode was placed in contact with the corneal surface and referenced to a second electrode placed within the orbit. The dc-ERG signal was amplified (dc-100 Hz), digitized, and stored offline. The duration of full-field flash stimuli was controlled using a mechanical shutter and flash luminance was controlled with neutral density filters. EOGs were recorded using subdermal platinum needle electrodes placed near the eye. In response to a 5-min light exposure, the dc-ERG of LE and SD rats included a distinct b-wave, after potential, c-wave, fast oscillation, and a slow potential of positive polarity the characteristics of which are consistent with a light peak.

In the vertebrate retina, vision is initiated and maintained by the photolysis and regeneration, respectively, of light-sensitive pigments in the disk membranes of the photoreceptor outer segments. This cyclical process depends on an exchange of retinoids between the photoreceptors and the retinal pigment epithelium (RPE). There is a great deal of indirect evidence that the transport of retinoids between these cellular compartments is mediated by the interphotoreceptor retinoid-binding protein (IRBP), a large glycoprotein synthesized in the photoreceptors and extruded into the interphotoreceptor matrix (IPM) that fills the subretinal space. Nevertheless, a number of in vitro experiments have demonstrated that an intermembranous transfer of retinoids can occur through an aqueous medium independent of any retinoid-binding protein. This led to the suggestion that IRBP may play the more passive role of an extracellular buffer, serving to prevent the degradation and potentially cytotoxic effects of free retinoids when large amounts are released into the IPM. We have studied the structural and functional properties of transgenic mice in which homologous recombination was used to delete the IRBP gene. Light- and electron-microscopic examination of the retinas of “knockout” (IRBP−/−) mice revealed a significant loss of photoreceptor nuclei, and profound changes in the structure and organization of the receptor outer segments. Consistent with these observations, electroretinographic recordings showed a marked reduction in response amplitude for both rod- and cone-mediated potentials. However, despite the histological and electrophysiological changes, there was no evidence of gross abnormalities in the visual cycle. After bleaching a significant fraction of the available rhodopsin, electroretinogram amplitude and rhodopsin density gradually increased toward their pre-bleach levels, and the rates of recovery were even more rapid than those seen in wild-type (IRBP+/+) mice.