T-type voltage-gated channels, Na+/Ca2+-exchanger, and calpain-2 promote photoreceptor cell death in inherited retinal degeneration.

Inherited retinal degenerations (IRDs) are a group of untreatable and commonly blinding diseases characterized by progressive photoreceptor loss. IRD pathology has been linked to an excessive activation of cyclic nucleotide-gated channels (CNGC) leading to Na+- and Ca2+-influx, subsequent activation of voltage-gated Ca2+-channels (VGCC), and further Ca2+ influx. However, a connection between excessive Ca2+ influx and photoreceptor loss has yet to be proven.Here, we used whole-retina and single-cell RNA-sequencing to compare gene expression between the rd1 mouse model for IRD and wild-type (wt) mice. Differentially expressed genes indicated links to several Ca2+-signalling related pathways. To explore these, rd1 and wt organotypic retinal explant cultures were treated with the intracellular Ca2+-chelator BAPTA-AM or inhibitors of different Ca2+-permeable channels, including CNGC, L-type VGCC, T-type VGCC, Ca2+-release-activated channel (CRAC), and Na+/Ca2+ exchanger (NCX). Moreover, we employed the novel compound NA-184 to selectively inhibit the Ca2+-dependent protease calpain-2. Effects on the retinal activity of poly(ADP-ribose) polymerase (PARP), sirtuin-type histone-deacetylase, calpains, as well as on activation of calpain-1, and - 2 were monitored, cell death was assessed via the TUNEL assay.While rd1 photoreceptor cell death was reduced by BAPTA-AM, Ca2+-channel blockers had divergent effects: While inhibition of T-type VGCC and NCX promoted survival, blocking CNGCs and CRACs did not. The treatment-related activity patterns of calpains and PARPs corresponded to the extent of cell death. Remarkably, sirtuin activity and calpain-1 activation were linked to photoreceptor protection, while calpain-2 activity was related to degeneration. In support of this finding, the calpain-2 inhibitor NA-184 protected rd1 photoreceptors.These results suggest that Ca2+ overload in rd1 photoreceptors may be triggered by T-type VGCCs and NCX. High Ca2+-levels likely suppress protective activity of calpain-1 and promote retinal degeneration via activation of calpain-2. Overall, our study details the complexity of Ca2+-signalling in photoreceptors and emphasizes the importance of targeting degenerative processes specifically to achieve a therapeutic benefit for IRDs. Video Abstract.

The Mongolian gerbil as an advanced model to study cone system physiology.

In this work, we introduce a diurnal rodent, the Mongolian gerbil (Meriones unguiculatus) (MG) as an alternative to study retinal cone system physiology and pathophysiology in mice. The cone system is of particular importance, as it provides high-acuity and color vision and its impairment in retinal disorders is thus especially disabling. Despite their nocturnal lifestyle, mice are currently the most popular animals to study cone-related diseases due to the high availability of genetically modified models. However, the potential for successful translation of any cone-related results is limited due to the substantial differences in retinal organization between mice and humans. Alternatively, there are diurnal rodents such as the MG with a higher retinal proportion of cones and a macula-like specialized region for improved visual resolution, the visual streak. The focus of this work was the evaluation of the MG's cone system functionality using full-field electroretinography (ERG), together with a morphological assessment of its retinal/visual streak organization via angiography, optical coherence tomography (OCT), and photoreceptor immunohistochemistry. We found that rod system responses in MGs were comparable or slightly inferior to mice, while in contrast, cone system responses were much larger, more sensitive, and also faster than those in the murine counterparts, and in addition, it was possible to record sizeable ON and OFF ERG components. Morphologically, MG cone photoreceptor opsins were evenly distributed throughout the retina, while mice show a dorsoventral M- and S-opsin gradient. Additionally, each cone expressed a single opsin, in contrast to the typical co-expression of opsins in mice. Particular attention was given to the visual streak region, featuring a higher density of cones, elongated cone and rod outer segments (OSs), and an increased thickness of the inner and outer retinal layers in comparison to peripheral regions. In summary, our data render the MG a supreme model to investigate cone system physiology, pathophysiology, and to validate potential therapeutic strategies in that context.

Mural Serum Response Factor (SRF) Deficiency Provides Insights into Retinal Vascular Functionality and Development.

Serum response factor (SRF) controls the expression of muscle contraction and motility genes in mural cells (MCs) of the vasculature. In the retina, MC-SRF is important for correct angiogenesis during development and the continuing maintenance of the vascular tone. The purpose of this study was to provide further insights into the effects of MC SRF deficiency on the vasculature and function of the mature retina in SrfiMCKO mice that carry a MC-specific deletion of Srf. Retinal morphology and vascular integrity were analyzed in vivo via scanning laser ophthalmoscopy (SLO), angiography, and optical coherence tomography (OCT). Retinal function was evaluated with full-field electroretinography (ERG). We found that retinal blood vessels of these mutants exhibited different degrees of morphological and functional alterations. With increasing severity, we found vascular bulging, the formation of arteriovenous (AV) anastomoses, and ultimately, a retinal detachment (RD). The associated irregular retinal blood pressure and flow distribution eventually induced hypoxia, indicated by a negative ERG waveform shape. Further, the high frequency of interocular differences in the phenotype of individual SrfiMCKO mice points to a secondary nature of these developments far downstream of the genetic defect and rather dependent on the local retinal context.

Inherited Retinal Degeneration: PARP-Dependent Activation of Calpain Requires CNG Channel Activity.

Inherited retinal degenerations (IRDs) are a group of blinding diseases, typically involving a progressive loss of photoreceptors. The IRD pathology is often based on an accumulation of cGMP in photoreceptors and associated with the excessive activation of calpain and poly (ADP-ribose) polymerase (PARP). Inhibitors of calpain or PARP have shown promise in preventing photoreceptor cell death, yet the relationship between these enzymes remains unclear. To explore this further, organotypic retinal explant cultures derived from wild-type and IRD-mutant mice were treated with inhibitors specific for calpain, PARP, and voltage-gated Ca2+ channels (VGCCs). The outcomes were assessed using in situ activity assays for calpain and PARP and immunostaining for activated calpain-2, poly (ADP-ribose), and cGMP, as well as the TUNEL assay for cell death detection. The IRD models included the Pde6b-mutant rd1 mouse and rd1*Cngb1-/- double-mutant mice, which lack the beta subunit of the rod cyclic nucleotide-gated (CNG) channel and are partially protected from rd1 degeneration. We confirmed that an inhibition of either calpain or PARP reduces photoreceptor cell death in rd1 retina. However, while the activity of calpain was decreased by the inhibition of PARP, calpain inhibition did not alter the PARP activity. A combination treatment with calpain and PARP inhibitors did not synergistically reduce cell death. In the slow degeneration of rd1*Cngb1-/- double mutant, VGCC inhibition delayed photoreceptor cell death, while PARP inhibition did not. Our results indicate that PARP acts upstream of calpain and that both are part of the same degenerative pathway in Pde6b-dependent photoreceptor degeneration. While PARP activation may be associated with CNG channel activity, calpain activation is linked to VGCC opening. Overall, our data highlights PARP as a target for therapeutic interventions in IRD-type diseases.

The Y227N mutation affects bestrophin-1 protein stability and impairs sperm function in a mouse model of Best vitelliform macular dystrophy.

Human bestrophin-1 (BEST1) is an integral membrane protein known to function as a Ca2+-activated and volume-regulated chloride channel. The majority of disease-associated mutations in BEST1 constitute missense mutations and were shown in vitro to lead to a reduction in mutant protein half-life causing Best disease (BD), a rare autosomal dominant macular dystrophy. To further delineate BEST1-associated pathology in vivo and to provide an animal model useful to explore experimental treatment efficacies, we have generated a knock-in mouse line (Best1Y227N). Heterozygous and homozygous mutants revealed no significant ocular abnormalities up to 2 years of age. In contrast, knock-in animals demonstrated a severe phenotype in the male reproductive tract. In heterozygous Best1Y227N males, Best1 protein was significantly reduced in testis and almost absent in homozygous mutant mice, although mRNA transcription of wild-type and knock-in allele is present and similar in quantity. Degradation of mutant Best1 protein in testis was associated with adverse effects on sperm motility and the capability to fertilize eggs. Based on these results, we conclude that mice carrying the Best1 Y227N mutation reveal a reproducible pathologic phenotype and thus provide a valuable in vivo tool to evaluate efficacy of drug therapies aimed at restoring Best1 protein stability and function.

A retinal model of cerebral malaria.

Malaria is a causative factor in about 500.000 deaths each year world-wide. Cerebral malaria is a particularly severe complication of this disease and thus associated with an exceedingly high mortality. Malaria retinopathy is an ocular manifestation often associated with cerebral malaria, and presumably shares a substantial part of its pathophysiology. Here, we describe that indeed murine malaria retinopathy reproduced the main hallmarks of the corresponding human disease. In the living animal, we were able to follow the circulation and cellular localization of malaria parasites transgenically labelled with GFP via non-invasive in vivo retinal imaging. We found that malaria parasites cross the blood-retinal-barrier and infiltrate the neuroretina, concomitant with an extensive, irreversible, and long-lasting retinal neurodegeneration. Furthermore, anti-malarial treatment with dihydroartemisinin strongly diminished the load of circulating parasites but resolved the symptoms of the retinopathy only in part. In summary, we introduce here a novel preclinical model for human cerebral malaria that is much more directly accessible for studies into disease pathophysiology and development of novel treatment approaches. In vivo retinal imaging may furthermore serve as a valuable tool for the early diagnosis of the human disease.

Optimized Subretinal Injection Technique for Gene Therapy Approaches.

Gene therapy for inherited eye diseases requires local viral vector delivery by intraocular injection. Since large animal models are lacking for most of these diseases, genetically modified mouse models are commonly used in preclinical proof-of-concept studies. However, because of the relatively small mouse eye, adverse effects of the subretinal delivery procedure itself may interfere with the therapeutic outcome. The method described here aims to provide the details relevant to perform a transscleral pars plana virus-mediated gene transfer to achieve an optimized therapeutic effect in the small mouse eye.

Cystoid edema, neovascularization and inflammatory processes in the murine Norrin-deficient retina.

Mutations in the Norrin (NDP) gene cause severe developmental blood vessel defects in the retina leading to congenital blindness. In the retina of Ndph-knockout mice only the superficial capillary network develops. Here, a detailed characterization of this mouse model at late stages of the disease using in vivo retinal imaging revealed cystoid structures that closely resemble the ovoid cysts in the inner nuclear layer of the human retina with cystoid macular edema (CME). In human CME an involvement of Müller glia cells is hypothesized. In Ndph-knockout retinae we could demonstrate that activated Müller cells were located around and within these cystoid spaces. In addition, we observed extensive activation of retinal microglia and development of neovascularization. Furthermore, ex vivo analyses detected extravasation of monocytic cells suggesting a breakdown of the blood retina barrier. Thus, we could demonstrate that also in the developmental retinal vascular pathology present in the Ndph-knockout mouse inflammatory processes are active and may contribute to further retinal degeneration. This observation delivers a new perspective for curative treatments of retinal vasculopathies. Modulation of inflammatory responses might reduce the symptoms and improve visual acuity in these diseases.

Assessment of the Absolute Excitatory Level of the Retina by Flicker ERG.

Electroretinography (ERG) is important for functional diagnostics of the retina. Types of information about retinal function obtainable by ERG differ depending on recording conditions, e.g., a combination of light stimulus and adaptation. In terms of stimulation, single-flash and flicker stimuli are frequently used because response properties have been well investigated, allowing an assessment of fundamental retinal functionality; for example, how photoreceptors and bipolar cells, including signal transmission between them, are affected under pathological conditions. Usually, ERGs are recorded with a nonzero lower cutoff frequency of amplifiers to avoid certain artifacts, and additionally, responses are averaged over time so that non-event-related signals are cancelled out. However, the improved signal quality is associated with a loss of information. Especially in steady-state flicker ERG, information about the absolute baseline of recordings is missing because the prestimulus baseline is not included on the recording trace as well as because a zero response is obtained in all cases in which the signal baseline stays constant for a sufficient amount of time. In other words, it is impossible to tell from the conventional flicker ERG whether a zero signal is obtained under conditions of maximal or no excitation of the visual system. In this chapter, we describe a direct current ERG protocol (featuring a lower cutoff frequency of zero) with repetitive single flashes mimicking conventional flicker that contains a defined onset. Using this recording protocol, it is possible to assess not only the absolute excitatory level of the retina but also the development of steady-state responses from the single flash response.

Patients and animal models of CNGß1-deficient retinitis pigmentosa support gene augmentation approach.

Retinitis pigmentosa (RP) is a major cause of blindness that affects 1.5 million people worldwide. Mutations in cyclic nucleotide-gated channel ß 1 (CNGB1) cause approximately 4% of autosomal recessive RP. Gene augmentation therapy shows promise for treating inherited retinal degenerations; however, relevant animal models and biomarkers of progression in patients with RP are needed to assess therapeutic outcomes. Here, we evaluated RP patients with CNGB1 mutations for potential biomarkers of progression and compared human phenotypes with those of mouse and dog models of the disease. Additionally, we used gene augmentation therapy in a CNGß1-deficient dog model to evaluate potential translation to patients. CNGB1-deficient RP patients and mouse and dog models had a similar phenotype characterized by early loss of rod function and slow rod photoreceptor loss with a secondary decline in cone function. Advanced imaging showed promise for evaluating RP progression in human patients, and gene augmentation using adeno-associated virus vectors robustly sustained the rescue of rod function and preserved retinal structure in the dog model. Together, our results reveal an early loss of rod function in CNGB1-deficient patients and a wide window for therapeutic intervention. Moreover, the identification of potential biomarkers of outcome measures, availability of relevant animal models, and robust functional rescue from gene augmentation therapy support future work to move CNGB1-RP therapies toward clinical trials.

In-Depth Functional Analysis of Rodents by Full-Field Electroretinography.

Full-field electroretinography (ERG) belongs to the gold-standard of electrophysiological test systems in ophthalmology and reflects the sum response of the entire retina to light stimulation. The assessment of the retinal function is a fundamental diagnostic technique not only in the clinical ophthalmology it is also indispensable in the ophthalmic research, in particular, in therapeutic approaches where the in vivo follow up of the benefit after treatment is absolutely necessary. Several current therapeutic approaches have demonstrated long-lasting amelioration in respective disease models and show promise for a successful translation to human patients. In this chapter we provide electroretinography protocols of experimental data which may serve as informative features for upcoming gene therapeutic approaches and clinical trials.

Advanced Ocular Injection Techniques for Therapy Approaches.

Treatment approaches for inherited eye diseases require local therapeutic molecule delivery by intraocular injection. One important factor that can influence the study outcome is the quality of intraocular administration. The intracompartmental structure (e.g., vitreous) of the eye allows a sustainable release of therapeutic biologicals using an intravitreal delivery. The protocol described here aims at providing the details relevant to perform a transscleral pars plana intravitreal transfer in small eyes using a genetically modified stem cell system. The fact that cells and therewith visually distinct particles are implanted, allows for the assessment of the implantation site and the distribution, and possibilities for temporal follow up studies-hence, valuable information becomes available which can be used to fine-tune the intravitreal delivery technique.

Gene Supplementation Rescues Rod Function and Preserves Photoreceptor and Retinal Morphology in Dogs, Leading the Way Toward Treating Human PDE6A-Retinitis Pigmentosa.

Mutations in the phosphodiesterase 6A gene (PDE6A) result in retinitis pigmentosa (RP) type 43 (RP43) and are responsible for about 4% of autosomal recessive RP. There is currently no treatment for this blinding condition. The aim of this project was to use a large-animal model to test a gene supplementation viral vector designed to be translated for use in a clinical trial for the treatment of RP43. Seven Pde6a-/- puppies were given sub-retinal injections of an adeno-associated viral vector (AAV) serotype 2/8 delivering human PDE6A cDNA under control of a short rhodopsin promoter (AAV8-PDE6A). Three puppies received ∼1 × 1011 vg in one eye and four puppies ∼5 × 1011 vg/per eye, with both eyes being injected in two animals. In vivo outcome measures included vision testing and electroretinography (ERG), as well as fundus and spectral domain-optical coherence tomography imaging. Some puppies were euthanized and their eyes processed for immunohistochemistry. All puppies had improved rod-mediated vision in the treated eye. ERGs showed improved rod-mediated responses in the higher-dose group but in only one of the lower-dose group animals. Receptor+ thickness was preserved and photoreceptor morphology improved in the treated retinal regions in all puppies. Treatment resulted in PDE6A transgene expression, accompanied by much increased levels of Pde6b, in rod outer segments in the injected retinal regions. There were several indications of improved retinal health in the PDE6A-expressing regions, including lack of abnormal cyclic guanosine monophosphate accumulation, appropriate rod opsin localization to the outer segments with a large reduction in mislocalization to other regions of the rod cell, and reduced Müller cell activation. Additionally, cone photoreceptors showed morphological improvement in the treated region, with normal-appearing inner and outer segments. AAV8-PDE6A gene supplementation therapy restored rod vision in Pde6a-/- puppies and preserved retinal morphology. These positive outcomes are an important step toward a human clinical trial to treat PDE6A-RP.

Gene Therapy Successfully Delays Degeneration in a Mouse Model of PDE6A-Linked Retinitis Pigmentosa (RP43).

Retinitis pigmentosa type 43 (RP43) is a blinding disease caused by mutations in the gene for rod phosphodiesterase 6 alpha (PDE6A). The disease process begins with a dysfunction of rod photoreceptors, subsequently followed by a currently untreatable progressive degeneration of the entire outer retina. Aiming at a curative approach via PDE6A gene supplementation, a novel adeno-associated viral (AAV) vector was developed for expression of the human PDE6A cDNA under control of the human rhodopsin promotor (rAAV8.PDE6A). This study assessed the therapeutic efficacy of rAAV8.PDE6A in the Pde6anmf363/nmf363-mutant mouse model of RP43. All mice included in this study were treated with sub-retinal injections of the vector at 2 weeks after birth. The therapeutic effect was monitored at 1 month and 6 months post injection. Biological function of the transgene was assessed in vivo by means of electroretinography. The degree of morphological rescue was investigated both in vivo using optical coherence tomography and ex vivo by immunohistological staining. It was found that the novel rAAV8.PDE6A vector resulted in a stable and efficient expression of PDE6A protein in rod photoreceptors of Pde6anmf363/nmf363 mice following treatment at both the short- and long-term time points. The treatment led to a substantial morphological preservation of outer nuclear layer thickness, rod outer segment structure, and prolonged survival of cone photoreceptors for at least 6 months. Additionally, the ERG analysis confirmed a restoration of retinal function in a group of treated mice. Taken together, this study provides successful proof-of-concept for the cross-species efficacy of the rAAV8.PDE6A vector developed for use in human patients. Importantly, the data show stable expression and rescue effects for a prolonged period of time, raising hope for future translational studies based on this approach.

Long-term consequences of developmental vascular defects on retinal vessel homeostasis and function in a mouse model of Norrie disease.

Loss of Norrin signalling due to mutations in the Norrie disease pseudoglioma gene causes severe vascular defects in the retina, leading to visual impairment and ultimately blindness. While the emphasis of experimental work so far was on the developmental period, we focus here on disease mechanisms that induce progression into severe adult disease. The goal of this study was the comprehensive analysis of the long-term effects of the absence of Norrin on vascular homeostasis and retinal function. In a mouse model of Norrie disease retinal vascular morphology and integrity were studied by means of in vivo angiography; the vascular constituents were assessed in detailed histological analyses using quantitative retinal morphometry. Finally, electroretinographic analyses were performed to assess the retinal function in adult Norrin deficient animals. We could show that the primary developmental defects not only persisted but developed into further vascular abnormalities and microangiopathies. In particular, the overall vessel homeostasis, the vascular integrity, and also the cellular constituents of the vascular wall were affected in the adult Norrin deficient retina. Moreover, functional analyses indicated to persistent hypoxia in the neural retina which was suggested as one of the major driving forces of disease progression. In summary, our data provide evidence that the key to adult Norrie disease are ongoing vascular modifications, driven by the persistent hypoxic conditions, which are ineffective to compensate for the primary Norrin-dependent defects.

AAV-Mediated Gene Supplementation Therapy in Achromatopsia Type 2: Preclinical Data on Therapeutic Time Window and Long-Term Effects.

Achromatopsia type 2 (ACHM2) is a severe, inherited eye disease caused by mutations in the CNGA3 gene encoding the α subunit of the cone photoreceptor cyclic nucleotide-gated (CNG) channel. Patients suffer from strongly impaired daylight vision, photophobia, nystagmus, and lack of color discrimination. We have previously shown in the Cnga3 knockout (KO) mouse model of ACHM2 that gene supplementation therapy is effective in rescuing cone function and morphology and delaying cone degeneration. In our preclinical approach, we use recombinant adeno-associated virus (AAV) vector-mediated gene transfer to express the murine Cnga3 gene under control of the mouse blue opsin promoter. Here, we provide novel data on the efficiency and permanence of such gene supplementation therapy in Cnga3 KO mice. Specifically, we compare the influence of two different AAV vector capsids, AAV2/5 (Y719F) and AAV2/8 (Y733F), on restoration of cone function, and assess the effect of age at time of treatment on the long-term outcome. The evaluation included in vivo analysis of retinal function using electroretinography (ERG) and immunohistochemical analysis of vector-driven Cnga3 transgene expression. We found that both vector capsid serotypes led to a comparable rescue of cone function over the observation period between 4 weeks and 3 months post treatment. In addition, a clear therapeutic effect was present in mice treated at 2 weeks of age as well as in mice treated at 3 months of age at the first assessment at 4 weeks after treatment. Importantly, the effect extended in both cases over the entire observation period of 12 months post treatment. However, the average ERG amplitude levels differed between the two groups, suggesting a role of the absolute age, or possibly, the associated state of the degeneration, on the achievable outcome. In summary, we found that the therapeutic time window of opportunity for AAV-mediated Cnga3 gene supplementation therapy in the Cnga3 KO mouse model extends at least to an age of 3 months, but is presumably limited by the condition, number and topographical distribution of remaining cones at the time of treatment. No impact of the choice of capsid on the therapeutic success was detected.

Murine Autoimmune Optic Neuritis Is Not Phenotypically Altered by the Retinal Degeneration 8 Mutation.

Purpose: To investigate whether the presence of the retinal degeneration 8 (rd8) mutation in C57BL/6 mice alters the phenotype of autoimmune optic neuritis (AON). Methods: C57BL/6J and C57BL/6N mice were genotyped for the rd8 mutation and fundus analyses and examination of retinal layer morphology were performed in vivo by scanning laser ophthalmoscopy and optical coherence tomography. Visual function was assessed by recording electroretinographs, and visual evoked potentials and retinae and optic nerves were assessed histopathologically. Retinal ganglion cell numbers were determined by retrograde labeling with fluorogold. Mice were then immunized with myelin oligodendrocyte glycoprotein 35-55 to induce AON before assessment of retinal ganglion cell degeneration, inflammatory infiltration of retinae and optic nerves, and demyelination. Furthermore, visual function was assessed by visual evoked potentials. Results: All C57BL/6N mice were homozygous for the mutation (Crb1rd8/rd8) and had pathology typical of the rd8 mutation; however, this was not seen in the C57BL/6J (Crb1wt/wt) mice. Following induction of AON, no differences were seen between the Crb1rd8/rd8 and Crb1wt/wt mice regarding disease parameters nor regarding inner retinal degeneration either in the retina as a whole or in the inferior nasal quadrant. Conclusions: The presence of the rd8 mutation in C57BL/6 mice does not affect the course of AON and should not provide a confounding factor in the interpretation of experimental results obtained in this model. However, it could be dangerous in other models of ocular pathology.

Disturbed Processing of Contextual Information in HCN3 Channel Deficient Mice.

Hyperpolarization-activated cyclic nucleotide-gated channels (HCNs) in the nervous system are implicated in a variety of neuronal functions including learning and memory, regulation of vigilance states and pain. Dysfunctions or genetic loss of these channels have been shown to cause human diseases such as epilepsy, depression, schizophrenia, and Parkinson's disease. The physiological functions of HCN1 and HCN2 channels in the nervous system have been analyzed using genetic knockout mouse models. By contrast, there are no such genetic studies for HCN3 channels so far. Here, we use a HCN3-deficient (HCN3-/-) mouse line, which has been previously generated in our group to examine the expression and function of this channel in the CNS. Specifically, we investigate the role of HCN3 channels for the regulation of circadian rhythm and for the determination of behavior. Contrary to previous suggestions we find that HCN3-/- mice show normal visual, photic, and non-photic circadian function. In addition, HCN3-/- mice are impaired in processing contextual information, which is characterized by attenuated long-term extinction of contextual fear and increased fear to a neutral context upon repeated exposure.

Differentiation of murine models of "negative ERG" by single and repetitive light stimuli.

PURPOSE: Marked attenuation of the single-flash electroretinographic (ERG) b-wave in the presence of a normal-amplitude or less-attenuated a-wave is commonly referred to as the "negative ERG." The purpose of this study was to investigate whether the disparate origins of the negative ERG in three murine models can be discriminated using flickering stimuli. METHODS: Three models were selected: (1) the Nyx (nob) mouse model of complete congenital stationary night blindness, (2) the oxygen-induced retinopathy (OIR) rat model of retinopathy of prematurity (ROP), and (3) the Rs1 knockout (KO) mouse model of X-linked juvenile retinoschisis. Directly after a dark-adapted, single-flash ERG luminance series, a flicker ERG frequency series (0.5-30 Hz) was performed at a fixed luminance of 0.5 log cd s/m(2). This series includes frequency ranges that are dominated by activity in (A) the rod pathways (below 5 Hz), (B) the cone ON-pathway (5-15 Hz), and (C) the cone OFF-pathway (above 15 Hz). RESULTS: All three models produced markedly attenuated single-flash ERG b-waves. In the Nyx (nob) mouse, which features postsynaptic deficits in the ON-pathways, the a-wave was normal and flicker responses were attenuated in ranges A and B, but not C. The ROP rat is characterized by inner-retinal ischemia which putatively affects both ON- and OFF-bipolar cell activity; flicker responses were reduced in all ranges (A-C). Notably, the choroid supplies the photoreceptors and is thought to be relatively intact in OIR, an idea supported by the nearly normal a-wave. Finally, in the Rs1 KO mouse, which has documented abnormality of the photoreceptor-bipolar synapse affecting both ON- and OFF-pathways, the flicker responses were attenuated in all ranges (A-C). The a-wave was also attenuated, likely as a consequence to schisms in the photoreceptor layer. CONCLUSION: Consideration of both single-flash and flickering ERG responses can discriminate the functional pathology of the negative ERG in these animal models of human disease.