Cardiac optogenetics: shining light on signaling pathways.

In the early 2000s, the field of neuroscience experienced a groundbreaking transformation with the advent of optogenetics. This innovative technique harnesses the properties of naturally occurring and genetically engineered rhodopsins to confer light sensitivity upon target cells. The remarkable spatiotemporal precision offered by optogenetics has provided researchers with unprecedented opportunities to dissect cellular physiology, leading to an entirely new level of investigation. Initially revolutionizing neuroscience, optogenetics quickly piqued the interest of the wider scientific community, and optogenetic applications were expanded to cardiovascular research. Over the past decade, researchers have employed various optical tools to observe, regulate, and steer the membrane potential of excitable cells in the heart. Despite these advancements, achieving control over specific signaling pathways within the heart has remained an elusive goal. Here, we review the optogenetic tools suitable to control cardiac signaling pathways with a focus on GPCR signaling, and delineate potential applications for studying these pathways, both in healthy and diseased hearts. By shedding light on these exciting developments, we hope to contribute to the ongoing progress in basic cardiac research to facilitate the discovery of novel therapeutic possibilities for treating cardiovascular pathologies.

Selective Block of Upregulated Kv1.3 Potassium Channels in ON-Bipolar Cells of the Blind Retina Enhances Optogenetically Restored Signaling.

Loss of photoreceptors in retinal degenerative diseases also impacts the inner retina: bipolar cell dendrites retract, neurons rewire, and protein expression changes. ON-bipolar cells (OBCs) represent an attractive target for optogenetic vision restoration. However, the above-described maladaptations may negatively impact the quality of restored vision. To investigate this question, we employed human post-mortem retinas and transgenic rd1_Opto-mGluR6 mice expressing the optogenetic construct Opto-mGluR6 in OBCs and carrying the retinal degeneration rd1 mutation. We found significant changes in delayed rectifier potassium channel expression in OBCs of degenerative retinas. In particular, we found an increase in Kv1.3 expression already in early stages of degeneration. Immunohistochemistry localized Kv1.3 channels specifically to OBC axons. In whole-cell patch-clamp experiments, OBCs in the degenerated murine retina were less responsive, which could be reversed by application of the specific Kv1.3 antagonist Psora-4. Notably, Kv1.3 block significantly increased the amplitude and kinetics of Opto-mGluR6-mediated light responses in OBCs of the blind retina and increased the signal-to-noise ratio of light-triggered responses in retinal ganglion cells. We propose that reduction in Kv1.3 activity in the degenerated retina, either by pharmacological block or by KCNA3 gene silencing, could improve the quality of restored vision.

The Bovine Ex Vivo Retina: A Versatile Model for Retinal Neuroscience.

Purpose: The isolated ex vivo retina is the standard model in retinal physiology and neuroscience. During isolation, the retina is peeled from the retinal pigment epithelium (RPE), which plays a key role in the visual cycle. Here we introduce the choroid-attached bovine retina as an in vivo-like model for retinal physiology. We find that-in the bovine eye-the choroid and retina can be peeled from the sclera as a single thin sheet. Importantly, the retina remains tightly associated with the RPE, which is sandwiched between the retina and the choroid. Furthermore, bovine tissue is readily available and cheap, and there are no ethical concerns related to the use of animals solely for research purposes. Methods: We combine multi-electrode array and single-cell patch-clamp recordings to characterize light responses in the choroid-attached bovine ex vivo retina. Results: We demonstrate robust and consistent light responses in choroid-attached preparations. Importantly, light responses adapt to different levels of background illumination and rapidly recover from photobleaching. The choroid-attached retina is also thin enough to permit targeted electrophysiological recording from individual retinal neurons using standard differential interference contrast microscopy. We also characterize light responses and membrane properties of bovine retinal ganglion cells and compare data obtained from bovine and murine retinas. Conclusions: The choroid-attached retinal model retains the advantages of the isolated retina but with an intact visual cycle and represents a useful tool to elucidate retinal physiology.

A visual opsin from jellyfish enables precise temporal control of G protein signalling.

Phototransduction is mediated by distinct types of G protein cascades in different animal taxa: bilateral invertebrates typically utilise the Gαq pathway whereas vertebrates typically utilise the Gαt(i/o) pathway. By contrast, photoreceptors in jellyfish (Cnidaria) utilise the Gαs intracellular pathway, similar to olfactory transduction in mammals1. How this habitually slow pathway has adapted to support dynamic vision in jellyfish remains unknown. Here we study a light-sensing protein (rhodopsin) from the box jellyfish Carybdea rastonii and uncover a mechanism that dramatically speeds up phototransduction: an uninterrupted G protein-coupled receptor - G protein complex. Unlike known G protein-coupled receptors (GPCRs), this rhodopsin constitutively binds a single downstream Gαs partner to enable G-protein activation and inactivation within tens of milliseconds. We use this GPCR in a viral gene therapy to restore light responses in blind mice.

Functional optimization of light-activatable Opto-GPCRs: Illuminating the importance of the proximal C-terminus in G-protein specificity.

Introduction: G-protein coupled receptors (GPCRs) are the largest family of human receptors that transmit signals from natural ligands and pharmaceutical drugs into essentially every physiological process. One main characteristic of G-protein coupled receptors is their ability to specifically couple with different families of G-proteins, thereby triggering specific downstream signaling pathways. While an abundance of structural information is available on G-protein coupled receptorn interactions with G-proteins, little is known about the G-protein coupled receptor domains functionally mediating G-protein specificity, in particular the proximal C-terminus, the structure which cannot be predicted with high confidentiality due to its flexibility. Methods: In this study, we exploited OptoGPCR chimeras between lightgated G-protein coupled receptors (opsins) and ligand-gated G-protein coupled receptors to systematically investigate the involvement of the C-terminus steering G-protein specificity. We employed rhodopsin-beta2-adrenoceptor and melanopsin-mGluR6 chimeras in second messenger assays and developed structural models of the chimeras. Results: We discovered a dominant role of the proximal C-terminus, dictating G-protein selectivity in the melanopsin-mGluR6 chimera, whereas it is the intracellular loop 3, which steers G-protein tropism in the rhodopsin-beta2-adrenoceptor. From the functional results and structural predictions, melanopsin and mGluR6 use a different mechanism to bovine rhodopsin and b2AR to couple to a selective G-protein. Discussion: Collectively, this work adds knowledge to the G-protein coupled receptor domains mediating G-protein selectivity, ultimately paving the way to optogenetically elicited specific G-protein signaling on demand.

Bipolar cell targeted optogenetic gene therapy restores parallel retinal signaling and high-level vision in the degenerated retina.

Optogenetic gene therapies to restore vision are in clinical trials. Whilst current clinical approaches target the ganglion cells, the output neurons of the retina, new molecular tools enable efficient targeting of the first order retinal interneurons, the bipolar cells, with the potential to restore a higher quality of vision. Here we investigate retinal signaling and behavioral vision in blind mice treated with bipolar cell targeted optogenetic gene therapies. All tested tools, including medium-wave opsin, Opto-mGluR6, and two new melanopsin based chimeras restored visual acuity and contrast sensitivity. The best performing opsin was a melanopsin-mGluR6 chimera, which in some cases restored visual acuities and contrast sensitivities that match wild-type animals. Light responses from the ganglion cells were robust with diverse receptive-field types, inferring elaborate inner retinal signaling. Our results highlight the potential of bipolar cell targeted optogenetics to recover high-level vision in human patients with end-stage retinal degenerations.

WNT Activation and TGFß-Smad Inhibition Potentiate Stemness of Mammalian Auditory Neuroprogenitors for High-Throughput Generation of Functional Auditory Neurons In Vitro.

Hearing loss affects over 460 million people worldwide and is a major socioeconomic burden. Both genetic and environmental factors (i.e., noise overexposure, ototoxic drug treatment and ageing), promote the irreversible degeneration of cochlear hair cells and associated auditory neurons, leading to sensorineural hearing loss. In contrast to birds, fish and amphibians, the mammalian inner ear is virtually unable to regenerate due to the limited stemness of auditory progenitors, and no causal treatment is able to prevent or reverse hearing loss. As of today, a main limitation for the development of otoprotective or otoregenerative therapies is the lack of efficient preclinical models compatible with high-throughput screening of drug candidates. Currently, the research field mainly relies on primary organotypic inner ear cultures, resulting in high variability, low throughput, high associated costs and ethical concerns. We previously identified and characterized the phoenix auditory neuroprogenitors (ANPGs) as highly proliferative progenitor cells isolated from the A/J mouse cochlea. In the present study, we aim at identifying the signaling pathways responsible for the intrinsic high stemness of phoenix ANPGs. A transcriptomic comparison of traditionally low-stemness ANPGs, isolated from C57Bl/6 and A/J mice at early passages, and high-stemness phoenix ANPGs was performed, allowing the identification of several differentially expressed pathways. Based on differentially regulated pathways, we developed a reprogramming protocol to induce high stemness in presenescent ANPGs (i.e., from C57Bl6 mouse). The pharmacological combination of the WNT agonist (CHIR99021) and TGFß/Smad inhibitors (LDN193189 and SB431542) resulted in a dramatic increase in presenescent neurosphere growth, and the possibility to expand ANPGs is virtually limitless. As with the phoenix ANPGs, stemness-induced ANPGs could be frozen and thawed, enabling distribution to other laboratories. Importantly, even after 20 passages, stemness-induced ANPGs retained their ability to differentiate into electrophysiologically mature type I auditory neurons. Both stemness-induced and phoenix ANPGs resolve a main bottleneck in the field, allowing efficient, high-throughput, low-cost and 3R-compatible in vitro screening of otoprotective and otoregenerative drug candidates. This study may also add new perspectives to the field of inner ear regeneration.

Functional Availability of ON-Bipolar Cells in the Degenerated Retina: Timing and Longevity of an Optogenetic Gene Therapy.

Degenerative diseases of the retina are responsible for the death of photoreceptors and subsequent loss of vision in patients. Nevertheless, the inner retinal layers remain intact over an extended period of time, enabling the restoration of light sensitivity in blind retinas via the expression of optogenetic tools in the remaining retinal cells. The chimeric Opto-mGluR6 protein represents such a tool. With exclusive ON-bipolar cell expression, it combines the light-sensitive domains of melanopsin and the intracellular domains of the metabotropic glutamate receptor 6 (mGluR6), which naturally mediates light responses in these cells. Albeit vision restoration in blind mice by Opto-mGluR6 delivery was previously shown, much is left to be explored in regard to the effects of the timing of the treatment in the degenerated retina. We performed a functional evaluation of Opto-mGluR6-treated murine blind retinas using multi-electrode arrays (MEAs) and observed long-term functional preservation in the treated retinas, as well as successful therapeutical intervention in later stages of degeneration. Moreover, the treatment decreased the inherent retinal hyperactivity of the degenerated retinas to levels undistinguishable from healthy controls. Finally, we observed for the first time micro electroretinograms (mERGs) in optogenetically treated animals, corroborating the origin of Opto-mGluR6 signalling at the level of mGluR6 of ON-bipolar cells.

Two Functional Classes of Rod Bipolar Cells in the Healthy and Degenerated Optogenetically Treated Murine Retina.

Bipolar cells have become successful targets for optogenetic gene therapies that restore vision after photoreceptor degeneration. However, degeneration was shown to cause changes in neuronal connectivity and protein expression, which may impact the quality of synthetically restored signaling. Further, the expression of an optogenetic protein may alter passive membrane properties of bipolar cells affecting signal propagation. We here investigated the passive membrane properties of rod bipolar cells in three different systems, the healthy retina, the degenerated retina, and the degenerated retina expressing the optogenetic actuator Opto-mGluR6. We found that, based on the shape of their current-voltage relations, rod bipolar cells in healthy and degenerated retinas form two clear functional groups (type 1 and type 2 cells). Depolarizing the membrane potential changed recorded current-voltage curves from type 1 to type 2, confirming a single cell identity with two functional states. Expression of Opto-mGluR6 did not alter the passive properties of the rod bipolar cell. With progressing degeneration, dominant outward rectifying currents recorded in type 2 rod bipolar cells decreased significantly. We demonstrate that this is caused by a downregulation of BK channel expression in the degenerated retina. Since this BK conductance will normally recover the membrane potential after RBCs are excited by open TRPM1 channels, a loss in BK will decrease high-pass filtering at the rod bipolar cell level. A better understanding of the changes of bipolar cell physiology during retinal degeneration may pave the way to optimize future treatment strategies of blindness.

Empowering Retinal Gene Therapy with a Specific Promoter for Human Rod and Cone ON-Bipolar Cells.

Optogenetic gene therapy holds promise to restore high-quality vision in blind patients and recently reached clinical trials. Although the ON-bipolar cells, the first retinal interneurons, make the most attractive targets for optogenetic vision restoration, they have remained inaccessible to human gene therapy due to the lack of a robust cell-specific promoter. We describe the design and functional evaluation of 770En_454P(hGRM6), a human GRM6 gene-derived, short promoter that drives strong and highly specific expression in both the rod- and cone-type ON-bipolar cells of the human retina. Expression also in cone-type ON-bipolar cells is of importance, since the cone-dominated macula mediates high-acuity vision and is the primary target of gene therapies. 770En_454P(hGRM6)-driven middle-wave opsin expression in ON-bipolar cells achieved lasting restoration of high visual acuity in the rd1 mouse model of late retinal degeneration. The new promoter enables precise manipulation of the inner retinal network and paves the way for clinical application of gene therapies for high-resolution optogenetic vision restoration, raising hopes of significantly improving the life quality of people suffering from blindness.

Emerging Approaches for Restoration of Hearing and Vision.

Impairments of vision and hearing are highly prevalent conditions limiting the quality of life and presenting a major socioeconomic burden. For a long time, retinal and cochlear disorders have remained intractable for causal therapies, with sensory rehabilitation limited to glasses, hearing aids, and electrical cochlear or retinal implants. Recently, the application of gene therapy and optogenetics to eye and ear has generated hope for a fundamental improvement of vision and hearing restoration. To date, one gene therapy for the restoration of vision has been approved, and ongoing clinical trials will broaden its application including gene replacement, genome editing, and regenerative approaches. Moreover, optogenetics, i.e., controlling the activity of cells by light, offers a more general alternative strategy. Over little more than a decade, optogenetic approaches have been developed and applied to better understand the function of biological systems, while protein engineers have identified and designed new opsin variants with desired physiological features. Considering potential clinical applications of optogenetics, the spotlight is on the sensory systems, particularly the eye and ear. Multiple efforts have been undertaken to restore lost or hampered function in the eye and ear. Optogenetic stimulation promises to overcome fundamental shortcomings of electrical stimulation, namely, poor spatial resolution and cellular specificity, and accordingly to deliver more detailed sensory information. This review aims to provide a comprehensive reference on current gene therapeutic and optogenetic research relevant to the restoration of hearing and vision. We will introduce gene-therapeutic approaches and discuss the biotechnological and optoelectronic aspects of optogenetic hearing and vision restoration.

Dynamic all-optical drug screening on cardiac voltage-gated ion channels.

Voltage-gated ion channels (VGCs) are prime targets for the pharmaceutical industry, but drug profiling on VGCs is challenging, since drug interactions are confined to specific conformational channel states mediated by changes in transmembrane potential. Here we combined various optogenetic tools to develop dynamic, high-throughput drug profiling assays with defined light-step protocols to interrogate VGC states on a millisecond timescale. We show that such light-induced electrophysiology (LiEp) yields high-quality pharmacological data with exceptional screening windows for drugs acting on the major cardiac VGCs, including hNav1.5, hKv1.5 and hERG. LiEp-based screening remained robust when using a variety of optogenetic actuators (ChR2, ChR2(H134R), CatCh, ChR2-EYFP-ßArchT) and different types of organic (RH421, Di-4-ANBDQPQ, BeRST1) or genetic voltage sensors (QuasAr1). The tractability of LiEp allows a versatile and precise alternative to state-of-the-art VGC drug screening platforms such as automated electrophysiology or FLIPR readers.

Present Molecular Limitations of ON-Bipolar Cell Targeted Gene Therapy.

Recent studies have demonstrated the safety and efficacy of ocular gene therapy based on adeno-associated viral vectors (AAVs). Accordingly, a surge in promising new gene therapies is entering clinical trials, including the first optogenetic therapy for vision restoration. To date, optogenetic therapies for vision restoration target either the retinal ganglion cells (GCs) or presynaptic ON-bipolar cells (OBCs). Initiating light responses at the level of the OBCs has significant advantages over optogenetic activation of GCs. For example, important neural circuitries in the inner retina, which shape the receptive fields of GCs, remain intact when activating the OBCs. Current drawbacks of AAV-mediated gene therapies targeting OBCs include (1) a low transduction efficiency, (2) off-target expression in unwanted cell populations, and (3) a poor performance in human tissue compared to the murine retina. Here, we examined side-by-side the performance of three state-of-the art AAV capsid variants, AAV7m8, AAVBP2, and AAV7m8(Y444F) in combination with the 4xGRM6-SV40 promoter construct in the healthy and degenerated mouse retina and in human post-mortem retinal explants. We find that (1) the 4xGRM6-SV40 promoter is not OBC specific, (2) that all AAV variants possess broad cellular transduction patterns, with differences between the transduction patterns of capsid variants AAVBP2 and AAV7m8 and, most importantly, (3) that all vectors target OBCs in healthy tissue but not in the degenerated rd1 mouse model, potentially limiting the possibilities for an OBC-targeted optogenetic therapy for vision restoration in the blind.

Generation of Otic Sensory Neurons from Mouse Embryonic Stem Cells in 3D Culture.

The peripheral hearing process taking place in the cochlea mainly depends on two distinct sensory cell types: the mechanosensitive hair cells and the spiral ganglion neurons (SGNs). The first respond to the mechanical stimulation exerted by sound pressure waves on their hair bundles by releasing neurotransmitters and thereby activating the latter. Loss of these sensorineural cells is associated with permanent hearing loss. Stem cell-based approaches aiming at cell replacement or in vitro drug testing to identify potential ototoxic, otoprotective, or regenerative compounds have lately gained attention as putative therapeutic strategies for hearing loss. Nevertheless, they rely on efficient and reliable protocols for the in vitro generation of cochlear sensory cells for their implementation. To this end, we have developed a differentiation protocol based on organoid culture systems, which mimics the most important steps of in vivo otic development, robustly guiding mouse embryonic stem cells (mESCs) toward otic sensory neurons (OSNs). The stepwise differentiation of mESCs toward ectoderm was initiated using a quick aggregation method in presence of Matrigel in serum-free conditions. Non-neural ectoderm was induced via activation of bone morphogenetic protein (BMP) signaling and concomitant inhibition of transforming growth factor beta (TGFß) signaling to prevent mesendoderm induction. Preplacodal and otic placode ectoderm was further induced by inhibition of BMP signaling and addition of fibroblast growth factor 2 (FGF2). Delamination and differentiation of SGNs was initiated by plating of the organoids on a 2D Matrigel-coated substrate. Supplementation with brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) was used for further maturation until 15 days of in vitro differentiation. A large population of neurons with a clear bipolar morphology and functional excitability was derived from these cultures. Immunostaining and gene expression analysis performed at different time points confirmed the transition trough the otic lineage and final expression of the key OSN markers. Moreover, the stem cell-derived OSNs exhibited functional electrophysiological properties of native SGNs. Our established in vitro model of OSNs development can be used for basic developmental studies, for drug screening or for the exploration of their regenerative potential.

Chronic activation of the D156A point mutant of Channelrhodopsin-2 signals apoptotic cell death: the good and the bad.

Channelrhodopsin-2 (ChR2) has become a celebrated research tool and is considered a promising potential therapeutic for neurological disorders. While making its way into the clinic, concerns about the safety of chronic ChR2 activation have emerged; in particular as the high-intensity blue light illumination needed for ChR2 activation may be phototoxic. Here we set out to quantify for the first time the cytotoxic effects of chronic ChR2 activation. We studied the safety of prolonged illumination on ChR2(D156A)-expressing human melanoma cells as cancer cells are notorious for their resistance to killing. Three days of illumination eradicated the entire ChR2(D156A)-expressing cell population through mitochondria-mediated apoptosis, whereas blue light activation of non-expressing control cells did not significantly compromise cell viability. In other words, chronic high-intensity blue light illumination alone is not phototoxic, but prolonged ChR2 activation induces mitochondria-mediated apoptosis. The results are alarming for gain-of-function translational neurological studies but open the possibility to optogenetically manipulate the viability of non-excitable cells, such as cancer cells. In a second set of experiments we therefore evaluated the feasibility to put melanoma cell proliferation and apoptosis under the control of light by transdermally illuminating in vivo melanoma xenografts expressing ChR2(D156A). We show clear proof of principle that light treatment inhibits and even reverses tumor growth, rendering ChR2s potential tools for targeted light-therapy of cancers.

Embryonic Cell Grafts in a Culture Model of Spinal Cord Lesion: Neuronal Relay Formation Is Essential for Functional Regeneration.

Presently there exists no cure for spinal cord injury (SCI). However, transplantation of embryonic tissue into spinal cord (SC) lesions resulted in axon outgrowth across the lesion site and some functional recovery, fostering hope for future stem cell therapies. Although in vivo evidence for functional recovery is given, the exact cellular mechanism of the graft support remains elusive: either the grafted cells provide a permissive environment for the host tissue to regenerate itself or the grafts actually integrate functionally into the host neuronal network reconnecting the separated SC circuits. We tested the two hypotheses in an in vitro SC lesion model that is based on propagation of activity between two rat organotypic SC slices in culture. Transplantation of dissociated cells from E14 rat SC or forebrain (FB) re-established the relay of activity over the lesion site and thus, provoked functional regeneration. Combining patch-clamp recordings from transplanted cells with network activity measurements from the host tissue on multi-electrode arrays (MEAs) we here show that neurons differentiate from the grafted cells and integrate into the host circuits. Optogenetic silencing of neurons developed from transplanted embryonic mouse FB cells provides clear evidence that they replace the lost neuronal connections to relay and synchronize activity between the separated SC circuits. In contrast, transplantation of neurospheres (NS) induced neither the differentiation of mature neurons from the grafts nor an improvement of functional regeneration. Together these findings suggest, that the formation of neuronal relays from grafted embryonic cells is essential to re-connect segregated SC circuits.

Optogenetic user's guide to Opto-GPCRs.

Optogenetics has taken biomedical research by storm. The power and precision at which light-gated ion channels control cellular excitability in diverse biological systems has convinced researchers of an optical future. Growing interest in optical methods has sparked the development of multiple new optogenetic tools, which allow precise control of numerous cellular processes. Among these new tools are the light-activatable G-protein coupled receptors (GPCRs) or Opto-GPCRs. The extent of the GPCR family, which in humans alone encompasses approximately 800 different proteins, and the immense therapeutic potential of Opto-GPCRs predict a big future for this juvenile field. Here the different approaches taken to design Opto-GPCRs are reviewed, outlining the advantages and disadvantages of each method for physiological and potential clinical application.

Variable phenotypic expressivity in inbred retinal degeneration mouse lines: A comparative study of C3H/HeOu and FVB/N rd1 mice.

PURPOSE: Recent advances in optogenetics and gene therapy have led to promising new treatment strategies for blindness caused by retinal photoreceptor loss. Preclinical studies often rely on the retinal degeneration 1 (rd1 or Pde6b(rd1)) retinitis pigmentosa (RP) mouse model. The rd1 founder mutation is present in more than 100 actively used mouse lines. Since secondary genetic traits are well-known to modify the phenotypic progression of photoreceptor degeneration in animal models and human patients with RP, negligence of the genetic background in the rd1 mouse model is unwarranted. Moreover, the success of various potential therapies, including optogenetic gene therapy and prosthetic implants, depends on the progress of retinal degeneration, which might differ between rd1 mice. To examine the prospect of phenotypic expressivity in the rd1 mouse model, we compared the progress of retinal degeneration in two common rd1 lines, C3H/HeOu and FVB/N. METHODS: We followed retinal degeneration over 24 weeks in FVB/N, C3H/HeOu, and congenic Pde6b(+) seeing mouse lines, using a range of experimental techniques including extracellular recordings from retinal ganglion cells, PCR quantification of cone opsin and Pde6b transcripts, in vivo flash electroretinogram (ERG), and behavioral optokinetic reflex (OKR) recordings. RESULTS: We demonstrated a substantial difference in the speed of retinal degeneration and accompanying loss of visual function between the two rd1 lines. Photoreceptor degeneration and loss of vision were faster with an earlier onset in the FVB/N mice compared to C3H/HeOu mice, whereas the performance of the Pde6b(+) mice did not differ significantly in any of the tests. By postnatal week 4, the FVB/N mice expressed significantly less cone opsin and Pde6b mRNA and had neither ERG nor OKR responses. At 12 weeks of age, the retinal ganglion cells of the FVB/N mice had lost all light responses. In contrast, 4-week-old C3H/HeOu mice still had ERG and OKR responses, and we still recorded light responses from C3H/HeOu retinal ganglion cells until the age of 24 weeks. These results show that genetic background plays an important role in the rd1 mouse pathology. CONCLUSIONS: Analogous to human RP, the mouse genetic background strongly influences the rd1 phenotype. Thus, different rd1 mouse lines may follow different timelines of retinal degeneration, making exact knowledge of genetic background imperative in all studies that use rd1 models.