Optic neuropathies are a major cause of visual impairment due to retinal ganglion cell (RGC) degeneration. Human induced-pluripotent stem cells (iPSCs) represent a powerful tool for studying both human RGC development and RGC-related pathological mechanisms. Because RGC loss can be massive before the diagnosis of visual impairment, cell replacement is one of the most encouraging strategies. The present work describes the generation of functional RGCs from iPSCs based on innovative 3D/2D stepwise differentiation protocol. We demonstrate that targeting the cell surface marker THY1 is an effective strategy to select transplantable RGCs. By generating a fluorescent GFP reporter iPSC line to follow transplanted cells, we provide evidence that THY1-positive RGCs injected into the vitreous of mice with optic neuropathy can survive up to 1 month, intermingled with the host RGC layer. These data support the usefulness of iPSC-derived RGC exploration as a potential future therapeutic strategy for optic nerve regeneration.
Optogenetics has revolutionized neurosciences by allowing fine control of neuronal activity. An important aspect for this control is assessing the activation and/or adjusting the stimulation, which requires imaging the entire volume of optogenetically-induced neuronal activity. An ideal technique for this aim is fUS imaging, which allows one to generate brain-wide activation maps with submesoscopic spatial resolution. However, optical stimulation of the brain with blue light might lead to non-specific activations at high irradiances. fUS imaging of optogenetic activations can be obtained at these wavelengths using lower light power (< 2mW) but it limits the depth of directly activatable neurons from the cortical surface. Our main goal was to report that we can detect specific optogenetic activations in V1 even in deep layers following stimulation at the cortical surface. Here, we show the possibility to detect deep optogenetic activations in anesthetized rats expressing the red-shifted opsin ChrimsonR in V1 using fUS imaging. We demonstrate the optogenetic specificity of these activations and their neuronal origin with electrophysiological recordings. Finally, we show that the optogenetic response initiated in V1 spreads to downstream (LGN) and upstream (V2) visual areas.
Complete congenital stationary night blindness (cCSNB) due to mutations in TRPM1 , GRM6 , GPR179 , NYX , or leucine-rich repeat immunoglobulin-like transmembrane domain 3 ( LRIT3 ) is an incurable inherited retinal disorder characterized by an ON-bipolar cell (ON-BC) defect. Since the disease is non-degenerative and stable, treatment could theoretically be administrated at any time in life, making it a promising target for gene therapy. Until now, adeno-associated virus (AAV)-mediated therapies lead to significant functional improvements only in newborn cCSNB mice. Here we aimed to restore protein localization and function in adult Lrit3 −/ − mice. LRIT3 localizes in the outer plexiform layer and is crucial for TRPM1 localization at the dendritic tips of ON-BCs and the electroretinogram (ERG)-b-wave. AAV2-7m8- Lrit3 intravitreal injections were performed targeting either ON-BCs, photoreceptors (PRs), or both. Protein localization of LRIT3 and TRPM1 at the rod-to-rod BC synapse, functional rescue of scotopic responses, and ON-responses detection at the ganglion cell level were achieved in a few mice when ON-BCs alone or both PRs and ON-BCs, were targeted. More importantly, a significant number of treated adult Lrit3 −/− mice revealed an ERG b-wave recovery under scotopic conditions, improved optomotor responses, and on-time ON-responses at the ganglion cell level when PRs were targeted. Functional rescue was maintained for at least 4 months after treatment.
Over the last 15 years, optogenetics has changed fundamental research in neuroscience and is now reaching toward therapeutic applications. Vision restoration strategies using optogenetics are now at the forefront of these new clinical opportunities. But applications to human patients suffering from retinal diseases leading to blindness raise important concerns on the long-term functional expression of optogenes and the efficient signal transmission to higher visual centers. Here, we demonstrate in non-human primates continued expression and functionality at the retina level ∼20 months after delivery of our construct. We also performed in vivo recordings of visually evoked potentials in the primary visual cortex of anesthetized animals. Using synaptic blockers, we isolated the in vivo cortical activation resulting from the direct optogenetic stimulation of primate retina. In conclusion, our work indicates long-term transgene expression and transmission of the signal generated in the macaque retina to the visual cortex, two important features for future clinical applications.
Tauopathies, including Alzheimer’s disease, are characterized by retinal ganglion cell loss associated with amyloid and phosphorylated tau deposits. We investigated the functional impact of these histopathological alterations in the murine P301S model of tauopathy. Visual impairments were demonstrated by a decrease in visual acuity already detectable at 6 months, the onset of disease. Visual signals to the cortex and retina were delayed at 6 and 9 months, respectively. Surprisingly, the retinal output signal was delayed at the light onset and advanced at the light offset. This antagonistic effect, due to a dysfunction of the cone photoreceptor synapse, was associated with changes in the expression of the vesicular glutamate transporter and a microglial reaction. This dysfunction of retinal glutamatergic synapses suggests a novel interpretation for visual deficits in tauopathies and it highlights the potential value of the retina for the diagnostic assessment and the evaluation of therapies in Alzheimer’s disease and other tauopathies.
Optogenetic activation of neurons [1] have greatly contributed to our understanding of how neural circuits operate, and holds huge promise in the field of neural prosthetics, particularly in sensory restoration. The discovery of new channelrhodopsins, Chrimson -which is 45 nm more red-shifted than any previously discovered or engineered channelrhodopsin -and its mutant ChrimsonR with faster kinetics [2] made this technology available for medical applications. However, a detailed model that would be able to accurately reproduce the membrane potential dynamics in cells transfected with ChrimsonR under light stimulation is missing. We address this issue by developing the first model for the electrochemical behavior of ChrimsonR that predicts its conductance in response to arbitrary light stimulation. Our model captures ON and OFF dynamics of the protein for stimuli with frequencies up to 100 Hz and their relationship with the brightness, as well as its activation curve, the steady-state amplitude of the response as a function of light intensity. Additionally, we capture a slow adaptation mechanism at a Introduction 1 This paper studies the activation dynamics of ChrimsonR, a channelrhodopsin which is 2 currently the best candidate for optogenetic-based sensory restoration in human 3 patients, due to its peak spectral sensitivity in the red at 590 nm, which is 45 nm more 4 red-shifted than all previously known channelrhodopsins [3]. However, design of 5 light-stimulation protocols for optogenetics-based vision restoration would greatly 6 benefit from a model of the channel's behavior, as its dynamics will influence the 7 PLOS 1/25ChrimsonR mediated temporal dynamics of membrane potential in transfected neurons. 8To address this issue, here we introduce for the first time the relevant kinetics and the 9 expected behavior of the channel. In particular, we propose a predictive model of the 10 conductance of a population of channels expressed in a given cell in response to 11 arbitrary illumination stimuli spanning the whole dynamic range of the protein. 12Upon photostimulation, channelrhodopsins modify their conformation and form a 13 water-filled pore allowing some ions to flow through the channel. The illumination 14 signal therefore drives the conductance of the cell's membrane, which then induces a 15 current through the membrane, the subsequent modification of the membrane potential 16 and the possible triggering of action potentials. A predictive model of the 17 channelrhodopsin's conductance provides the mapping between the controlled 18 illumination and the physiologically relevant variable. 19The main features of this transformation that we capture are the ON and OFF 20 dynamics (and their relationship to the brightness value of the stimulus); the activation 21 curve, the steady-state amplitude of the response as a function of light intensity as well 22 as a slow adaptation mechanism leading to a progressive decrease in the amplitude of 23 the response. These features can be captured using a five-state Markov k...
Optogenetic stimulation of the primary visual cortex (V1) is a promising therapy for sight restoration, but it remains unclear what total cerebral volume is activated after surface stimulation. In this study, we expressed the red-shifted opsin ChrimsonR in excitatory neurons within V1 in rats, and used the fine spatial resolution provided by functional ultrasound imaging (fUS) over the whole depth of the brain to investigate the brain response to focal surface stimulation. We observed optogenetic activation of a high proportion of the volume of V1. Extracellular recordings confirmed the neuronal origin of this activation. Moreover, neuronal responses were even located in deep layers under conditions of low irradiance, spreading to the LGN and V2, consistent with a normal visual information process. This study paves the way for the use of optogenetics for cortical therapies, and highlights the value of coupling fUS with optogenetics.
Various therapeutic strategies for vision restoration have been developed, including retinal prostheses [1:4], stem cell transplantation [5:8] and optogenetic therapies [9,10,19,11:18]. In optogenetic therapy, the residual retinal neurons surviving the pathological degenerative process are rendered light-sensitive. Using this approach, we targeted the retinal ganglion cells (RGCs) through the in vivo expression of an ectopic light-sensitive ion channel, ChrimsonR [13] coupled to the fluorescent reporter tdTomato. The application of this strategy to blind patients [20] suffering from retinal dystrophies raises important concerns about the long-term functional expression of efficient signal transmission to higher brain centers (i.e. the visual cortex). We have previously shown that the transfected retina displays high spatiotemporal resolution ex vivo, compatible with the perception of highly dynamic visual scenes at light levels suitable for use in humans. Other studies have provided evidence of retinal activation in vivo [17]. Here, we demonstrate, in non-human primates, sustained functional efficacy ~20 months after delivery of an AAV2.7m8-ChrimsonR-tdTomato vector similar to that currently undergoing clinical evaluation. Our results reveal a persistence of expression in the perifovea, mediating information transfer to higher brain centers. Indeed, we recorded visually evoked potentials in the primary visual cortex of anesthetized animals in response to optogenetic retinal activation. We used an intravitreal injection of synaptic blockers to isolate the cortical component resulting from the in vivo optogenetic stimulation of primate RGCs. Our findings demonstrate the long-term functional efficacy of optogenetic retinal information transfer to the brain in vivo.
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