Long-term expression of melanopsin and channelrhodopsin causes no gross alterations in the dystrophic dog retina.

Several preclinical studies have investigated the potential of algal channelrhodopsin and human melanopsin as optogenetic tools for vision restoration. In the present study, we assessed the potentially deleterious effects of long-term expression of these optogenes on the diseased retina in a large animal model of retinal degeneration, the RPE65-deficient Briard dog model of Leber congenital amaurosis. Intravitreal injection of adeno-associated virus vectors expressing channelrhodopsin and melanopsin had no effect on retinal thickness over a 16-month period post injection. Our data support the safety of the optogenetic approach for the treatment of blindness.

First-principles study of the interaction between NO and large carbonaceous clusters modeling the soot surface.

First-principles calculations are performed to characterize the NO adsorption on large carbonaceous clusters modeling the surface of soot. Adsorption on the face and on the edges of perfect and defective clusters is considered in the calculations. It is shown that the first situation corresponds to physisorption and requires taking into account long-range dispersion interactions in the calculations. In contrast, interaction of NO with the unsaturated edge of a defective cluster leads preferentially to a C-N rather than to a C-O chemical binding. This indicates that soot may be an efficient sink for NO in the troposphere only if it contains a high number of unsaturated carbon atoms. From a more fundamental point of view, this study also clearly evidences that quantum calculations have to be carefully conducted when considering the interaction between radical species and carbonaceous surfaces. Problems encountered with the choice of the functional used in density functional theory approaches as well as with the size of the basis set, spin multiplicity, and spin contamination have to be systematically addressed before any relevant conclusion can be drawn.

Dysfunction of the glutamatergic photoreceptor synapse in the P301S mouse model of tauopathy.

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 therapy for retinitis pigmentosa.

Retinitis pigmentosa (RP) refers to a diverse group of progressive, hereditary diseases of the retina that lead to incurable blindness and affect two million people worldwide. Artificial photoreceptors constructed by gene delivery of light-activated channels or pumps ('optogenetic tools') to surviving cell types in the remaining retinal circuit has been shown to restore photosensitivity in animal models of RP at the level of the retina and cortex as well as behaviorally. The translational potential of this optogenetic approach has been evaluated using in vitro studies involving post-mortem human retinas. Here, we review recent developments in this expanding field and discuss the potential and limitations of optogenetic engineering for the treatment of RP.

Retinitis pigmentosa: rod photoreceptor rescue by a calcium-channel blocker in the rd mouse.

Retinitis pigmentosa is an inherited degenerative disease of photoreceptors leading to blindness. A well-characterized model for this disease is provided by the retinal degeneration mouse, in which the gene for the rod cGMP phosphodiesterase is mutated, as in some affected human families. We report that D-cis-diltiazem, a calcium-channel blocker that also acts at light-sensitive cGMP-gated channels, rescued photoreceptors and preserved visual function in the retinal degeneration mouse. The long record of diltiazem prescription in cardiology should facilitate the design of clinical trials for some forms of retinitis pigmentosa.

Functional ultrasound imaging of the spreading activity following optogenetic stimulation of the rat visual cortex.

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.

Tryptophan-Mediated Interactions between Tristetraprolin and the CNOT9 Subunit Are Required for CCR4-NOT Deadenylase Complex Recruitment.

The zinc-finger protein tristetraprolin (TTP) binds to AU-rich elements present in the 3' untranslated regions of transcripts that mainly encode proteins of the inflammatory response. TTP-bound mRNAs are targeted for destruction via recruitment of the eight-subunit deadenylase complex "carbon catabolite repressor protein 4 (CCR4)-negative on TATA-less (NOT)," which catalyzes the removal of mRNA poly-(A) tails, the first obligatory step in mRNA decay. Here we show that a novel interaction between TTP and the CCR4-NOT subunit, CNOT9, is required for recruitment of the deadenylase complex. In addition to CNOT1, CNOT9 is now included in the identified CCR4-NOT subunits shown to interact with TTP. We find that both the N- and C-terminal domains of TTP are involved in an interaction with CNOT9. Through a combination of SPOT peptide array, site-directed mutagenesis, and bio-layer interferometry, we identified several conserved tryptophan residues in TTP that serve as major sites of interaction with two tryptophan-binding pockets of CNOT9, previously found to interact with another modulator GW182. We further demonstrate that these interactions are also required for recruitment of the CCR4-NOT complex and TTP-directed decay of an mRNA containing an AU-rich element in its 3'-untranslated region. Together the results reveal new molecular details for the TTP-CNOT interaction that shape an emerging mechanism whereby TTP targets inflammatory mRNAs for deadenylation and decay.

Subretinal electrode implantation in the P23H rat for chronic stimulations.

BACKGROUND: In age related macular degeneration and inherited dystrophies, preservation of retinal ganglion cells has been demonstrated. This finding has led to the development of various models of subretinal or epiretinal implant in order to restore vision. This study addresses the development of a polyimide subretinal electrode platform in the dystrophic P23H rat in vivo. METHODS: A technique was developed for implanting a subretinal electrode into the subretinal space and stabilising the distal extremity of the cabling on the rat cranium in order to allow future electrical stimulations of the retina. RESULTS: In vivo imaging of the retina with the scanning laser ophthalmoscope demonstrated reabsorption of the surgically induced retinal detachment and the absence of major tissue reactions. These in vivo observations were confirmed by retinal histology. The extraocular fixation system on the rat cranium was effective in stabilising the distal connector for in vivo stimulation. CONCLUSION: This study demonstrates that a retinal implant can be introduced into the subretinal space of a dystrophic rat with a stable external connection for repeatable electrical measurements and stimulation. This in vivo model should therefore allow us to evaluate the safety and efficacy of electrical stimulations on dystrophic retina.

Excessive activation of serotonin (5-HT) 1B receptors disrupts the formation of sensory maps in monoamine oxidase a and 5-ht transporter knock-out mice.

Deficiency in the monoamine degradation enzyme monoamine oxidase A (MAOA) or prenatal exposure to the monoamine uptake inhibitor cocaine alters behavior in humans and rodents, but the mechanisms are unclear. In MAOA knock-out mice, inhibiting serotonin synthesis during development can prevent abnormal segregation of axons in the retinogeniculate and somatosensory thalamocortical systems. To investigate this effect, we crossed MAOA knock-outs with mice lacking the serotonin transporter 5-HTT or the 5-HT1B receptor, two molecules present in developing sensory projections. Segregation was abnormal in 5-HTT knock-outs and MAOA/5-HTT double knock-outs but was normalized in MAOA/5-HT1B double knock-outs and MAOA/5-HTT/5-HT1B triple knock-outs. This demonstrates that the 5-HT1B receptor is a key factor in abnormal segregation of sensory projections and suggests that serotonergic drugs represent a risk for the development of these projections. We also found that the 5-HT1B receptor has an adverse developmental impact on beam-walking behavior in MAOA knock-outs. Finally, because the 5-HT1B receptor inhibits glutamate release, our results suggest that visual and somatosensory projections must release glutamate for proper segregation.

Rod-cone interactions: developmental and clinical significance.

During the last decade, numerous research reports have considerably improved our knowledge about the physiopathology of retinal degenerations. Three non-mutually exclusive general areas dealing with therapeutic approaches have been proposed; gene therapy, pharmacology and retinal transplantations. The first approach involving correction of the initial mutation, will need a great deal of time and further development before becoming a therapeutic tool in human clinical practice. The observation that cone photoreceptors, even those seemingly unaffected by any described anomaly, die secondarily to rod disappearance related to mutations expressed specifically in the latter, led us to study the interactions between these two photoreceptor populations to search for possible causal links between rod degeneration and cone death. These in vivo and in vitro studies suggest that paracrine interactions between both cell types exist and that rods are necessary for continued cone survival. Since the role of cones in visual perception is essential, pending the identification of the factors mediating these interactions underway, rod replacement by transplantation and/or neuroprotection by trophic factors or alternative pharmacological means appear as promising approaches for limiting secondary cone loss in currently untreatable blinding conditions.

Advanced glycation end products can induce glial reaction and neuronal degeneration in retinal explants.

BACKGROUND/AIMS: Neuronal degeneration has been reported to occur in diabetic retinopathy before the onset of detectable microvascular abnormalities. To investigate whether advanced glycation end products (AGE) could be directly responsible for retinal neurodegeneration, retinal explants were incubated with glycated bovine serum albumin (BSA). METHODS: Retinal explants obtained from non-diabetic adult rats were incubated 4 days with or without 200 mug/ml glycated BSA. Neural apoptosis was quantified by terminal dUTP nick end labelling (TUNEL) binding and immunostaining with anti-cleaved caspase-3 antibody. Expression of glial fibrillary acidic protein (GFAP) was localised by immunofluorescence. RESULTS: TUNEL and cleaved caspase-3 positive cells increased significantly by 2.2-fold and 2.5-fold in retinal explants incubated in glycated BSA (p<0.05), respectively. The ganglion cell layer was the most sensitive retinal layer to the glycated BSA. Neuronal degeneration was confirmed by the increased GFAP labelling in Müller glial cells from retinal explants treated with glycated BSA. CONCLUSION: These results suggest that AGE could induce retinal neurodegeneration in the absence of blood perfusion. Cells in the ganglion cell layer appeared to be the most sensitive as in diabetic retinopathy and its animal models. AGE toxicity could therefore contribute to the early pathological mechanisms of diabetic retinopathy.

[Blindness and visual rehabilitation]. / Cécité et réhabilitation visuelle.

Blindness and visual impairment are a major public health problem all over the world and in all societies. A large amount of basic science and clinical research aims to rehabilitate patients and help them become more independent. Various methods are explored from cell and molecular therapy to prosthetic interfaces. We review the various treatment alternatives, describing their results and their limitations.

Incorporation of chromaffin granule membranes into large-size vesicles suitable for patch-clamp recording.

Incubation of chromaffin granules with excess liposomes at pH 6.0 resulted in the formation of cell-size structures, which were purified by centrifugation on sucrose gradients. Experiments with fluorescein-labeled granules indicated incorporation of granule membrane to these structures. The preparation contained various vesicular structures with a diameter up to 15 micron. The largest elements were studied by the 'patch-clamp' technique. 'Cell-attached' and 'whole-cell' recordings indicated the presence of currents corresponding to unitary conductances ranging from 100 to 500 pS.

Adult human retinal neurons in culture: Physiology of horizontal cells.

PURPOSE: Adult postmortem human retinal neurons in long-term monolayer cultures were recorded to characterize the voltage- and transmitter-gated currents in putative human horizontal cells (HCs). METHODS: Enzymatically and mechanically dissociated human retinal cells were seeded on polylysine and laminin- coated coverslips. Cells were identified by immunocytochemistry with cell type-specific antibodies and recorded with the patch-clamp technique. RESULTS: Immunostaining and responses to voltage steps confirmed the survival of various retinal cell types. Horizontal cells were identified by their specific glutamate-modulated anomalous rectifier K+ current conductance. This identification was further confirmed by subsequent immunolabeling of dye-labeled recorded cells with an anti-parvalbumin antibody that selectively stained HCs in frozen human retinal sections. Horizontal cells generated voltage-gated currents classically observed in HCs from fish to mammals: a transient outward K+ current, a sustained outward K+ current, and an L-type (Ca2+ current. Na+ currents were observed in only a few HCs. As in other species, glutamate, gamma-aminobutyric acid (GABA), and glycine generated responses mediated by the activation of kainate/(RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), GABA(A), and glycine receptors, respectively. CONCLUSIONS: Various human retinal cell populations survive in vitro as indicated by immunolabeling with specific cell markers and by the diversity of responses to voltage steps. Human HCs exhibited extensive physiological similarities to HCs from other vertebrate species and a maintained expression of parvalbumin. These results constitute a comprehensive analysis of voltage- and transmitter-gated currents in a primate retinal neuron and validate the use of long-term monolayer culture of adult human neurons as a novel in vitro model for the study of human vision.

Differential distribution of dystrophins in rat retina.

PURPOSE: Duchenne muscular dystrophy is frequently associated with a reduced amplitude of b-wave under scotopic conditions in the electroretinogram. This suggests that the dystrophin gene-encoded proteins play a role in retinal neurotransmission. The abnormal neurotransmission has been attributed to altered expressions of C-terminal products of the dystrophin gene in the outer plexiform layer, where photoreceptor cells form synapses with secondary neurons. The present study was undertaken to determine the cellular distribution of each member of the dystrophin superfamily in rat retina. METHODS: Examined in the study were the developmental pattern of dystrophins in rat retinae that exhibit inherited progressive photoreceptor degeneration; dystrophins messengers expression in the outer and the inner retina of normal rats, prepared by mechanical fractionation through the outer plexiform layer; and immunolocalization of dystrophin proteins and utrophin in normal and degenerated adult rat retinae, with several antibodies prepared against specific regions of the dystrophin superfamily. RESULTS: The results showed that Dp260 is exclusively localized in photoreceptor cells; Dp140 seems to be present in perivascular astrocytes; the exon 78 spliced isoform of Dp71 and the unspliced form are located in Müller glial cells and in perivascular astrocytes, respectively. Müller glial cells also contain utrophin. CONCLUSIONS: Although the role of these membrane cytoskeletal proteins remains to be elucidated in retina, the results support the hypothesis that b-wave reduction may be caused by molecular anomalies of C-terminal products of the dystrophin gene expressed in both neuron and glial cells.

Selective excitotoxic degeneration of adult pig retinal ganglion cells in vitro.

PURPOSE: Excitotoxicity is proposed to play a prominent role in retinal ganglion cell (RGC) death ensuing from diseases such as glaucoma and ischemia, but cell culture studies have used tissue from newborn rodents, yielding conflicting data that implicate either N-methyl D-aspartate (NMDA) or non-NMDA glutamate (Glu) receptor-mediated pathways. Excitotoxic RGC death was examined in vitro in this study, using adult pigs, a large-animal model for human retina. METHODS: Adult pig retina (and for comparative purposes young and adult rat retina) were dissociated and maintained in monolayer culture. Medium was supplemented with Glu or pharmacologic agonists or antagonists, and surviving RGCs and other retinal neurons were quantified using specific immunolabeling methods. Electrophysiological responses to externally applied Glu of RGCs in culture were recorded using whole-cell patch-clamp techniques. RESULTS: Application of Glu led to selective, dose-dependent losses in large RGCs (maximal 37% decrease at 1 mM; median effective dose [ED50], approximately 80 microM) and neurite damage in surviving RGCs. Application of Glu agonists and Glu receptor subclass antagonists showed that large RGC death was mediated through both NMDA and non-NMDA receptor pathways. Small RGCs, amacrine cells, and all other retinal neurons were resistant to Glu-induced death. By comparison, rat retinal cultures displayed heightened RGC vulnerability to Glu, mediated exclusively by non-NMDA receptor-mediated pathways. Amacrine cells were unaffected by NMDA but were very sensitive to kainate application (>90% loss). Other retinal neurons were unaffected by any treatment. CONCLUSIONS: The molecular pathways underlying excitotoxic RGC death in vitro (non-NMDA or NMDA-preferring Glu receptors) vary among species and developmental stages. The selective elimination of adult pig large RGCs by NMDA receptor-mediated pathways more closely resembles human and animal glaucoma in vivo than other published culture models, providing a simplified experimental system for investigating the pharmacologic and toxicologic bases of glaucoma-like neuronal death.

Glial cell line-derived neurotrophic factor induces histologic and functional protection of rod photoreceptors in the rd/rd mouse.

PURPOSE: To evaluate the neuroprotective potential of glial cell line-derived neurotrophic factor (GDNF) in the retinal degeneration (rd/rd) mouse model of human retinitis pigmentosa. METHODS: Subretinal injections of GDNF were made into rd/rd mice at 13 and 17 days of age and electroretinograms (ERGs) recorded at 22 days. Control mice received saline vehicle injections or underwent no procedure. At 23 days of age, retinas from treated and control mice were fixed and processed for wholemount immunohistochemistry using an anti-rod opsin antibody, and rod numbers were estimated using an unbiased stereological systematic random approach. Subsequent to counting, immunolabeled retinas were re-embedded and sectioned in a transverse plane and the numbers of rods recalculated. RESULTS: Although ERGs could not be recorded from sham-operation or nonsurgical rd/rd mice at 22 days of age, detectable responses (both a- and b-waves) were observed in 4 of 10 GDNF-treated mice. Stereological assessment of immunolabeled rods at 23 days showed that control rd/rd retinas contained 41,880+/-3,890 (mean +/- SEM; n = 6), phosphate-buffered saline (PBS)-injected retinas contained 61,165+/-4,932 (n = 10; P < 0.001 versus control retinas) and GDNF-injected retinas contained 89,232+/-8,033 (n = 10; P < 0.001 versus control retinas, P < 0.002 versus PBS). This increase in rod numbers after GDNF treatment was confirmed by cell counts obtained from frozen sections. CONCLUSIONS: GDNF exerts both histologic and functional neuroprotective effects on rod photoreceptors in the rd/rd mouse. Thus rescue was demonstrated in an animal model of inherited retinal degeneration in which the gene defect was located within the rods themselves, similar to most forms of human retinitis pigmentosa. GDNF represents a candidate neurotrophic factor for palliating some forms of hereditary human blindness.

Dye-induced photopermeabilization and photodegeneration: a lesion technique useful for neuronal tracing.

Using as a neural system the fly retina, which is visually accessible in vivo, we describe a lesion technique that takes advantage of the photodynamic damage produced by extrinsic dyes. Contrary to the photo-inactivation technique described by Miller and Selverston (1979), this technique does not involve intracellular injection, since the dye is applied to the extracellular space of the tissue. This treatment was found to trigger neuronal degeneration and cell permeabilization in fly photoreceptor neurones. We coined the names 'photodegeneration' and 'photopermeabilization' for these two phenomena. While the technique can be used to delete given neurones from the neural circuit after several days' survival time, it was found to produce adequate cytoplasmic labelling for anatomical studies with both light and electron microscopy. Since the area occupied by the degenerating cells is restricted to the light spot imaged onto the nervous tissue, the resolution with this lesion technique can range from single cells to whole neuronal populations. The remarkable precision of the 'photolesions' produced in this way makes this technique a powerful tool for physiological and anatomical investigations on real neural networks, whenever these can be made optically accessible in vivo or in situ.

Dye-induced 'photo-degeneration' and 'photo-permeabilization' of mammalian neurons in vivo.

Dyes are known to induce neuronal 'photo-degeneration' and 'photo-permeabilization' in fly photoreceptor cells in vivo. In the present study, we attempted to generalize this photodynamic damage to vertebrate neurons, using the rat retina, a brain part which is optically accessible in vivo. After intravitreal injection of the photosensitizing dye Rose Bengal (RB), irradiation of the retina of a living rat with a T-shaped microbeam was found to induce striking 'optograms' which could be observed on the excised retina. The T-shaped pattern which was to be seen in the translucent retina under transmitted light was attributed to neuronal degeneration of the neurons irradiated in the presence of RB, as attested by classical degenerative features such as a cytoplasmic darkening or a drastic swelling. The T-shaped pattern could also be observed on adding the dye Lucifer yellow to the extracellular space of the retina either in vitro or in vivo, showing that the cells irradiated in the presence of RB became permeable. These structural reactions were observed in the cells in the inner nuclear layer (INL) and ganglion cell layer (GCL), in the processes in both plexiform layers, and in the ganglion cell axons crossing this area, whereas the photoreceptors in the outer retina appeared to be undamaged. From these reactions, due to photo-degeneration and photo-permeabilization, it was possible to identify the photodynamic damage to the nervous system histologically at the macroscopic, cellular and ultrastructural levels. In view of its accuracy and reproducibility, the photo-lesion technique holds great potential as a tool for investigating various nervous systems.

'Photo-degeneration' of neurones after extracellular dye application.

Irradiation in the presence of a dye applied to the extracellular space was found to trigger neuronal degeneration in fly photoreceptor neurones. This phenomenon endowed the selected cells with a dark and fine-grained label such that they could be traced and scrutinized for synaptic specializations. It also initiated their phagocytotic removal from the neural network. Various states of degeneration fitting classical descriptions could be achieved by acting upon the light dose. The phenomenon seems to rely on photosensitization of the cell by the dye. The simplicity and precision with which this phenomenon can be induced makes 'photo-degeneration' an exciting prospective tool for combined anatomical and physiological studies on (natural) neural networks and may provide a new line of medical applications.