Publications by authors named "Antoine Chaffiol"

23 Publications

  • Page 1 of 1

Control of Microbial Opsin Expression in Stem Cell Derived Cones for Improved Outcomes in Cell Therapy.

Front Cell Neurosci 2021 18;15:648210. Epub 2021 Mar 18.

Institut de la Vision, Sorbonne Université, Paris, France.

Human-induced pluripotent stem cell (hiPSC) derived organoids have become increasingly used systems allowing 3D-modeling of human organ development, and disease. They are also a reliable source of cells for transplantation in cell therapy and an excellent model to validate gene therapies. To make full use of these systems, a toolkit of genetic modification techniques is necessary to control their activity in line with the downstream application. We have previously described adeno-associated viruse (AAV) vectors for efficient targeting of cells within human retinal organoids. Here, we describe biological restriction and enhanced gene expression in cone cells of such organoids thanks to the use of a 1.7-kb L-opsin promoter. We illustrate the usefulness of implementing such a promoter to enhance the expression of the red-shifted opsin Jaws in fusion with a fluorescent reporter gene, enabling cell sorting to enrich the desired cell population. Increased Jaws expression after transplantation improved light responses promising better therapeutic outcomes in a cell therapy setting. Our results point to the importance of promoter activity in restricting, improving, and controlling the kinetics of transgene expression during the maturation of hiPSC retinal derivatives. Differentiation requires mechanisms to initiate specific transcriptional changes and to reinforce those changes when mature cell states are reached. By employing a cell-type-specific promoter we put transgene expression under the new transcriptional program of mature cells.
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http://dx.doi.org/10.3389/fncel.2021.648210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8012682PMC
March 2021

Optogenetic therapy: high spatiotemporal resolution and pattern discrimination compatible with vision restoration in non-human primates.

Commun Biol 2021 Jan 27;4(1):125. Epub 2021 Jan 27.

Sorbonne Université, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, F-75012, Paris, France.

Vision restoration is an ideal medical application for optogenetics, because the eye provides direct optical access to the retina for stimulation. Optogenetic therapy could be used for diseases involving photoreceptor degeneration, such as retinitis pigmentosa or age-related macular degeneration. We describe here the selection, in non-human primates, of a specific optogenetic construct currently tested in a clinical trial. We used the microbial opsin ChrimsonR, and showed that the AAV2.7m8 vector had a higher transfection efficiency than AAV2 in retinal ganglion cells (RGCs) and that ChrimsonR fused to tdTomato (ChR-tdT) was expressed more efficiently than ChrimsonR. Light at 600 nm activated RGCs transfected with AAV2.7m8 ChR-tdT, from an irradiance of 10 photons.cm.s. Vector doses of 5 × 10 and 5 × 10 vg/eye transfected up to 7000 RGCs/mm in the perifovea, with no significant immune reaction. We recorded RGC responses from a stimulus duration of 1 ms upwards. When using the recorded activity to decode stimulus information, we obtained an estimated visual acuity of 20/249, above the level of legal blindness (20/400). These results lay the groundwork for the ongoing clinical trial with the AAV2.7m8 - ChR-tdT vector for vision restoration in patients with retinitis pigmentosa.
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http://dx.doi.org/10.1038/s42003-020-01594-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7840970PMC
January 2021

Generation of a Transplantable Population of Human iPSC-Derived Retinal Ganglion Cells.

Front Cell Dev Biol 2020 27;8:585675. Epub 2020 Oct 27.

Institut de la Vision, Sorbonne Université, INSERM, CNRS, Paris, France.

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.
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http://dx.doi.org/10.3389/fcell.2020.585675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7652757PMC
October 2020

cAMP-Dependent Co-stabilization of Axonal Arbors from Adjacent Developing Neurons.

Cell Rep 2020 10;33(1):108220

Sorbonne Université, Inserm, CNRS, Institut de la Vision, 17 rue Moreau, Paris 75012, France. Electronic address:

Axonal arbors in many neuronal networks are exuberant early during development and become refined by activity-dependent competitive mechanisms. Theoretical work proposed non-competitive interactions between co-active axons to co-stabilize their connections, but the demonstration of such interactions is lacking. Here, we provide experimental evidence that reducing cyclic AMP (cAMP) signaling in a subset of retinal ganglion cells favors the elimination of thalamic projections from neighboring neurons, pointing to a cAMP-dependent interaction that promotes axon stabilization.
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http://dx.doi.org/10.1016/j.celrep.2020.108220DOI Listing
October 2020

Restoration of visual function by transplantation of optogenetically engineered photoreceptors.

Nat Commun 2019 10 4;10(1):4524. Epub 2019 Oct 4.

Sorbonne Université, Institut de la Vision, INSERM, CNRS, 75012, Paris, France.

A major challenge in the treatment of retinal degenerative diseases, with the transplantation of replacement photoreceptors, is the difficulty in inducing the grafted cells to grow and maintain light sensitive outer segments in the host retina, which depends on proper interaction with the underlying retinal pigment epithelium (RPE). Here, for an RPE-independent treatment approach, we introduce a hyperpolarizing microbial opsin into photoreceptor precursors from newborn mice, and transplant them into blind mice lacking the photoreceptor layer. These optogenetically-transformed photoreceptors are light responsive and their transplantation leads to the recovery of visual function, as shown by ganglion cell recordings and behavioral tests. Subsequently, we generate cone photoreceptors from human induced pluripotent stem cells, expressing the chloride pump Jaws. After transplantation into blind mice, we observe light-driven responses at the photoreceptor and ganglion cell levels. These results demonstrate that structural and functional retinal repair is possible by combining stem cell therapy and optogenetics.
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http://dx.doi.org/10.1038/s41467-019-12330-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6778196PMC
October 2019

Optogenetic Light Sensors in Human Retinal Organoids.

Front Neurosci 2018 2;12:789. Epub 2018 Nov 2.

Institut de la Vision, Sorbonne Université, INSERM, CNRS, Paris, France.

Optogenetic technologies paved the way to dissect complex neural circuits and monitor neural activity using light in animals. In retinal disease, optogenetics has been used as a therapeutic modality to reanimate the retina after the loss of photoreceptor outer segments. However, it is not clear today which ones of the great diversity of microbial opsins are best suited for therapeutic applications in human retinas as cell lines, primary cell cultures and animal models do not predict expression patterns of microbial opsins in human retinal cells. Therefore, we sought to generate retinal organoids derived from human induced pluripotent stem cells (hiPSCs) as a screening tool to explore the membrane trafficking efficacy of some recently described microbial opsins. We tested both depolarizing and hyperpolarizing microbial opsins including CatCh, ChrimsonR, ReaChR, eNpHR 3.0, and Jaws. The membrane localization of eNpHR 3.0, ReaChR, and Jaws was the highest, likely due to their additional endoplasmic reticulum (ER) release and membrane trafficking signals. In the case of opsins that were not engineered to improve trafficking efficiency in mammalian cells such as CatCh and ChrimsonR, membrane localization was less efficient. Protein accumulation in organelles such as ER and Golgi was observed at high doses with CatCh and ER retention lead to an unfolded protein response. Also, cytoplasmic localization was observed at high doses of ChrimsonR. Our results collectively suggest that retinal organoids derived from hiPSCs can be used to predict the subcellular fate of optogenetic proteins in a human retinal context. Such organoids are also versatile tools to validate other gene therapy products and drug molecules.
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http://dx.doi.org/10.3389/fnins.2018.00789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6224345PMC
November 2018

Characterization and Transplantation of CD73-Positive Photoreceptors Isolated from Human iPSC-Derived Retinal Organoids.

Stem Cell Reports 2018 09 9;11(3):665-680. Epub 2018 Aug 9.

Institut de la Vision, Sorbonne Université, INSERM, CNRS, 17, Rue Moreau, Paris 75012, France. Electronic address:

Photoreceptor degenerative diseases are a major cause of blindness for which cell replacement is one of the most encouraging strategies. For stem cell-based therapy using human induced pluripotent stem cells (hiPSCs), it is crucial to obtain a homogenous photoreceptor cell population. We confirmed that the cell surface antigen CD73 is exclusively expressed in hiPSC-derived photoreceptors by generating a fluorescent cone rod homeobox (Crx) reporter hiPSC line using CRISPR/Cas9 genome editing. We demonstrated that CD73 targeting by magnetic-activated cell sorting (MACS) is an effective strategy to separate a safe population of transplantable photoreceptors. CD73+ photoreceptor precursors can be isolated in large numbers and transplanted into rat eyes, showing capacity to survive and mature in close proximity to host inner retina of a model of photoreceptor degeneration. These data demonstrate that CD73+ photoreceptor precursors hold great promise for a future safe clinical translation.
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http://dx.doi.org/10.1016/j.stemcr.2018.07.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6135113PMC
September 2018

Mini-Review: Cell Type-Specific Optogenetic Vision Restoration Approaches.

Adv Exp Med Biol 2018 ;1074:69-73

Sorbonne Université, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, Paris, France.

The expression of light-sensitive microbial opsins is a promising mutation-independent approach to restore vision in retinal degenerative diseases. Using viral vectors, optogenetic tools can be genetically expressed in various subpopulations of retinal neurons. The choice of cell type depends on the availability of surviving retinal cells. If cones are still alive but they lack outer segments, they can be targeted with optogenetic inhibitors, such as halorhodopsin. Alternatively, it is possible to bypass the photoreceptors and to target bipolar cells. In late-stage degeneration, when bipolar cells degenerate, "artificial photoreceptors" can be made from retinal ganglion cells, but with this approach, upstream retinal processing cannot be utilized. However, when ganglion cells are stimulated directly, higher brain regions might be able to compensate for some loss of retinal processing, which is indicated by clinical studies with epiretinal implants, where patients can perform simple visual tasks. Finally, optogenetics in combination with neuroprotective approaches could serve as a valuable strategy to restore the function of remaining cells, as well as to rescue retinal neurons from progressive degeneration.
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http://dx.doi.org/10.1007/978-3-319-75402-4_9DOI Listing
May 2019

Noninvasive gene delivery to foveal cones for vision restoration.

JCI Insight 2018 01 25;3(2). Epub 2018 Jan 25.

Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, Paris, France.

Intraocular injection of adeno-associated viral (AAV) vectors has been an evident route for delivering gene drugs into the retina. However, gaps in our understanding of AAV transduction patterns within the anatomically unique environments of the subretinal and intravitreal space of the primate eye impeded the establishment of noninvasive and efficient gene delivery to foveal cones in the clinic. Here, we establish new vector-promoter combinations to overcome the limitations associated with AAV-mediated cone transduction in the fovea with supporting studies in mouse models, human induced pluripotent stem cell-derived organoids, postmortem human retinal explants, and living macaques. We show that an AAV9 variant provides efficient foveal cone transduction when injected into the subretinal space several millimeters away from the fovea, without detaching this delicate region. An engineered AAV2 variant provides gene delivery to foveal cones with a well-tolerated dose administered intravitreally. Both delivery modalities rely on a cone-specific promoter and result in high-level transgene expression compatible with optogenetic vision restoration. The model systems described here provide insight into the behavior of AAV vectors across species to obtain safety and efficacy needed for gene therapy in neurodegenerative disorders.
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http://dx.doi.org/10.1172/jci.insight.96029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5821199PMC
January 2018

Dopamine Regulation of GABA Receptors Contributes to Light/Dark Modulation of the ON-Cone Bipolar Cell Receptive Field Surround in the Retina.

Curr Biol 2017 Sep 24;27(17):2600-2609.e4. Epub 2017 Aug 24.

Department of Neuroscience, The Ohio State University College Of Medicine, Columbus, OH 43210, USA. Electronic address:

Cone bipolar cells are interneurons that receive synaptic input from cone photoreceptor cells and provide the output of the first synaptic layer of the retina. These cells exhibit center-surround receptive fields, a prototype of lateral inhibition between neighboring sensory cells in which stimulation of the receptive field center excites the cell whereas stimulation of the surrounding region laterally inhibits the cell. This fundamental sensory coding mechanism facilitates spatial discrimination and detection of stimulus edges. However, although it is well established that the receptive field surround is strongest when ambient or background illumination is most intense, e.g., at midday, and that the surround is minimal following maintained darkness, the synaptic mechanisms that produce and modulate the surround have not been resolved. Using electrical recording of bipolar cells under experimental conditions in which the cells exhibited surround light responses, and light and electron microscopic immunocytochemistry, we show in the rabbit retina that bright-light-induced activation of dopamine D receptors located on ON-center cone bipolar cell dendrites increases the expression and activity of GABA receptors on the dendrites of the cells and that surround light responses depend on endogenous GABA receptor activation. We also show that maintained darkness and D receptor blockade following maintained illumination and D receptor activation result in minimal GABA receptor expression and activity and greatly diminished surrounds. Modulation of the D/GABA receptor signaling pathway of ON-cBC dendrites by the ambient light level facilitates detection of spatial details on bright days and large dim objects on moonless nights.
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http://dx.doi.org/10.1016/j.cub.2017.07.063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5595654PMC
September 2017

A New Promoter Allows Optogenetic Vision Restoration with Enhanced Sensitivity in Macaque Retina.

Mol Ther 2017 11 20;25(11):2546-2560. Epub 2017 Jul 20.

INSERM U968, Institut de la Vision, 75012 Paris, France; UMRS968, Institut de la Vision, Sorbonne Universités, Pierre et Marie Curie University (UPMC) University Paris 06, 75012 Paris, France; Centre National de la Recherche Scientifique (CNRS) UMR7210, Institut de la Vision, 75012 Paris, France. Electronic address:

The majority of inherited retinal degenerations converge on the phenotype of photoreceptor cell death. Second- and third-order neurons are spared in these diseases, making it possible to restore retinal light responses using optogenetics. Viral expression of channelrhodopsin in the third-order neurons under ubiquitous promoters was previously shown to restore visual function, albeit at light intensities above illumination safety thresholds. Here, we report (to our knowledge, for the first time) activation of macaque retinas, up to 6 months post-injection, using channelrhodopsin-Ca-permeable channelrhodopsin (CatCh) at safe light intensities. High-level CatCh expression was achieved due to a new promoter based on the regulatory region of the gamma-synuclein gene (SNCG) allowing strong expression in ganglion cells across species. Our promoter, in combination with clinically proven adeno-associated virus 2 (AAV2), provides CatCh expression in peri-foveolar ganglion cells responding robustly to light under the illumination safety thresholds for the human eye. On the contrary, the threshold of activation and the proportion of unresponsive cells were much higher when a ubiquitous promoter (cytomegalovirus [CMV]) was used to express CatCh. The results of our study suggest that the inclusion of optimized promoters is key in the path to clinical translation of optogenetics.
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http://dx.doi.org/10.1016/j.ymthe.2017.07.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5675708PMC
November 2017

Generation of Storable Retinal Organoids and Retinal Pigmented Epithelium from Adherent Human iPS Cells in Xeno-Free and Feeder-Free Conditions.

Stem Cells 2017 05 20;35(5):1176-1188. Epub 2017 Feb 20.

Institut de la Vision, Sorbonne Universités, INSERM, CNRS UMR 7210, UPMC Univ Paris 06, Paris, France.

Human induced pluripotent stem cells (hiPSCs) are potentially useful in regenerative therapies for retinal disease. For medical applications, therapeutic retinal cells, such as retinal pigmented epithelial (RPE) cells or photoreceptor precursors, must be generated under completely defined conditions. To this purpose, we have developed a two-step xeno-free/feeder-free (XF/FF) culture system to efficiently differentiate hiPSCs into retinal cells. This simple method, relies only on adherent hiPSCs cultured in chemically defined media, bypassing embryoid body formation. In less than 1 month, adherent hiPSCs are able to generate self-forming neuroretinal-like structures containing retinal progenitor cells (RPCs). Floating cultures of isolated structures enabled the differentiation of RPCs into all types of retinal cells in a sequential overlapping order, with the generation of transplantation-compatible CD73 photoreceptor precursors in less than 100 days. Our XF/FF culture conditions allow the maintenance of both mature cones and rods in retinal organoids until 280 days with specific photoreceptor ultrastructures. Moreover, both hiPSC-derived retinal organoids and dissociated retinal cells can be easily cryopreserved while retaining their phenotypic characteristics and the preservation of CD73 photoreceptor precursors. Concomitantly to neural retina, this process allows the generation of RPE cells that can be effortlessly amplified, passaged, and frozen while retaining a proper RPE phenotype. These results demonstrate that simple and efficient retinal differentiation of adherent hiPSCs can be accomplished in XF/FF conditions. This new method is amenable to the development of an in vitro GMP-compliant retinal cell manufacturing protocol allowing large-scale production and banking of hiPSC-derived retinal cells and tissues. Stem Cells 2017;35:1176-1188.
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http://dx.doi.org/10.1002/stem.2586DOI Listing
May 2017

Red-shifted channelrhodopsin stimulation restores light responses in blind mice, macaque retina, and human retina.

EMBO Mol Med 2016 11 2;8(11):1248-1264. Epub 2016 Nov 2.

INSERM U968, Paris, France

Targeting the photosensitive ion channel channelrhodopsin-2 (ChR2) to the retinal circuitry downstream of photoreceptors holds promise in treating vision loss caused by retinal degeneration. However, the high intensity of blue light necessary to activate channelrhodopsin-2 exceeds the safety threshold of retinal illumination because of its strong potential to induce photochemical damage. In contrast, the damage potential of red-shifted light is vastly lower than that of blue light. Here, we show that a red-shifted channelrhodopsin (ReaChR), delivered by AAV injections in blind rd1 mice, enables restoration of light responses at the retinal, cortical, and behavioral levels, using orange light at intensities below the safety threshold for the human retina. We further show that postmortem macaque retinae infected with AAV-ReaChR can respond with spike trains to orange light at safe intensities. Finally, to directly address the question of translatability to human subjects, we demonstrate for the first time, AAV- and lentivirus-mediated optogenetic spike responses in ganglion cells of the postmortem human retina.
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http://dx.doi.org/10.15252/emmm.201505699DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5090658PMC
November 2016

The Thioredoxin Encoded by the Rod-Derived Cone Viability Factor Gene Protects Cone Photoreceptors Against Oxidative Stress.

Antioxid Redox Signal 2016 06 12;24(16):909-23. Epub 2016 May 12.

1 INSERM , U968, Paris, France .

Aims: Rod-derived cone viability factor long (RdCVFL) is an enzymatically active thioredoxin encoded by the nucleoredoxin-like-1 (Nxnl1) gene. The second product of the gene, RdCVF, made by alternative splicing is a novel trophic factor secreted by rods that protects cones in rodent models of retinitis pigmentosa, the most prevalent inherited retinal disease. It acts on cones by stimulating aerobic glycolysis through its interaction with a complex containing basigin-1 and the glucose transporter GLUT1. We studied the role of Nxnl1 in cones after its homologous recombination using a transgenic line expressing Cre recombinase under the control of a cone opsin promoter.

Results: We show that the cones of these mice are dysfunctional and degenerate by 8 months of age. The age-related deficit in cones is exacerbated in young animals by exposure to high level of oxygen. In agreement with this phenotype, we found that the cones express only one of the two Nxnl1 gene products, the thioredoxin RdCVFL. Administration of RdCVFL to the mouse carrying a deletion of the Nxnl1 gene in cones reduces the damage produced by oxidative stress. Silencing the expression of RdCVFL in cone-enriched culture reduces cell viability, showing that RdCVFL is a cell-autonomous mechanism of protection.

Innovation: This novel mode of action is certainly relevant for the therapy of retinitis pigmentosa since the delivery into cones of the rd10 mouse, a recessive model of the disease, rescues cones.

Conclusion: Our work highlights the duality of the Nxnl1 gene, which protects the cones by two distinct mechanisms. Antioxid. Redox Signal. 24, 909-923.
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http://dx.doi.org/10.1089/ars.2015.6509DOI Listing
June 2016

Heterogeneity and convergence of olfactory first-order neurons account for the high speed and sensitivity of second-order neurons.

PLoS Comput Biol 2014 Dec 4;10(12):e1003975. Epub 2014 Dec 4.

Institut National de la Recherche Agronomique (INRA), Unité Mixte de Recherche 1392 Institut d'Ecologie et des Sciences de l'Environnement de Paris, Versailles, France; Laboratoire Lorrain de Recherche en Informatique et ses Applications (LORIA), Unité Mixte de Recherche 7503, Centre National de la Recherche Scientifique (CNRS), Vandœuvre-lès-Nancy, France.

In the olfactory system of male moths, a specialized subset of neurons detects and processes the main component of the sex pheromone emitted by females. It is composed of several thousand first-order olfactory receptor neurons (ORNs), all expressing the same pheromone receptor, that contact synaptically a few tens of second-order projection neurons (PNs) within a single restricted brain area. The functional simplicity of this system makes it a favorable model for studying the factors that contribute to its exquisite sensitivity and speed. Sensory information--primarily the identity and intensity of the stimulus--is encoded as the firing rate of the action potentials, and possibly as the latency of the neuron response. We found that over all their dynamic range, PNs respond with a shorter latency and a higher firing rate than most ORNs. Modelling showed that the increased sensitivity of PNs can be explained by the ORN-to-PN convergent architecture alone, whereas their faster response also requires cell-to-cell heterogeneity of the ORN population. So, far from being detrimental to signal detection, the ORN heterogeneity is exploited by PNs, and results in two different schemes of population coding based either on the response of a few extreme neurons (latency) or on the average response of many (firing rate). Moreover, ORN-to-PN transformations are linear for latency and nonlinear for firing rate, suggesting that latency could be involved in concentration-invariant coding of the pheromone blend and that sensitivity at low concentrations is achieved at the expense of precise encoding at high concentrations.
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http://dx.doi.org/10.1371/journal.pcbi.1003975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4256018PMC
December 2014

Reactive searching and infotaxis in odor source localization.

PLoS Comput Biol 2014 Oct 16;10(10):e1003861. Epub 2014 Oct 16.

CNRS, LORIA, UMR 7503, Vandoeuvre-les-Nancy, France.

Male moths aiming to locate pheromone-releasing females rely on stimulus-adapted search maneuvers complicated by a discontinuous distribution of pheromone patches. They alternate sequences of upwind surge when perceiving the pheromone and cross- or downwind casting when the odor is lost. We compare four search strategies: three reactive versus one cognitive. The former consist of pre-programmed movement sequences triggered by pheromone detections while the latter uses Bayesian inference to build spatial probability maps. Based on the analysis of triphasic responses of antennal lobe neurons (On, inhibition, Off), we propose three reactive strategies. One combines upwind surge (representing the On response to a pheromone detection) and spiral casting, only. The other two additionally include crosswind (zigzag) casting representing the Off phase. As cognitive strategy we use the infotaxis algorithm which was developed for searching in a turbulent medium. Detection events in the electroantennogram of a moth attached to a robot indirectly control this cyborg, depending on the strategy in use. The recorded trajectories are analyzed with regard to success rates, efficiency, and other features. In addition, we qualitatively compare our robotic trajectories to behavioral search paths. Reactive searching is more efficient (yielding shorter trajectories) for higher pheromone doses whereas cognitive searching works better for lower doses. With respect to our experimental conditions (2 m from starting position to pheromone source), reactive searching with crosswind zigzag yields the shortest trajectories (for comparable success rates). Assuming that the neuronal Off response represents a short-term memory, zigzagging is an efficient movement to relocate a recently lost pheromone plume. Accordingly, such reactive strategies offer an interesting alternative to complex cognitive searching.
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http://dx.doi.org/10.1371/journal.pcbi.1003861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4211930PMC
October 2014

Targeting channelrhodopsin-2 to ON-bipolar cells with vitreally administered AAV Restores ON and OFF visual responses in blind mice.

Mol Ther 2015 Jan 6;23(1):7-16. Epub 2014 Aug 6.

1] INSERM, U968, Paris, France [2] Sorbonne Universités, UPMC Univ Paris 06, UMR_S 968, Institut de la Vision, Paris, France [3] CNRS, UMR_7210, Paris, France.

Most inherited retinal dystrophies display progressive photoreceptor cell degeneration leading to severe visual impairment. Optogenetic reactivation of retinal neurons mediated by adeno-associated virus (AAV) gene therapy has the potential to restore vision regardless of patient-specific mutations. The challenge for clinical translatability is to restore a vision as close to natural vision as possible, while using a surgically safe delivery route for the fragile degenerated retina. To preserve the visual processing of the inner retina, we targeted ON bipolar cells, which are still present at late stages of disease. For safe gene delivery, we used a recently engineered AAV variant that can transduce the bipolar cells after injection into the eye's easily accessible vitreous humor. We show that AAV encoding channelrhodopsin under the ON bipolar cell-specific promoter mediates long-term gene delivery restricted to ON-bipolar cells after intravitreal administration. Channelrhodopsin expression in ON bipolar cells leads to restoration of ON and OFF responses at the retinal and cortical levels. Moreover, light-induced locomotory behavior is restored in treated blind mice. Our results support the clinical relevance of a minimally invasive AAV-mediated optogenetic therapy for visual restoration.
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http://dx.doi.org/10.1038/mt.2014.154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4270733PMC
January 2015

Pheromone modulates plant odor responses in the antennal lobe of a moth.

Chem Senses 2014 Jun 5;39(5):451-63. Epub 2014 May 5.

UMR 1392 Institut d'Ecologie et des Sciences de l'Environnement de Paris, INRA, 78000 Versailles, France and Université d'Angers, Laboratoire Récepteurs et Canaux Ioniques Membranaires, UPRES-EA 2647, USC INRA 1330, SFR 4207 QUASAV, UFR Sciences, 2 Boulevard Lavoisier, 49045 Angers, France,

In nature, male moths are exposed to a complex plant odorant environment when they fly upwind to a sex pheromone source in their search for mates. Plant odors have been shown to affect responses to pheromone at various levels but how does pheromone affects plant odor perception? We recorded responses from neurons within the non-pheromonal "ordinary glome ruli" of the primary olfactory center, the antennal lobe (AL), to single and pulsed stimulations with the plant odorant heptanal, the pheromone, and their mixture in the male moth Agrotis ipsilon. We identified 3 physiological types of neurons according to their activity patterns combining excitatory and inhibitory phases. Both local and projection neurons were identified in each physiological type. Neurons with excitatory responses to heptanal responded also frequently to the pheromone and showed additive responses to the mixture. Moreover, the neuron's ability of resolving successive pulses generally improved with the mixture. Only some neurons with combined excitatory/inhibitory, or purely inhibitory responses to heptanal, also responded to the pheromone. Although individual mixture responses were not significantly different from heptanal responses in these neurons, pulse resolution was improved with the mixture as compared with heptanal alone. These results demonstrate that the pheromone and the general odorant subsystems interact more intensely in the moth AL than previously thought.
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http://dx.doi.org/10.1093/chemse/bju017DOI Listing
June 2014

Multiphasic on/off pheromone signalling in moths as neural correlates of a search strategy.

PLoS One 2013 17;8(4):e61220. Epub 2013 Apr 17.

UMR7503, Laboratoire Lorrain de Recherche en Informatique et ses Applications, Centre National de la Recherche Scientifique, Vandoeuvre-lès-Nancy, France.

Insects and robots searching for odour sources in turbulent plumes face the same problem: the random nature of mixing causes fluctuations and intermittency in perception. Pheromone-tracking male moths appear to deal with discontinuous flows of information by surging upwind, upon sensing a pheromone patch, and casting crosswind, upon losing the plume. Using a combination of neurophysiological recordings, computational modelling and experiments with a cyborg, we propose a neuronal mechanism that promotes a behavioural switch between surge and casting. We show how multiphasic On/Off pheromone-sensitive neurons may guide action selection based on signalling presence or loss of the pheromone. A Hodgkin-Huxley-type neuron model with a small-conductance calcium-activated potassium (SK) channel reproduces physiological On/Off responses. Using this model as a command neuron and the antennae of tethered moths as pheromone sensors, we demonstrate the efficiency of multiphasic patterning in driving a robotic searcher toward the source. Taken together, our results suggest that multiphasic On/Off responses may mediate olfactory navigation and that SK channels may account for these responses.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0061220PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3629186PMC
November 2013

Plant odour stimuli reshape pheromonal representation in neurons of the antennal lobe macroglomerular complex of a male moth.

J Exp Biol 2012 May;215(Pt 10):1670-80

INRA, UMR 1272 Physiologie de l'Insecte: Signalisation et Communication, F-78000 Versailles, France.

Male moths are confronted with complex odour mixtures in a natural environment when flying towards a female-emitted sex pheromone source. Whereas synergistic effects of sex pheromones and plant odours have been observed at the behavioural level, most investigations at the peripheral level have shown an inhibition of pheromone responses by plant volatiles, suggesting a potential role of the central nervous system in reshaping the peripheral information. We thus investigated the interactions between sex pheromone and a behaviourally active plant volatile, heptanal, and their effects on responses of neurons in the pheromone-processing centre of the antennal lobe, the macroglomerular complex, in the moth Agrotis ipsilon. Our results show that most of these pheromone-sensitive neurons responded to the plant odour. Most neurons responded to the pheromone with a multiphasic pattern and were anatomically identified as projection neurons. They responded either with excitation or pure inhibition to heptanal, and the response to the mixture pheromone + heptanal was generally weaker than to the pheromone alone, showing a suppressive effect of heptanal. However, these neurons responded with a better resolution to pulsed stimuli. The other neurons with either purely excitatory or inhibitory responses to all three stimuli did not exhibit significant differences in responses between stimuli. Although the suppression of the pheromone responses in AL neurons by the plant odour is counter-intuitive at first glance, the observed better resolution of pulsed stimuli is probably more important than high sensitivity to the localization of a calling female.
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http://dx.doi.org/10.1242/jeb.066662DOI Listing
May 2012

Automatic spike train analysis and report generation. An implementation with R, R2HTML and STAR.

J Neurosci Methods 2009 Jun 24;181(1):119-44. Epub 2009 Feb 24.

Laboratoire de Physiologie Cérébrale, CNRS UMR 8118, UFR biomédicale de l'université Paris-Descartes, 45 Rue des Saints-Pères, 75006 Paris, France.

Multi-electrode arrays (MEA) allow experimentalists to record extracellularly from many neurons simultaneously for long durations. They therefore often require that the data analyst spends a considerable amount of time first sorting the spikes, then doing again and again the same basic analysis on the different spike trains isolated from the raw data. This spike train analysis also often generates a considerable amount of figures, mainly diagnostic plots, that need to be stored (and/or printed) and organized for efficient subsequent use. The analysis of our data recorded from the first olfactory relay of an insect, the cockroach Periplaneta americana, has led us to settle on such "routine" spike train analysis procedures: one applied to spontaneous activity recordings, the other used with recordings where an olfactory stimulation was repetitively applied. We have developed a group of functions implementing a mixture of common and original procedures and producing graphical or numerical outputs. These functions can be run in batch mode and do moreover produce an organized report of their results in an HTML file. A R package: Spike Train Analysis with R (STAR) makes these functions readily available to the neurophysiologists community. Like R, STAR is open source and free. We believe that our basic analysis procedures are of general interest but they can also be very easily modified to suit user specific needs.
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http://dx.doi.org/10.1016/j.jneumeth.2009.01.037DOI Listing
June 2009

SIMONE: a realistic neural network simulator to reproduce MEA-based recordings.

IEEE Trans Neural Syst Rehabil Eng 2008 Apr;16(2):149-60

CEA/LETI, Minatec, Grenoble, France.

Contemporary multielectrode arrays (MEAs) used to record extracellular activity from neural tissues can deliver data at rates on the order of 100 Mbps. Such rates require efficient data compression and/or preprocessing algorithms implemented on an application specific integrated circuit (ASIC) close to the MEA. We present SIMONE (Statistical sIMulation Of Neuronal networks Engine), a versatile simulation tool whose parameters can be either fixed or defined by a probability distribution. We validated our tool by simulating data recorded from the first olfactory relay of an insect. Different key aspects make this tool suitable for testing the robustness and accuracy of neural signal processing algorithms (such as the detection, alignment, and classification of spikes). For instance, most of the parameters can be defined by a probabilistic distribution, then tens of simulations may be obtained from the same scenario. This is especially useful when validating the robustness of the processing algorithm. Moreover, the number of active cells and the exact firing activity of each one of them is perfectly known, which provides an easy way to test accuracy.
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http://dx.doi.org/10.1109/TNSRE.2007.914467DOI Listing
April 2008

Prior classical olfactory conditioning improves odour-cued flight orientation of honey bees in a wind tunnel.

J Exp Biol 2005 Oct;208(Pt 19):3731-7

Laboratoire de Physiologie Cérébrale, CNRS UMR 8118, Paris, France.

Odours are key cues used by the honey bee in various situations. They play an important role in sexual attraction, social behaviour and location of profitable food sources. Here, we were interested in the role of odours in orientation at short distance, for instance the approach flight to a floral patch or in close proximity to the hive entrance. Using a newly designed wind tunnel, we investigated the orientation behaviour of the bee towards two different odours: a social odour and a floral component, linalool. We then tested the effect of prior olfactory conditioning (conditioning of the proboscis extension reflex) on subsequent flight orientation. We showed that both stimuli induced orientated behaviour (orientated flights, circling around the odour source) in up to 70% of the worker bees, social odour being slightly more attractive than the linalool. We found thereafter that orientation performance towards the floral compound can be significantly enhanced by prior classical olfactory learning. This type of information transfer, from a Pavlovian associative context to an orientation task, might allow future foragers to acquire, within the hive, relevant information about the odours and food they will encounter during their later foraging bouts.
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http://dx.doi.org/10.1242/jeb.01796DOI Listing
October 2005