european retina meeting 2017 - ERM 2017

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EUROPEAN RETINA MEETING 2017 October 5 -7, 2017 Paris, France

Organisers: Serge Picaud, Alvaro Rendon (Institut de la Vision, France) Petri Ala-Laurila (University of Helsinki, Finland)

Very glad to have been generously supported by

OUR SPONSORS

CONTENTS Page Introduction Organizers of the ERM 2017 The venue The Gala event Young researchers award Program Programme du symposium en français TALKS The Human and Primate Retina Session Retinal Diseases and Therapies Session Retinal Impact on Eye Development and Myopia Session Retinal Circuit Session Tools Against Retinal Diseases Session Light Adaptation Session POSTERS Poster Session I: Retinal Diseases and Therapies Poster Session II: Retinal Diseases and Therapies Poster Session III: Retinal Circuits Poster Session IV: Retinal Circuits Poster Session V: Human Primate Retina, Myopia, Light Adaptation, Others

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61 99 135 171 205

TALKS Symposium en français

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Companies & Sponsors

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INTRODUCTION We are glad and honored to have the opportunity to host the 6th edition of the European Retina Meeting. The ERM started in Frankfurt exactly 10 years ago in 2007 at the invitation by Pr Wässle. A decade is not a particularly long time, but during this time, the ERM has changed and reached a very important turning point. The ERM is leaving its “childhood” and is entering into a new and more confident phase. The ERM event is reaching a new dimensionality and maturity without losing its original innocence. The meeting still has its original excitation of the beginning and is willing to offer a suitable and fair playground especially for young scientists. We are immensely thankful to our helpers, supporters and sponsors who have given us the possibility to organize this edition in Paris − a wonderful town, rich in history, and always a bubbly, amazing and surprising place with its unique vibe. I hope you will have time to feel the French inspiration, energy and beautiful memories both in science and in life. The Gala should offer you a flashback in French history. We are immensely thankful to YOU. Yes YOU! You as a speakers, presenters, sponsors and participants. Without YOU and your presence, none of this would have been possible. Welcome to Paris and the ERM 2017 meeting! The organisers: Serge Picaud (Institut de la Vision, France) Alvaro Rendon (Institut de la Vision, France) Emanuela De Luca (Fondation Voir et Entendre, France) Petri Ala-Laurila (University of Helsinki, Finland)

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EUROPEAN RETINA MEETING 2017 October 5-7, 2017, Paris, France

Organisers: Serge Picaud , Alvaro Rendon (Institut de la Vision, France) Petri Ala-Laurila (University of Helsinki, Finland)

Executive Committee : Agathe Alviset Peggy Chambaz William Deschamps Elisabeth Dubus Laure Guibbal Christine Hamayon Corentin Joffrois Diep Nguyen Soda Thiam Manon Valet

www.erm2017.eu

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THE VENUE The European Retina Meeting 2017, will be held at the International House of the University City, in the South of Paris very close to the Montsouris Park. The mission of the International House of the University City, since its inception, consists in promoting international mobility for students and researchers for one purpose: to be a catalyst for the Universalist values of peace and solidarity. The Campus offers to the 12,000 students, doctoral candidates, young researchers and young artists from 140 nationalities, the best possible conditions of reception and lodging. The address of the venue is 17 Boulevard Jourdan, 75014 Paris You will easily reach the venue by RER B or tram T3a. The venue is just in front of the “Cité Universitaire” RER B stop

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THE GALA EVENT The gala dinner, in the evening of October 5th, will take place in one of the most exclusive and representative places in Paris, the Hôtel de Ville (City Hall), situated in the heart of Paris. The entrance will be on the back of the building, exactly at 3 Rue de Lobeau To reach the Hôtel de Ville from the meetings venue, just take the RER B, from the "University City" until the "Châtelet" stop. From the "Châtelet" stop, walk 5 minutes to reach the City Hall. We are very glad and also a bit proud, to have the opportunity to celebrate the ERM 2017 with you in this wonderful frame!

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YOUNG RESEARCHERS AWARDS By means of the generosity of our sponsors, we will be able to attribute 2 awards to the young scientists (PhD students and PostDoc fellows). There will be 2 categories: 1) oral presentation 2) poster presentation The scientific scope as well as the form of the presentation will have an impact in the evaluation for the awards attribution. The winners will benefit of a registration and flight tickets to participate in the ARVO 2018 !

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PROGRAM Thursday October 5th 8h – 9h

Registration

9h-9h05

Introduction by Serge Picaud The Human and Primate Retina – Session Chair: John Dowling

9h05 – 9h25

John Dowling (Molecular and Cellular Biology Harvard University, Cambridge, MA) Reconstructing the Human Fovea

9h25 – 9h45

Raunak Sinha (Physiology and Biophysics, University of Washington School of Medicine, USA) Transformation of visual signals in the fovea

9h45 – 10h05

Szabó Arnold (Semmelweis University, Department of Human Morphology and Developmental Biology, Budapest, Hungary) Long-term organotypic culture model of the adult human retina

10h05 – 10h15

Alexandra Tikidji-Hamburyan (Stanford University, CA, USA) Sampling of cone inputs by major ganglion cell types in primate retina Retinal Diseases and Therapies – Session I Chair: Homaira Nawabi

10h15 – 10h35

Florian Sennlaub (Institut de la Vision, Paris, France) Genetic AMD-risk factors promote pathogenic subretinal inflammation

10h35 – 10h55

Przemyslaw Sapieha (University of Montreal, Montréal, Canada) Cellular Senescence and Dormancy in Retinopathy

10h55 – 11h25

Coffee Break

11h25 – 11h45

Marius Ader (Technische Universität, Center for Regenerative Therapies Dresden, Germany) Photoreceptor transplantation: marker-free identification of photoreceptors by mechanical phenotyping

11h45 – 12h05

Homaira Nawabi (Neuroscience Institute, Grenoble, France) Axon Regeneration in the visual system

12h05 – 12h25

Andrew Huberman (Neurobiology & Ophthalmology, Stanford School of Medicine, Stanford, CA, USA) Visual system regeneration. Breaking and re-creating brain circuits for seeing

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12h25 – 12h45

Short Presentations of 7 of our sponsors* (about 3 minutes each)

12h45 – 14h30

Lunch Break and Poster Session I: Retinal Diseases and Therapies Retinal Diseases and Therapies – Session II Chair: Nicolas Cuenca

14h30 – 14h50

Fabio Benfenati (Centre for Synaptic Neuroscience and Technology, Istituto Italiano di Tecnologia, Genova, IT) A fully organic retinal prosthesis restores vision in a rat model of degenerative blindness

14h50 – 15h10

Deniz Dalkara (Institut de la Vision, Paris, France) Optogenetics for vision restoration - translation from mice to primates

15h10 – 15h20

Alexander Kolesnikov (Washington University School of Medicine in St. Louis, USA) Rhodopsin expression increases the resistance of mammalian M-cones to retinoid deficiency in LCA model

15h20 – 15h30

Paola Vagni (LNE, École polytechnique fédérale de Lausanne, Swisstzerland) Preventing visual function loss in the rd10 mouse model of retinitis pigmentosa using gene editing

15h30 – 15h40

Nicolás Cuenca (Departamento de Fisiología, Genética y Microbiología Universidad de Alicante, Spain) Impairment of dopaminergic circuitries, ganglion cells loss and Lewy bodies containing p-alpha synuclein were found in human retinas in Parkinson`s disease

15h40 – 16h10

8 Quick-fire presentations (3 min to introduce the posters) 1) Luba Astakhova Two convenient experimental models of photoreceptor degeneration for screening of molecular photoswitches 2) Juliette Varin Development of a gene therapy approach for cCSNB when mutations in GRM6 and LRIT3 are involved 3) Elisa Castaldi Visual BOLD response in late-blind subjects with Argus II retinal prosthesis 4) Mirella Telles Salgueiro Barboni ON/OFF asymmetrical dysfunction of retinal mechanisms in Duchenne muscular dystrophy patients 5) Ulisse Bocchero Early steps of photoreceptor degeneration in a model of retinitis pigmentosa 6) Martina Biagioni Unexpected prevalence of inflammatory response in a mouse model of Retinitis Pigmentosa: looking toward therapeutic potential 7) Samuel Mills The contribution of microglia to early vascular dysfunction in diabetic retinopathy 8) Matt Rutar Dynamic interplay of innate and adaptive immunity during sterile retinal inflammation: Insights from the transcriptome

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16h10 – 17h45

Coffee Break and Poster Session II : Retinal Diseases and Therapies

19:30

Gala cocktail buffet at the Hotel de Ville

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Friday October 6th Retinal Impact on Eye Develoment and Myopia - Session Chair: Machelle Pardue 9h– 9h20

David Copenhagen (UCSF School of Medicine, Ophthalmology, San Francisco CA, USA) Melanopsin-based photoreception in fetal and newborn mice: actions on behavior and both vascular and neural development in the eye

9h20 – 9h40

Frank Schaeffel (Neurobiology of the Eye, University of Tübingen, Germany) Retinal control of myopia - lenses, light and atropine

9h40 – 10h00

Machelle Pardue (Biomedical Engineering, Emory University, Atlanta, GA, USA) Contributions of the three photoreceptor pathways to refractive eye growth and myopia in mice

10h00 – 10h30

Coffee Break – Hanging of the Session III Posters Retinal Circuits – Session I Chair: Katrin Franke

10h30 – 10h50

Leon Lagnado (School of Life Sciences, University of Sussex, Brighton, UK) How do ribbon synapses encode visual information?

10h50 – 11h10

Greg Schwartz (Northwestern University Feinberg School of Medicine, Chicago,USA) A self regulating gap junction network of amacrine cells releases nitric oxide in the retina

11h10 – 11h30

Daniel Kerschensteiner (Neuroscience, and Biomedical Engineering, Washington University School of Medicine, USA) Dissecting motion processing circuits in the retina

11h30 – 11h50

Katrin Franke (Ophthalmic Research, University of Tübingen, Germany) Functional diversity in the mouse inner retina

11h50 – 12h00

Jeffrey Diamond (National Institute of Neurological Disorders and Stroke U.S. National Institutes of Health, USA) Synaptic transfer between ON and OFF visual channels mediated by AII amacrine cells in the mouse retina

12h00 – 12h10

Lena Nemitz (University of Oldenburg, Germany) Development of the photoreceptor ribbon synapse in the absence of horizontal cells

12h10 – 12h20

Lea Ankri (Weizmann Institute of Science, Rehovot, Israel) Complexity and dynamics of inhibitory circuits shape the directional code of the retina

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12h20 – 14h20

Lunch Break and Poster Session III: Retinal Circuits Retinal Circuits – Session II Chair: Mrinalini Hoon

14h20 – 15h00

10 Quickfire presentations (3 min to introduce your own poster) 1) Morven A Cameron Photoreceptor inputs for light-induced dopamine release in the mouse retina 2) Maria M. Arietti Circadian control of cone kinetics 3) Gloria Colombo Quantitative analysis of microglial morphology and cell type interaction during retinal postnatal development 4) Benjamin Sivyer Specific inhibitory pathways mediate saccadic suppression in direction-selective ganglion cells 5) Mohammad Khani Linear and nonlinear integration of chromatic stimuli in retinal circuitry 6) Yanli Ran Spatial integration in mouse retinal ganglion cell dendrites 7) Rebekah Warwick Response properties of retinal ganglion cells and their underlying circuits vary with retinal location 8) Gerrit Hilgen Functional characterisation of parvalbumin-expressing cells in the mouse retina 9) Norma Kühn Synergistic decoding of complex texture motion from populations of directionselective ganglion cells 10) Yang Yue Encoding natural images by gap junctions in retinal rod photoreceptors through a large-scale network model

15h00 – 15h10

Mrinalini Hoon (Dept of Biological Structure University of Washington, USA) Role of the GABAA α3 receptor as a developmental organizer of retinal inhibitory synapses

15h10 – 15h20

David Krizaj (Moran Eye Institute, University of Utah School of Medicine, USA) Endocannabinoids modulate RGC physiology through parallel modulation of TRPV1 and cannabinoid receptors

15h20 – 15h30

Tom Baden (School of Life Sciences, University of Sussex, UK) Zebrafish colour vision: anisotropic retinal circuits match asymmetric spectral content in natural light

15h30 – 15h40

Nina Milosavljevic (Medicine and Health The University of Manchester, UK) Probing the intraretinal influences of ipRGCs using chemogenetic manipulation

15h40 – 15h50

Stephen C. Massey (University of Texas at Houston, USA) Rod/Cone coupling in the mouse retina

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15h50 – 16h00

Giulia Spampinato (Institut de la Vision , Paris, France) Probing the retinal circuit by combining two photon holographic stimulation and multi electrode recordings

16h00 – 17h45

Coffee Break and Poster Session IV: Retinal Circuits

Saturday 7th October Tools against retinal diseases – Session Chair: Botond Roska 9h– 9h20

Botond Roska (Friedrich Miescher Institute for Biomedical research, Basel,Swisstzerland) Tools for studying retinal circuits and disease

9h20 – 9h40

Joana Neves (The Buck Institute for Research on Aging, Novato, CA, USA) MANF as an immune modulatory intervention to improve retinal regenerative therapies in aging Light Adaptation - Session Chair: Petri Ala-Laurila

9h40 – 10h00

Thomas Münch (Werner Reichardt Centre for Integrative Neuroscience, Tübingen, Germany) Adaptation of retinal processing in a dynamically changing environment

10h00 – 10h20

Greg Field (Neurobiology, Duke University School of Medicine, USA) Light adaptation and correlated activity in the rodent retina.

10h20 – 10h40

Petri Ala-Laurila (Department of Biosciences, University of Helsinki, Finland) Is mouse vision more sensitive during the night ?

10h40 – 12h30

Coffee Break + Poster Session V : Human Primate Retina + Myopia + Light Adaption + others

12h30 – 13h00

Conclusions and presentation of the next meetings (2019 – 2021), winners of the young researchers awards, by Serge Picaud, Petri Ala-Laurili, Andrew Huberman

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Jeudi 5 Octobre – Symposium en français Innovations et Solutions thérapeutiques President de la session: Robert Duvoisin (Oregon Health & Science University, Portland U.S.A.) 14h00 – 14h30

Les prothèses rétiniennes de nouvelle génération + discussion Paul-Henri Prevot (Institut de la Vision, Paris) Yannick Lemer (Fondation Ophtalmologique A. de Rothschild, Paris)

14h30 – 15h00

Restauration visuelle par thérapie optogénétique + discussion Antoine Chaffiol (Institut de la Vision, Paris)

15h00 – 15h30

Production de rétines in vitro à partir de cellules pluripotentes humaines : un nouvel outil thérapeutique + discussion Sacha Reichman (Institut de la Vision, Paris)

15h30 – 16h00

Approches ciblant le remplacement de l’EPR et les essais cliniques en cours + discussion Karim Ben M Barek (Institut de la Vision, Paris)

16h00 – 16h20

Pause-Café

16h20 – 16h50

Imagerie et nouveaux critères d’évaluations médicaux + discussion Michel Paques / Kate Grieve (Institut de la Vision et CHNO des 15-20, Paris)

16h50 – 17h10

Dystrophie rétinienne : la survie des cônes et le RdCVF + discussion Emmanuelle Clérin (Institut de la Vision, Paris)

17h10 – 17h30

Impact des troubles visuels sur la vie journalière des patients atteints de dystrophies rétiniennes + discussion Saddek Mohand-Saïd (Institut de la Vision et CHNO des 15-20, Paris)

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TALKS THE HUMAN AND PRIMATE RETINA SESSION 1) Reconstructing the Human Fovea John Dowling Molecular and Cellular Biology Harvard University*, Cambridge, MA

With connectomics methods now available, it is possible to reconstruct pieces of neural tissue to the synapse level. To this end, we are reconstructing a human fovea obtained from a young man who died because of a tragic accident. We have focused on the central-most cones that mediate the highest-acuity vision in the blue cone-free region of the fovea. Using the Automatic Tape Ultramicrotome (AUTM), we have cut approximately 4,000 serial sections at 60 nm thickness. So far we have imaged by scanning electron microscopy 1500 sections at 6 nm resolution from the central cone inner segments to their cone pedicles and associated second-order bipolar and horizontal cells. Contrary to the classic view, we have found the pedicles of the central-most 200 cones sit almost directly below the inner segments in the center of the foveal pit. These cone pedicles contact each other via teleodendria that appear to form gap junctions with each other. The synaptic organization of the pedicles appears similar to parafoveal cone pedicles, which form invaginating or semi-invaginating ribbon contacts with ON midget and diffuse bipolar cells, and flat, basal, contacts with midget and diffuse OFF bipolar cells. In addition horizontal cell processes form the lateral elements in the pedicle invaginations. Another novel finding is the discovery of a second type of glial ell in the central fovea. This cell extends only to the level of the cone pedicles and not to the external limiting membrane. *This research is a collaboration involving Dennis Dacey, Rachel Wong and Orin Packer (University of Washington) and Jeffrey Lichtman, Richard Schalek and john Dowling (Harvard University)

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2) Transformation of visual signals in the fovea Raunak Sinha, Mrinalini Hoon, Jacob Baudin, Fred Rieke Physiology and Biophysics, University of Washington School of Medicine, USA Howard Hughes Medical Institute, University of Washington, USA Dept. of Biological Structure, University of Washington, USA

Our visual perception is dominated by a specialized region in the retina called the fovea. Consequently our perceptual sensitivity varies remarkably across the visual field. Foveal vision, which comprises the central 1-2 degrees of our visual field has the highest spatial and chromatic resolution but cannot detect rapidly changing visual inputs such as flickering light. Past in vivo recordings of foveal output signals suggest that perceptual specializations of foveal vision originate largely in the retina itself rather than in subsequent cortical circuits. Nonetheless, we know very little about the cellular and synaptic basis of these functional specializations due to a lack of intracellular recordings from foveal neurons. In this talk, I will describe the first direct comparisons of the physiological properties of foveal and peripheral retinal neurons and a detailed structure-function correlation in the fovea. These experiments reveal how differences in the cellular and synaptic operation of foveal and peripheral retina can account for well-established perceptual differences between foveal and peripheral vision.

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3) Long-term organotypic culture model of the adult human retina Arnold Szabo Department of Anatomy, Histology and Embryology, Semmelweis University, Budapest, Hungary

Corneal transplantation replaces diseased or damaged tissue with healthy tissue from an organ donor. While the surgery to harvest transplantable corneas from recently deceased donors removes the whole eye, the rest of the tissue is typically disposed of. We developed a culture technique which maintains retinas from those eyes in excellent condition for more than three months, enabling research on human retinal tissue that was previously impossible. Human eyes were collected through scheduled multi-organ donations. Following enucleation and dissection of the eye ball, organotypic retina cultures were prepared within 1-2 hours after the circulation arrest in the donor. The retinal pieces with or without attached retinal pigment epithelium (RPE) and choroid were cultured for up to 100 days in a specific, serum-free, chemically defined medium which has been optimized for the human retina. Both isolated neural retina and retina-RPE-choroid co-cultures were astonishingly well preserved morphologically with low inter-sample variability. Every major cell type survived and all retinal layers were maintained even after twelve weeks. Outer segments could be detected in high numbers in both cultures, but the quality and the density of outer segments were superior in retina-RPE-choroid cocultures. Cones did not undergo severe apoptosis and a mean density of 50005500 cones/mm2 were measured even in long-term cultures. Subpopulations of bipolar, horizontal and amacrine cells showed close to normal morphology. The stratification of the inner plexiform layer remained recognizable. Only the number of surviving ganglion cells showed a significant decrease in long-term cultures, but ganglion cells were still present even after 84 days. Synaptic structures showed close to normal morphology on samples stained against synapthophysin, while prominent telodendria on cone pedicles indicated intact gap junctions. Our results show that the adult human retina can be maintained in an appropriate culture system for at least three months. By long-term culturing, both acute and chronic effects of pharmacological compounds could be tested directly on human tissue in a cost- and time-effective manner. Further, the long culture time allows the administration of viral vectors and opens new strategies for developing and testing gene therapeutic approaches and can help to reduce the use of animals both in academic and industrial research.

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4) Sampling of cone inputs by major ganglion cell types in primate retina Alexandra Tikidji-Hamburyan1, Nishal Shah1, Colleen Rhoades1, Nora Brackbill1, Georges Goetz1, Alexander Sher2, Alan Litke2 & E.J. Chichilnisky1 1 Stanford University, 2University of California Santa Cruz Introduction: Spatial receptive fields (RFs) of retinal ganglion cells (RGCs) are shaped by their sampling of cone inputs, via retinal interneurons. Our goal is to understand the spatial sampling of cones performed by individual cells and by neighboring cells of the same type. We tested the hypotheses that (1) individual RFs exhibit a difference of Gaussian (DOG) spatial profile at single cone resolution, and (2) sharing of cone inputs with neighboring cells of the same type is consistent in different RGC types. Methods: Large-scale multi-electrode recordings of mid-peripheral RGCs of the four major types (ON and OFF parasol and midget) were obtained from macaque retinas ex vivo. Most of the RGC populations examined formed nearly complete mosaics. The location and strength of input of individual cones to the RF of each RGC were estimated using high-resolution white noise stimuli. This provided a spatial map of the input from ~1500 cones to mosaics of ~170 RGCs of the four major types over a ~500 µm region of retina. A low-resolution RF measurement was also obtained using coarser white noise stimuli. Results: (1) On average, RFs of all four RGC types exhibited spatial profiles consistent with the DOG model, as expected. However, the profiles of individual cells, measured at single cone resolution, deviated strongly from this model. Qualitatively, these deviations manifested as individual weak cone inputs, or patches of weak cone inputs, intermingled with individual strong cone inputs, or patches of strong cone inputs. Examination of simultaneously recorded RGCs of other types indicated that weak cone inputs were not entirely attributable to cone damage. (2) In most cases, midget cells shared a smaller fraction of cone inputs with immediate neighbors of the same type than did parasol cells. This finding differs from results obtained previously using coarse RF measurements. However, the degree of cone sharing varied between preparations, particularly in midget cells. Conclusions: (1) spatial RFs of midget and parasol cells deviate strongly from the DOG model at single cone resolution, and (2) midget cells generally share fewer cones with their neighbors than parasol cells. In general, measurements at single cone resolution reveal spatial properties of cone sampling not observed with more standard coarse measurements.

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RETINAL DISEASES AND THERAPIES SESSION 5) Genetic AMD-risk factors promote pathogenic subretinal inflammation Florian Sennlaub Institut de la Vision, Paris, France

Age-related macular degeneration (AMD) is a highly heritable major cause of blindness characterized by subretinal inflammation. Of all genetic factors, variants of Complement factor H (CFH) are associated with greatest linkage to AMD. Using loss of function genetics and orthologous models of AMD, we provide mechanistic evidence that deficiency in CFH completely prevents pathogenic subretinal accumulation of mononuclear phagocytes (MP) and accelerates resolution of inflammation. We show that MP-persistence arises secondary to binding of CFH to CD11b/CD18, which obstructs physiologically-occurring thrombospsondin-1 (TSP-1)-CD47-mediated elimination of MPs from the subretinal space. The AMD-associated CFH402H isoform markedly increased this inhibitory effect on microglial cells, indicating a causal link to disease etiology. Pharmacological activation of CD47 accelerated resolution of both subretinal and peritoneal inflammation, which may be exploited in the therapy for chronic inflammatory diseases, including AMD.

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6) Cellular Senescence and Dormancy in Retinopathy Przemyslaw Sapieha, Malika Oubaha, Agnieszka Dejda, Khalil Miloudi, Frédérique Pilon, Frédérick A Mallette Department of Ophthalmology, Maisonneuve-Rosemont Hospital Research Centre University of Montreal, Montréal, Canada

Pathological retinal neovascularization is the hallmark of primary blinding diseases across all age groups, yet surprisingly little is known about the causative factors. These diseases include diabetic retinopathy and retinopathy of prematurity where progressive decay of retinal vasculature yields zones of neural ischemia. These avascular zones and the hypoxic neurons and glia that reside in them are the source of pro-angiogenic factors that mediate destructive pre-retinal angiogenesis. Central neurons such as retinal ganglion cells (RGCs), which are directly apposed to degenerating vasculature in ischemic retinopathies, require stable metabolic supply for proper function. However, we unexpectedly found that RGCs are resilient to hypoxia/ischemia and a generally compromised metabolic supply and instead of degenerating, trigger protective mechanisms of cellular senescence and RGCs enter a state of cellular dormancy. Paradoxically, while potentially favoring neuronal survival, the senescent state of RGCs is incompatible with vascular repair as they adopt a senescence-associated secretory phenotype (SASP) that provokes release of a secretome of inflammatory cytokines that drives paracrine senescence and further exacerbates pathological angiogenesis. The mechanisms that lead to retinal cellular senescence and dormancy as well as the therapeutic potential of targeting these pathways will be discussed.

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7) Photoreceptor transplantation: Marker-free identification of photoreceptors by mechanical phenotyping Marius Ader1, Tiago Santos-Ferreira1, Maik Herbig2, Oliver Otto2, 5, Madalena Carido1, 3, Mike O Karl1, 3, Stylianos Michalakis4, Jochen Guck2 1

CRTD/Center for Regenerative Therapies Dresden, Technische Universität Dresden, Fetscherstrasse 105, 01307 Dresden, Germany 2 Biotechnology Center, Technische Universität Dresden, Tatzberg 47-49, 01307 Dresden, Germany 3 German Center for Neurodegenerative Diseases (DZNE), Dresden, Germany 4 Center for Integrated Protein Science Munich (CiPSM), Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-Universität München, Munich, Germany 5 Centre for Innovation Competence: Humoral Immune Reactions in Cardiovascular Diseases (HIKE), University of Greifswald, Greifswald, Germany

Photoreceptor transplantation represents a promising approach for the treatment of dysfunctional or lost photoreceptors in retinal diseases as demonstrated in preclinical animal studies. Vision improvement might be achieved either by transfer of cytoplasmic material from healthy donor to dysfunctional host photoreceptors or by cell replacement approaches. Furthermore, 3D cell culture protocols have been established that allow the production of retinal organoids to obtain high numbers of transplantable donor photoreceptors. Improved transplantation success and depletion of contaminating cells in donor cell suspensions is currently achieved by photoreceptor enrichment prior engraftment using flow cytometry or magnetic activated cell sorting. However, such purification methods require genetic modifications or the use of specific cell surface binding antibodies. An attractive alternative for donor cell enrichment is the use of inherent physical properties for cell identification. Therefore, we characterized mechanical parameters of rod photoreceptors isolated at different developmental stages from the mouse retina or derived from retinal organoids using real-time deformability cytometry, a high throughput, contactless microfluidic technique that measures morphological and rheological parameters such as size and elasticity of single cells. We provide evidence that rods become smaller and softer as development proceeds and that their mechanical properties are sufficient to identify them from other retinal cells, thus allowing future marker-free sorting of transplantable rod photoreceptors.

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8) Axon Regeneration in the visual system Homaira Nawabi Grenoble Institute of Neurosciences- Inserm U1216, Université Grenoble Alpes

Central nervous system (CNS) impairment leads to irreversible loss of cognitive and motor functions, as mature neurons do not regenerate. Thus, understanding the detailed mechanisms of axonal growth and repair remains one of the greatest neurobiological challenges. We are using the visual system as a window to the brain and spinal cord to understand the molecular mechanisms underlying adult neuronal growth and circuit formation. Those features are absolutely crucial to reach our ultimate goal of functional recovery. We recently used quantitative proteomics on purified adult retina ganglion cells to uncover the injury signature specifically within those neurons. This approach reveals two new critical factors involved in axon regeneration. First, we highlight that the transcription factor cmyc is essential for regeneration and that a combination of signaling pathways (mTOR, JAK/STAT and c-myc) promotes long distance regeneration. We also found a critical structural protein, doublecortin like kinase 2 (DCLK2) is essential for the very step of axonal growth: the formation of a new growth cone and participates both to neuroprotection and regeneration. Our studies open up new perspectives in the field of CNS regeneration such as circuit formation, axon guidance and recovery of lost functions.

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9) Visual system regeneration. Breaking and re-creating brain circuits for seeing Andrew Huberman Neurobiology & Ophthalmology, Stanford School of Medicine, Stanford, CA, USA

Mammalian retinal neurons do not regenerate after injury. I will present data describing how gene therapy combined with enhancements to neural activity can encourage regeneration of retinal ganglion cells (RGCs) in the adult animal. I will also address how axon guidance molecules that were present during development may be up-regulated after injury and serve to either enhance or restrict retargeting of RGC axons to their correct targets. Finally, I will review the design and preliminary results of a human pre-clinical trial aimed at regenerating RGCs in glaucoma patients.

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10) A fully organic retinal prosthesis restores vision in a rat model of degenerative blindness Maya-Vetencourt JF1, Ghezzi D1, Antognazza MR2, Colombo E1, Mete M3, Feyen P1, Desii A2, Buschiazzo A4, Di Paolo M5, Di Marco S5, Ticconi F4, Emionite L6, Shmal D1, Marini C7, Donelli I8, Freddi G8, Maccarone R8, Bisti S5, Sanbuceti G4, Pertile G3, Lanzani G2, Benfenati F1,9 1

Centre for Synaptic Neuroscience and Technology, Istituto Italiano di Tecnologia, Genova, IT; 2Centre for NanoScience and Technology, Istituto Italiano di Tecnologia, Milan, IT; 3Ophthalmology Department, Sacro Cuore Hospital, Negrar, IT; 4Department of health Science, Nuclear Medicine, University of Genova, IT; 5Department of Biotechnology and Applied Clinical Science, University of L'Aquilla, IT; 6Animal Facility, National Institute Cancer Research, IRCCS AOU San Martino, Genova, IT; 7Institute of Molecular Bioimaging and Physiology, CNR, Milan, IT; 8Innovhub-SSI, Silk Division, Milan, IT; 9Department of Experimental Medicine, University of Genova, Genova, IT

The degeneration of photoreceptors in the retina is one of the major causes of adult blindness in humans. Unfortunately, no effective clinical treatments exist for the majority of retinal degenerative disorders. We exploited organic electronics, in particular photovoltaic semiconducting polymers, for the generation of a fully organic retinal prosthesis, to restore light sensitivity in degenerate retinas. We found that primary neurons grown onto a semiconductor polymer (P3HT) coating a conductive indium-tin oxide layer are depolarized and fire action potentials by appropriate light stimuli with a spatial resolution in the order of the neuronal cell body. Light stimulation of degenerate retinas placed on the organic polymer in subretinal configuration showed that a light stimulus in the daylight range of intensity elicited intense spiking activity in retinal ganglion cells to levels indistinguishable from those recorded in control retinas. We now report on the fabrication and functional validation of a fully organic prosthesis made of P3HT as a photosensitive semiconductive layer, PEDOT:PSS as a conductive layer and silk fibroin as a biocompatible passive substrate for long-term in vivo subretinal implantation in the eye of Royal College of Surgeons rats, a widely recognized model of Retinitis pigmentosa. Electrophysiological and behavioral analyses revealed a prosthesis-dependent recovery of light sensitivity and visual acuity that persisted up to 6-10 months after surgery. The rescue of the visual function was accompanied by an increase in the basal metabolic activity of the primary visual cortex, as demonstrated by PET imaging. At the same time, we are currently exploiting CVD graphene as a highly flexible conductive layer to replace 26

PEDOT:PSS with graphene and improve photosensitivity/charge separation for novel prosthesis design. We found that the addition of the graphene layer did not alter the high biocompatibility of the multi-layer device with respect to primary neurons. Interestingly, graphene enhanced the light-induced effects of the interface on the membrane potential and firing properties of primary neurons, suggesting a high potential for in vivo retina applications. Our results highlight the possibility of developing a new generation of organic, highly biocompatible and functionally autonomous prostheses for subretinal implants to treat degenerative blindness.

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11) Optogenetics for vision restoration- translation from mice to primates Antoine Chaffiol, Hanen Khabou, Romain Caplette, Marcela Garita, Céline Jaillard, Elena Brazhnikova, Mélissa Desrosiers, Elisabeth Dubus, Olivier Marre, Ernst Bamberg, Jens Duebel, José-Alain Sahel, Serge Picaud, and Deniz Dalkara INSERM, U968, Paris, F-75012, France; Sorbonne Universités, UPMC Univ Paris 06, UMR_S 968, Institut de la Vision, Paris, F75012, France; CNRS, UMR_7210, Paris, F-75012, France

Optogenetics is a biological technique, which uses light to control neurons genetically modified to express light-sensitive membrane proteins. It has become a widely used strategy in research with therapeutic applications ranging from cardiology to neurology. In terms of its clinical application, vision restoration in blind patients has come first due to the existence of an optical window to the retina and thanks to established success of gene therapy in the ocular compartment. Microbial type opsins as well as vertebrate opsins have shown promise in restoring visual responses in blind rodents and other small animals. However, it was not clear if such treatments can be translated to humans. The major challenges in translating optogenetics from mice to man are related to the lack of optimized viral vectors for targeting various populations of neurons in the primate retina and to the dose extrapolation, going from small animal eyes with minimal immune responses to a large primate eye with immune surveillance. High-level opsin expression is necessary to generate light responses in blind retinas and achieving such expression levels in a large primate eye may come at the expense of a prohibitive viral dose triggering immune responses to both the vector and the transgene. In order to deal with these translational challenges, we studied combinations of adeno-associated virus (AAV) vectors and promoters to target optogenetic proteins to different subsets of neurons in the non-human primate retina. Our data show that successful expression of optogenetic proteins in the primate retina is cell type dependent. Furthermore, we highlight safety and efficacy of optogenetics for cell types that can be efficiently targeted with vector promoter combinations optimized for primates. Challenges remain for targeting certain neuronal populations with existing AAV technologies and for testing functional outcomes in vivo.

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13) Rhodopsin expression increases the resistance of mammalian M-cones to retinoid deficiency in LCA model Alexander Kolesnikov1, Chloe Potter1, David Razafsky1, Andrew Hughes2, Joseph Corbo2, Didier Hodzic1, Vladimir Kefalov1 1

Ophthalmology and Visual Sciences and Pathology and Immunology, Washington University School of Medicine, Saint Louis, MO

2

Leber Congenital Amaurosis (LCA) is a severe retinal dystrophy causing child blindness. Mutations in retinoid isomerase RPE65 account for up to 16% of LCA cases and lead to early loss of cones. The mechanisms of this disease are poorly understood. Cone pigments are generally less stable and rely on the supply of visual chromophore. We tested the hypothesis that the low stability of cone opsin makes mammalian cones susceptible to degeneration under conditions of sustained chromophore deprivation in the absence of RPE65. We generated a knockin mouse model expressing more stable rod pigment in cones instead of their native M-opsin (RhoKI). Cone expression of rhodopsin was confirmed in RhoKI-Rho-/- animals by IHC and further quantified by qRT-PCR. To induce chromophore deficiency, we generated RhoKI-Rpe65-/-Gnat1-/- mice and their Rpe65-/-Gnat1-/- controls lacking rod signaling. We then compared their cone numbers and M-cone ex vivo ERG responses (in presence of exogenous 9-cisretinal) at the ages of 1 or 5 months. The total level of rhodopsin mRNA in RhoKI-Rho-/- retinas was 15-fold lower than that of native M-cone pigment in control (Rho-/-) mice. This caused ∼40–100-fold desensitization of M-cones in RhoKI animals. However, their maximal cone response amplitude and amount of cone transducin were normal. Rpe65-/-Gnat1/mice had rapidly progressing degeneration of nearly all ventral and most dorsal cones. Notably, there was a substantial preservation of mid-dorsal cones in 5month-old RhoKI-Rpe65-/-Gnat1-/- animals. Furthermore, RPE65-deficient cones expressing rhodopsin had a 2-fold higher maximal response and 10-fold higher sensitivity than age-matched controls. Following a chromophore treatment, the overall physiological function of RhoKI-Rpe65-/-Gnat1-/- cones was only 2–3-fold lower than in RhoKI-Gnat1-/- animals with normal chromophore supply. This is in stark contrast to Rpe65-/-Gnat1-/- cones whose sensitivity was several orders of magnitude lower than that in Gnat1-/- mice. Cones expressing rhodopsin are viable and have robust phototransduction. The substitution of cone M-opsin with even small amount of rhodopsin increases the survival of cones and their function under severe chromophore deficiency caused by the loss of RPE65. This opens the potential for respective gene-therapy based approach for treating LCA and related retinal pathologies. 29

14) Preventing visual function loss in the rd10 mouse model of retinitis pigmentosa using gene editing Paola Vagni1, Laura E Perlini2, Martina Parrini2, Andrea Contestabile2, Laura Cancedda2, and Diego Ghezzi1 1

Medtronic Chair in Neuroengineering, École Polytechnique Fédérale de Lausanne (EPFL) STI IBI-STI LNE, 1015 Lausanne, Switzerland. 2 Local micro-environment and Brain Development Laboratory, Neuroscience and Brain Technology Department, Istituto Italiano di Tecnologia (IIT), 16163 Genova, Italy.

Currently, there is no known cure for retinitis pigmentosa (RP). Even if some treatments can slow down the progression of the disease, none of them can effectively stop retinal degeneration. This study exploits the possibility of an early intervention in photoreceptor progenitors aiming at preventing cell death. For this purpose, we selected the rd10 mouse model, which carries a point mutation in a gene associated with human RP. We designed a CRISPR/Cas9 gene editing system to repair the mutation taking advantage of the increased activity of the homologous directed repair mechanism in dividing cells. The efficiency of the editing system (composed of guide RNA, Cas9, and DNA repair template) was first tested in vitro in neural progenitor cells derived from rd10 mice (52.8±11.1%, n=3). The constructs were then injected in vivo in the subretinal space of rd10 pups either at postnatal day (P) 3 (early treated, ET) or at P8 (late treated, LT); we also tried a P3-P8 combined treatment (multiple treated, MT). One eye was injected, while the other one was kept as internal control. The injection was followed by electroporation (electric field: 40 V/cm). Histological analysis of the eyes showed GFP expression in the photoreceptors layer starting from 2 days after electroporation. The visual acuity was measured at P30, P60 and P90 with the optomotor response test in ET, MT, LT, sham treated (ST), non-treated rd10 (NT), and WT (WT) mice. The treated eye showed a higher visual acuity than the control eye in ET, MT and LT for all the time points tested, despite a decreased visual acuity at P90 (p<0.01, one-tailed Student’s t-test). ET, MT, and LT mice had a significantly higher visual acuity compared to ST and NT mice for all the time points tested (p<0.01, one-way ANOVA + Tukey). Moreover, in order to test the integrity of the cortical visual pathway, the flash visually evoked potentials (fVEPs) were recorded from the visual cortex of ET, MT, LT, ST, NT, and WT mice at P90. We observed a partial recovery of the light-evoked response in the visual cortex of ET and LT mice (n.s and p<0.05; one-way ANOVA + Tukey), compared to ST and NT mice. Our results strongly suggest a positive effect of the CRISPR/Cas9-based therapy on photoreceptors survival in our model of RP. In the future, we would like to perform 30

additional morphological analyses to better understand the correlation between the injection site in the retina and the specificity of the targeted visual circuits.

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15) Impairment of dopaminergic circuitries, ganglion cells loss and Lewy bodies containing p-alpha synuclein were found in human retinas in Parkinson's desease N. Cuenca1 I. Ortuño-Lizarán1, X. Sánchez-Sáez1, G.E. Serrano2, D.G. Walker4, C.H. Adler3, T.G. Beach2 1

Department of Physiology, Genetics and Microbiology, University of Alicante, Spain Banner Sun Health Research Institute, Sun City, Arizona, USA 3 Mayo Clinic Arizona, Arizona, Scottsdale, USA 4 Arizona State University, Tempe, Arizona, USA 2

Purpose: Parkinson disease (PD) is one of the most common neurodegerative diseases and it is mainly characterized by movement disorders. Among a variety of secondary simptons visual alterations have been described, indicating that the retina could be affected by PD. The aim of this work is to characterize some of the cellular alterations that occur in PD retinas and could lead to the reported visual impairments Methods: Wholemount PD and control retinas and brains were immunostained against posphorylated-α-synuclein. The number of Lewy bodies in both tissues was compared with the Spearman correlation test. Flat retinas were also counterstained with Hoescht and cellular density in the ganglion cell layer (GCL) was calculated. Immunostain of dopaminergic and AII amacrine cells was done using antibodies against tyrosine hydroxilase and calretinin respectively. Cell morphology, density and synaptic connectivity were assessed. Results: Posphorylated-α-synuclein was found in the retina within ganglion cells, axons, dendrites and in structures resembling Lewy Bodies. Its load strongly correlated between retina and brain (r=0,91; p<0,001): those subjects with a high amount of Lewy bodies in the brain also had a high number of them in the retina, and vice-versa. Cells in the GCL were loss in the whole retinal length, from center to periphery, with a more marked decrease in the central retina (p<0,05). Dopaminergic cells in PD retinas had swollen and shorter dendrites and their plexus were reduced. Number of dopaminergic cells and synaptic contacts with AII amacrines were diminished (p<0,001), indicating alterations in the dopaminergic circuitries. Conclusions: Accumulation of p-α-synuclein, impairment of the dopaminergic circuit and loss of cells in the GCL indicate that PD affects the retina and may explain some of the visual alterations detected in patients. Also, the significant pα-synuclein correlation suggest that the synucleinopathy occurs in the retina in a way analogous to the brain and, because of that, the retina could act as a window 32

into the brain and be an useful tissue or biomarker to study disease presence and progression. Support: Michael J. Fox Foundation for Parkinson’s Research; Generalitat Valenciana Prometeo/2016/158, MINECO-FEDER-BFU2015-67139-R; MECD FPU 14/03166

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RETINAL IMPACT ON EYE DEVELOPMENT AND MYOPIA SESSION 16) Melanopsin-based photoreception in fetal and newborn mice: Actions on behavior and vascular development David Copenhagen1, Shruti Vemaraju2 , Jan Verweij1 , Shawnta Y. Chaney1, Anton Delwig1, William E. Miller2, Gabriele M. König3, Evi Kostenis3 , Sujata Rao2, Richard Lang2 1

University of California, San Francisco, 2University of Cincinnati, 3University of Bonn

A class of ganglion cells that express melanopsin are intrinsically photosensitive In adult mammals, including primates, light activation of these mRGCs can regulate reflexive and adaptive functions such as photoentrainment of circadian rhythms and light induced pupillary constriction. Developmentally, mRGCs are born well before rods and cones. In fetal mouse eyes mRGCs are born 9 days before birth, which is 19 days before visual signaling from rods and cones emerges. In human eyes, these mRGCs are born 22 weeks before birth. Given the possibility of visual signaling in the eye during embryonic and early postnatal maturation, we have addressed three issues: 1) Are mRGCs in the embryonic retina actually light responsive? 2) What are the transduction pathways activated by melanopsin in the embryonic eye? 3) Can visual signaling by mRGCs regulate eye development and behavioral responses to light? 1) We expressed the calcium indicator GCaMP3 and GCaMP6 in embryonic mRGCs by crossing melanopsin cre mice with Ai38 and Ai96 (floxed GCaMP3,6) mice. Light-induced calcium rises could be recorded in ex vivo retinas from fetal mice as young as embryonic day 16, directly demonstrating these mRGCs are light responsive at least 4 days before birth. 2) There are conflicting reports in adult mice whether melanopsin is coupled to the Gq/11 family of G-proteins. We find that fetal light responses are virtually abolished in two different Gq antagonists supporting a role for melanopsin coupling to Gq/11 G-proteins. 3) We find that light activation of melanopsin in fetal eyes regulates normal postnatal regression of hyaloid vasculature in the eye. Fetal mouse pups deprived of light until after birth exhibit delayed regression of hyaloid vasculature. Genetic deletion of melanopsin phenocopied the effects of light deprivation. Light elicits an aversive response in young mouse pups. At P6 mouse pups will turn away from blue light and issue ultrasonic vocalizations. At P1 mouse pups show increased random body movements to light. Body movements at P1 and both avoidance 34

locomotor movements and ultrasonic vocalizations at P6 are eliminated in melanopsin knock out mice. Thus, blue light activation of melanopsin in utero can regulate vascular development in the eye. Similarly blue light can produce anxiety-like responses at ages as young as one day after birth. These findings support the concept that light exposure of pregnant mothers during pregnancy, and lighting in NICUs, which house infants as young as gestational week 24 could have profound effects on early growth and emotional health of human infants.

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17) Retinal control of myopia - lenses, light and atropine Frank Schaeffel Section of Neurobiology of the Eye, Ophthalmic Research Institute, University of Tuebingen, Germany

To achieve high visual acuity, the image projected on the retina must be in good focus. Therefore, the position of the plane of focus must be matched during postnatal development to the position of the photoreceptor layer with a tolerance about 100 µm in the human eye. Experiments in animal models have shown that this process is controlled by the retina. The retina can detect not only the amount but also the sign of defocus, with an integration time of about 2 minutes (in the chicken model). The average defocus is used as error signal for visually feedbackcontrolled fine tuning of eye growth. Nevertheless, childrens’ eyes often grow too long during school times (1 mm longer = 2.7 diopters of myopia). Myopia is on the rise worldwide, linked to visual experience associated with extensive education, and it is expected that half of the world population will be myopic by 2050. Fortunately, several attempts to “tell the retina” that it should keep the eye shorter show some success. (1) Adding a secondary near focal plane by using multifocal contact lenses is interpreted by the retina as a too long eye and results in reduced myopia progression. (2) Exposing kids more often to outdoor lighting releases more dopamine from the retina (a known eye growth inhibitor), delays myopia onset and results therefore in lower endpoints of myopia when they are young adults. (3) Atropine, applied as eye drops at very low doses (0.01%) inhibits axial eye growth with little side effects by yet unknown mechanisms - for instance, atropine releases dopamine from the retina, as well as nitric oxide (a retinal light signal like dopamine), enhances contrast sensitivity and inhibits scleral growth directly - but apparently not via muscarinic mechanisms. (4) There are a number of other tricks to modify the defocus error signal. For instance, long wavelength light (>650 nm) makes the primate eye hyperopic (not myopic, as expected from chromatic aberration), and predominant ON stimulation has been shown to inhibit myopia in the model of the chicken.

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18) Contributions of the three photoreceptor pathways to refractive eye growth and myopia in mice Machelle T. Pardue1,2, Ranjay Chakraborty2,3, Erica Landis2,3, Hanna Park2,3, Richard A. Stone4, P. Michael Iuvone3,5 1

Wallace H. Coulter, Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA; 2Rehab R&D Center of Excellence, Atlanta VA Medical Center, Decatur, GA; 3Ophthalmology, Emory University School of Medicine, Atlanta, GA; 4 Ophthalmology, University of Pennsylvania, Philadelphia, PA; 5Pharmacology, Emory University School of Medicine, Atlanta, GA, United States

The specific retinal pathways that detect visual input and initiate the signaling for refractive eye growth are unknown. We evaluated the potential contribution of the three photoreceptor pathways: rods, cones, and intrinsically photosensitive retinal ganglion cells (ipRGCs) by evaluating refractive development and the response to form-deprivation (FD) in mice with mutations in these photoreceptors. Refractive development under normal laboratory conditions was measured biweekly from post-natal day (P) 28 to P112 in mice with non-functional rods (Gnat1-/-), cones (Gnat2-/-), or ipRGCs (Opn4-/-), compared to wild-type (WT) age-matched controls. Refraction was measured using an automated photorefractor, corneal curvature with keratometry, and ocular parameters (axial length, lens thickness, etc) with spectral-domain optical coherence tomography. Separate cohorts of mice wore a unilateral diffuser goggle to induce monocular FD myopia for three weeks starting at P28 and were tested weekly. Gnat1-/- mice had abnormal refractive development with no change in refractive error from P28-P84 when housed under normal laboratory conditions, while WT counterparts showed a shift towards hyperopia that plateaued about P42. Additionally, FD had no effect on Gnat1-/- mice, while WT mice showed significant myopic shifts. In contrast, Gnat2-/- mice showed a similar refractive development curve to WT controls under normal laboratory conditions. FD produced greater myopic shifts in Gnat2-/- mice compared to WT controls (p<0.04). With unimpaired visual input, Opn4-/- mice had abnormal refractive development with more myopic refractions than their WT counterparts from P28 to P56 and more hyperopic refractions than WT controls from P56 to P112. After FD, goggled Opn4/- mice showed a significantly greater myopic shift than WT controls (p<0.001). Our findings suggest that photoreceptor pathways differentially influence refractive development. Disruption of rod or ipRGC pathways significantly altered refractive development under normal and FD conditions, although in different directions. Surprisingly, disrupted cone pathways which are needed for high 37

acuity vision in primates did not affect refractive development under normal conditions and had minimal effects on the response to FD. We will discuss the potential role of dopamine as a stop signal for refractive eye growth and its regulation by these pathways.

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RETINAL CIRCUITS SESSION 19) How do ribbon synapses use multivesicular release to encode visual information? Ben James, Jose Moya-Diaz, Lea Darnet and Leon Lagnado School of Life Sciences, University of Sussex, Brighton, BN1 9QG, UK

The fundamental unit of information transfer between cells in a neural circuit is the synaptic vesicle containing neurotransmitter. In the retina, the output from ribbon synapses of bipolar cells (BCs) occurs both as single quanta and by a process of multivesicular release (MVR) in which two or more vesicles can be released simultaneously. What is the role of MVR in encoding a visual stimulus? To investigate this question we are using zebrafish larvae expressing the fluorescent glutamate sensor iGluSnFR, allowing us to monitor both uniquantal release and MVR in vivo using multiphoton microscopy (see also posters by Darnet et al., James et al. and Seibel et al.). We find that MVR is fundamental to the transmission of the visual signal from bipolar cells to inner retina : at high contrasts the large majority of release events from OFF terminals have amplitudes equivalent to two or more vesicles. To investigate the information encoded by MVR we began by characterising the temporal filters leading to uniquantal and multiquantal transmission by reverse correlating these events with a ‘white noise’ stimulus. Events with higher quantal content were triggered by intensity changes that were on average larger and faster than those triggering release of single vesicles. These results indicate that MVR encodes higher contrasts with higher temporal precision than uniquantal release. To investigate the role of MVR further, we used an approach based on information theory. Stimuli of varying contrasts were applied over multiple trials at a fixed frequency of 5 Hz and events occuring in 10 ms time-windows were counted. This short time-window was chosen so that only 1 event occured within any single sample. We then calculated the joint probablity p(r, s) between the stimulus of differing contrasts (s) and response (r). This procedure was repeated for responses categorized as single quanta and two, three four quanta etc. Events containing more vesicles conveyed more bits of information about the contrast of the stimulus. We then calculated how many bits were transmitted per vesicle and found that this also increased systematically with the number of vesicles released. For instance, vesicles contained within five-quantal events transmitted an average of twice as much information as single quantal events. 39

These results demonstrate that MVR increases both the temporal accuracy and efficiency with which synapses transmit visual information.

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20) An auto-regulated gap-junction network of amacrine cells controls nitric oxide release in the inner retina Jason Jacoby1, Amurta Nath2, Zachary Jessen3, Gregory Schwartz1,4,5 1

Department of Ophthalmology, Feinberg School of Medicine, Northwestern University Northwestern University Interdepartmental Neuroscience Program, Feinberg School of Medicine, Northwestern University 3 Medical Scientist Training Program, Feinberg School of Medicine, Northwestern University 4 Department of Physiology, Feinberg School of Medicine, Northwestern University 5 Department of Neurobiology, Weinberg College of Arts and Sciences, Northwestern University 2

Circuits throughout the nervous system are regulated by both fast inhibition and the slower effects of neuromodulators. In some circuits, neuromodulators change the effective coupling between neurons by altering the conductance of gap junctions. This motif is best studied in the mammalian retina, where dopamine and nitric oxide (NO) regulate separate gap junction networks. While dopaminergic amacrine cells, the dopamine source of the retina, are well studied, much less is known about the NO source of the retina. We characterized the light responses and voltage-gated calcium influx in NO-releasing amacrine cells (NOACs) in the mouse retina to reveal how these cells are specialized to release NO under particular stimulus conditions. Anatomical reconstructions revealed that NOACs form a massive, homologous gap junction network. We discovered that coupling in the NOAC network is itself regulated by NO. Cable modeling based on our anatomical and electrophysiological measurements suggested that the novel form of auto-regulation in the NOAC network serves to shape the amplitude and spatial spread of NO in the retina depending on light conditions.

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21) Dissecting motion processing circuits in the retina Daniel Kerschensteiner Neuroscience, and Biomedical Engineering, Washington University School of Medicine, USA

I will discuss work from my group on object motion sensitive (OMS) circuits in the mouse retina. We identified and characterized two amacrine cell types that serve complementary functions in these circuits, one amplifying ganglion cell responses to local (i.e. object) motion and the other suppressing ganglion cell responses to global motion stimuli, which occur during eye movements. We analyzed the mechanisms by which the two amacrine cell types distinguish local and global motion using targeted patch clamp recordings and functional two-photon imaging, and employed cell-type-specific silencing and deletion to study their importance for OMS signals sent to the brain and for behaviors relying on these signals.

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22) Visual feature extraction in the mouse inner retina Katrin Franke Bernstein Centre for Computational Neuroscience, Centre for Integrative Neuroscience, Institute for Ophthalmic Research, Tübingen, Germany

Visual processing begins in the retina: within only two synaptic layers, multiple parallel channels emerge, which relay highly processed visual information to different parts of the brain. The origin of this functional diversity lies in the retina’s second synaptic layer, the inner plexiform layer (IPL), where bipolar cells (BCs), amacrine cells (ACs) and retinal ganglion cells (RGCs) form complex interconnected networks. We use two-photon microscopy to record light stimulus-evoked calcium and glutamate population activity at different levels of the mouse retina. This allows following the complete visual signal across consecutive processing stages in a systematic way. In my presentation, I will summarise our recent findings on the functional diversity of BC (Franke, Berens et al., 2017) and RGC types (Baden, Berens, Franke et al., 2016) present in the mouse retina and focus on how individual visual information channels emerge within the inner retinal network.

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23) Synaptic transfer between ON and OFF visual channels mediated by AII amacrine cells in the mouse retina Cole W. Graydon1, Evan E. Lieberman2, Nao Rho2, Kevin L. Briggman3,4, Joshua H. Singer2, and Jeffrey S. Diamond1 1

Synaptic Physiology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892 USA 2 Department of Biology, University of Maryland, College Park, MD 20742 USA 3 Circuit Dynamics and Connectivity Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892 USA 4 Department of Biomedical Optics, Max Planck Institute for Medical Research, Heidelberg 69120, Germany

In the mammalian retina, AII amacrine cells relay rod signals to the ON and OFF cone pathway. The synaptic inputs to AIIs from (ON) rod bipolar cells have been studied extensively, as have the biophysical properties of AIIs, but AII outputs to ON and OFF cone bipolar cells, mediated by electrical and chemical synapses, respectively, are poorly understood. Here, we have evoked AII-mediated synaptic inputs in ON and OFF cone bipolar cells by stimulating individual rod bipolar cells presynaptic to the AIIs. We find that AIIs transmit different components of the rod bipolar cell input to ON and OFF cone bipolar cells. Anatomical reconstruction of synaptic connectivity within this circuit indicates that AIIs preferentially contact a subset of OFF cone bipolar cells. These results identify synaptic and circuit features that likely distinguish ON and OFF signals during night vision.

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24) Development of the photoreceptor ribbon synapse in the absence of horizontal cells Lena Nemitz1,2, Jasmin Segelken1,2, Konrad Schultz1, Karin Dedek1,3,4, Ulrike Janssen-Bienhold1,2,4 1

Neurobiology, Department of Neuroscience, University of Oldenburg, Germany Visual Neuroscience, Department of Neuroscience, University of Oldenburg, Germany 3 Neurosensorics, Institute for Biology and Environmental Studies, University of Oldenburg, Germany 4 Research Center Neurosensory Science, University of Oldenburg, Germany 2

Horizontal cells represent a class of interneurons in the mammalian retina that form triad synapses with photoreceptors and ON bipolar cells in the outer plexiform layer (OPL). During retinal development, two horizontal cell processes invaginate into the photoreceptor terminal and occupy the positions lateral to the presynaptic ribbon. Subsequently, one or two central ON bipolar cell dendrites are added and complete the triad configuration. To study the role of horizontal cells in the formation of the photoreceptor ribbon synapse, we used a mouse line which expresses the primate diphtheria toxin receptor (DTR) under the control of the connexin57 (Cx57) promoter. This mouse line enabled us to specifically ablate horizontal cells during early postnatal development via the injection of diphtheria toxin (DT) at postnatal day 4 (P4) and P5. Immunohistochemical analysis at P8 revealed rod and cone terminals in the OPL of DT-treated Cx57+/+ and Cx57+/DTR mice. Labeling for the synaptic ribbon marker CtBP2 showed a punctate pattern within this layer in both genotypes. However, the morphology of the rod/rod bipolar cell synapse was affected by early postnatal horizontal cell ablation. At P15, when the formation of triad synapses is largely completed in wild-type mice, Cx57+/DTR mice showed abnormal ribbon structures, a reduced number of photoreceptor terminals in the OPL and ectopic photoreceptors terminals and bipolar cell dendrites in the outer nuclear layer. Rod spherules only partially retained their association with rod bipolar cells. A strong reduction in the immunoreactivity for the mGluR6 macromolecular complex suggests that synaptic transmission between photoreceptor terminals and ON bipolar cells is disturbed. In contrast, GluR5-positive structures were present at the base of cone pedicles indicating that flat contacts between cones and OFF bipolar cells are formed in the absence of horizontal cells. To evaluate the effects of early postnatal horizontal cell ablation on retinal function, we measured electroretinograms from DT-treated Cx57+/+ und Cx57+/DTR mice at P53. Consistent with our immunohistochemical findings, b-wave amplitudes of horizontal cell45

ablated mice were severely reduced under scotopic conditions demonstrating that the retinal ON pathway is impaired. In summary, our results suggest that horizontal cells affect the establishment of synaptic contacts between photoreceptors and ON bipolar cells in the OPL. We gratefully acknowledge funding from the Deutsche Forschungsgemeinschaft (grant DE1154/3-1 to KD and UJB; RTG 1885/1 stipend to LN)

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25) Complexity and dynamics of inhibitory circuits shape the directional code of the retina Lea Ankri, Nathali Kaushansky and Michal Rivlin Weizmann Institute of Science, Rehovot, Israel

Direction selective retinal ganglion cells (DSGCs) fire robustly in response to motion in one (preferred) direction, and poorly to motion in the opposite (null) direction. Starburst amacrine cells (SACs) have been shown to mediate this computation via two mechanisms – they form asymmetric connections onto DSGCs; and their processes display directional preference. Surprisingly, we have found that DSGCs can overcome the circuit’s anatomy and reverse their directional preference following the presentation of a short repetitive visual stimulus. Here, we use this reversal in DSGC directional preference as an experimental model to discover new mechanisms that contribute to the computation of motion direction in the retina. Using two-photon targeted patch clamp techniques and various moving stimuli, we verify that SACs’ processes display directional preference, with larger response amplitudes to centrifugal (away from cell soma) vs. centripetal (towards cell soma) motion. Following repetitive stimulation, SACs lose this directional preference and display similar response amplitudes in both directions. Instead, their response phase changes to support the reversed computation in DSGCs. As the directional preference of SAC processes is thought to be most significant to DSGCs’ responses during local motion, we continue to explore whether repetitive stimulation changes DSGCs’ directional preference during global motion. Global motion stimulation uncovers multiple response phases in DSGCs that may be tuned to opposite directions. Following repetitive stimulation the null-tuned response phases are strengthened, therefore the weights of these phases contribute more to the overall response. Our results reveal new mechanisms that act in orchestra to support the computation of motion direction in DSGCs, and add to recent findings demonstrating that the computations performed by anatomically-defined neuronal circuits can be altered by dynamic circuit mechanisms.

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26) Role of the GABAA α3 receptor as a developmental organizer of retinal inhibitory synapses Mrinalini Hoon, Clare Gamlin, Raunak Sinha, Will Grimes, Wan-Qing Yu, Adam Bleckert, Fred Rieke and Rachel O Wong University of Washington, USA

Distinct α-subunits within the GABAA receptor pentamer dictate functional characteristics of the receptor. In the mature vertebrate retina, GABAergic inhibition onto the axon terminals of bipolar cells is predominantly mediated by GABAAα1 receptors. However, we recently found that these synapses undergo a developmental switch in α-subunit expression before eye--opening: GABAAα3 receptors show high expression transiently during early development, which is downregulated as GABAAα1 receptor expression increases just before eyeopening. Using transgenic approaches, we asked whether: (i) downregulation of GABAAα3 receptors and upregulation of GABAAα1 receptors are inter--dependent, or regulated separately, and (ii) whether these synaptic changes occur at the same contact or signify the remodeling of synaptic connectivity during development. To answer these questions, we focused on a well characterized and stereotypic retinal synapse, between the GABAergic A17 amacrine cell and the rod bipolar cell. We demonstrate that the A17-rod bipolar synapses accrue GABAAα3 before eye--opening, which is replaced by GABAAα1 as the circuit matures. Using GABAAα3 knockout mice, we found that GABAAα3 receptor expression during development is necessary for the emergence of GABAAα1 receptors on rod bipolar terminals as GABAAα1 expression on retinal bipolar axon terminals is severely downregulated in the GABAAα3 knockout. In contrast, deleting GABAAα1 specifically from retinal bipolar cells does not affect the developmental downregulation of GABAAα3 receptors. We then asked whether GABAAα3 influences the developmental recruitment of other inhibitory receptor types in the retina. Indeed, mouse ON--sustained alpha ganglion cells co--express receptor clusters containing both GABAAα3 and Glycine receptor α1 subunits. Deleting GABAAα3 during retinal development impairs the recruitment of glycine receptor α1 subunits to these unusual ‘mixed’ synapses. Thus, GABAAα3 plays an unexpected and crucial role in establishing the appropriate receptor types and complex postsynaptic arrangements at diverse retinal inhibitory microcircuits.

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27) Endocannabinoids modulate RGC physiology through parallel modulation of TRPV1 and cannabinoid receptors David Krizaj1, Andrew O. Jo1, Jennifer M. Noel2, Jan Verweij3, Daniel A. Ryskamp1, David R. Copenhagen3, Maureen A. McCall2 and Monika Lakk1 1

Dept. of Ophthalmology & Visual Sciences, University of Utah, Salt Lake City UT, USA; Dept. of Anatomical Sciences, University of Louisville, Louisville KY, USA; 3 Dept. of Ophthalmology, University of California San Francisco, San Francisco CA, USA 2

Endocannabinoids (eCBs) and their receptors are strongly expressed in the retina, modulate night vision and were implicated in the etiology of blinding diseases (glaucoma, ischemia, diabetic retinopathy) but it is not known how they modulate retinal circuits. We show that anandamide (N-arachidonoyl ethanolamide, AEA) and 2arachidonoyl glycerol (2-AG), the two most prominent endogenous retinal eCBs, potently regulate RGC calcium homeostasis and excitability through parallel stimulation of TRPV1 (transient receptor potential vanilloid isoform 1) and CB1Rs (cannabinoid receptor type 1). Exposure to eCBs transiently elevated [Ca2+]i in a subset of RGCs. This effect that was absent in RGCs from TRPV1 knockout animals, inhibited by TRPV1 antagonists and suppressed by preincubation with 2-AG which predominantly targets CB1Rs. An increase in [cAMP]i blocked the effect of 2-AG whereas exposure to pertussis toxin and cAMP inhibitors mimicked the effect of CB1R activation on TRPV1-mediated calcium influx. Analysis of TRPV1 expression using transgenic Trpv1Cre:Ai9 reporter mice and animals injected with AAV1.CAG.Flex.tdTomato revealed that TRPV1 expression is toxin and cAMP inhibitors mimicked the effect of CB1R activation on TRPV1-mediated calcium influx. Analysis of TRPV1 expression using transgenic Trpv1Cre:Ai9 and Trpv1Cre :AAV1.CAG.Flex. tdTomato reporter mice showed expression in a subset of RGCs, all of which expressed CB1Rs. TRPV1 :tdTomato+ cells showed nonuniform distribution with peak density in the mid-periphery. Dendritic stratification, somatic size and expression of specific molecular markers identified the main expressors of TRPV1 to be M1 & M2 ipRGCs and medium-field B3 RGCs. Calcium imaging from Opn4Cre+/- Ai96 GCaMP3 reporter mice confirmed that TRPV1 agonists modulate the light response in subsets of the intrinsically photosensitive cells targeting the suprachiasmatic nucleus (SCN) but not the shell of the optokinetic nucleus (OPN) or the dorsal lateral geniculate nucleus (LGN). These results suggest that activity-dependent eCB release modulates the visual 49

signal gain in RGCs through parallel stimulation of nociceptive TRPV1 channels and CB1R GPCRs, resulting in dynamic time-dependent gating of calcium homeostasis and RGC output bandwidth. The findings are the first to identify endogenous retinal activators of TRPV1, localize the channel to identified subsets of RGCs and to implicate an identified TRP channel isoform in the modulation of one aspect of intrinsically photosensitive signaling (circadian photoentrainment vs. pupillary reflex).

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28) Zebrafish colour vision: anisotropic retinal circuits match asymmetric spectral content in natural light Maxime Zimmermann1, Noora E Nevala1, Takeshi Yoshimatsu1, Daniel Osorio1, Dan-Eric Nilsson2, Philipp Berens3,4 and Tom Baden1,3,4 1

School of Life Sciences, University of Sussex, UK; Lund Vision Group, University of Lund, Sweden; 3 Institute of Ophthalmic Research, University of Tuebingen, Germany; 4 Bernstein Centre for Computational Neuroscience, University of Tuebingen, Germany 2

Sensory systems evolve to best serve animals’ requirements. Driven by the statistics of the natural physical world an animal inhabits, yet constrained by preexisting circuit motifs and metabolic cost, sensory networks tune their available neuronal real estate to prioritise behaviourally important computations. In vision, specialisations tend to come in hand with specific regions in visual space. Here we show how achromatic and chromatic retinal circuits of larval zebrafish are both anatomically and functionally highly anisotropic to best process “colour” around a chromatically rich natural visual horizon, while prioritising UV- and red-biased achromatic contrasts on the sky and ground, respectively. Moreover, we suggest how zebrafish UV vision not only serve to expand spectral sampling, but more directly aides to spot prey and avoid predators. First, hyperspectral scans of the zebrafish underwater natural visual world in shallow freshwater streams and ponds of the Indian subcontinent revealed a striking chromatic hotspot around the underwater horizon, separated by a spectral gradient from short- to long wavelengths with decreasing visual elevation. Next, whole-eye quantification of genetically labelled cone distributions revealed that out of the zebrafish’s 4-cone and 1-rod complement, only green cones showed no major anisotropy. All other photoreceptor types systematically varied with retinal position in a pattern consistent with the distribution of natural light. Next, we used 2-photon in vivo imaging of bipolar cells expressing GCaMP6f to survey inner retinal chromatic responses. Recordings were taken across the entire depth of the IPL and at all retinal positions around a fixed eccentricity. This revealed that chromatic and achromatic bipolar cell responses fell into distinct layers of the IPL, and their nature varied systematically with the corresponding position in visual space. Like natural light, chromatic circuits dominated the visual horizon, but were more achromatic and UV-biased above yet red-biased below the animal.

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29) Probing the intraretinal influences of ipRGCs using chemogenetic manipulation Nina Milosavljevic1, Annette E. Allen1, Riccardo Storchi1, Jasmina CehajicKapetanovic1,2, Robert J. Lucas1 1

Faculty of Biology, Medicine and Health Manchester Royal Eye Hospital, CMFT, Manchester Academic Health Sciences Centre

2

Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) are thought to provide the retina’s ability to measure background light intensity (irradiance). In order to explore the intraretinal influence of ipRGCs we adopted a chemogenetic approach to selectively and acutely activate ipRGCs. Using this approach we were able to determine the effect of activating ipRGCs on the retinal light response by electroretinography (ERG) and relate this to retinal cell activation by c-Fos mapping. Melanopsin-Cre mice were intravitreally injected with a Cre-recombinasedependent viral vector to express engineered excitatory chemogenetic hM3Dq receptors in ipRGCs. We previously showed that using this approach and applying a drug that specifically activates hM3Dq receptors (clozapine N-oxide), we can recreate the effects of bright light to this cell class only (ipRGCs) in dark-housed mice1. Flash ERG was performed across the 9-fold intensity range on hM3Dq mice before and after chemogenetic activation of ipRGCs. Chemogenetic activation of ipRGCs suppressed a- and b-wave amplitudes of the scotopic ERG across the flash intensity range in hM3Dq compared to control mice. Examination of the normalized irradiance-response functions revealed a shift in b-wave but not awave sensitivity. No changes in a- and b-wave implicit times were detected. To identify cells that are excited following ipRGC activation we used c-Fos immunoreactivity as an early marker of neuronal activation. C-Fos mapping in the hM3Dq retinas revealed a significant number of cells that receive ipRGCs signalling. These c-Fos+ cells were localized mostly in the ganglion cell layer but some were found in the inner nuclear layer. Acute and selective activation of ipRGCs modulates the amplitude of both a- and b-waves of the scotopic ERG, indicating that the influence of this ganglion cell class on the retinal physiology extends to the photoreceptors as well as their downstream pathways. This event is associated with a significant number of excited retinal cells (c-Fos+) following ipRGCs chemogenetic activation. This rare cell class (~5%of RGCs) has a wide ranging influence on retinal physiology. 52

30) Rod/Cone coupling in the mouse retina Stephen C. Massey1, Friso Postma2, Zhijing Zhang1, Sean Youn3, Shivani Raman3, Eduardo Silveyra1, Nan Ge Jin1, David L. Paul2 and Christophe P. Ribelayga1 1

Ruiz Department of Ophthalmology & Visual Science, The University of Texas at Houston, McGovern Medical School, Houston, TX 2 Department of Neurobiology, Harvard University, Medical School, Boston, MA 3 Undergraduate Program, Rice University, Houston, TX

Background: In the retina, photoreceptors are electrically coupled via gap junctions, forming a functional network that regulates the early steps of vision. Gap junctions in the outer plexiform layer (OPL) may be due to rod/rod, cone/cone or rod/cone coupling. The gap junction-forming protein connexin36 (Cx36) is expressed in cones but its presence in rods remains controversial. To differentiate between the possible coupling patterns, we have created rod- and cone-specific Cx36 knock-out mice for comparison with wild type (WT) and pan Cx36 knock-out lines. We used high-resolution imaging and paired recordings to evaluate these mouse lines. Methods: To generate photoreceptor type-specific Cx36 knock out lines, we used a mouse line with a loxP-flanked allele of the Cx36 gene (Cx36f/f) crossed with either a rod- or cone-cre line. Whole-mounts or sections of mouse retinas were reacted with antibodies against Cx36, cone arrestin (to label cones), and vesicular glutamate transporter 1 (vGlut1, to label rod spherules). The distribution of Cx36 in the OPL was assessed by 5-channel confocal microscopy. Paired recordings were made using a perforated patch technique. Results: In the WT, Cx36 was strongly associated with cone telodendria, mainly at points of contact between cones and rods. Cx36 labeling in the OPL was reduced by more than 90% in both the rod-Cx36-/- and the cone-Cx36-/- lines. In the rodCx36-/- line, a few remaining Cx36 plaques were found at contacts between cone telodendria but in the cone-Cx36-/- line, Cx36 was virtually absent from the photoreceptor terminals. The junctional conductance between pairs of adjacent rods was  150 pS in WT mice and close to 0 pS in pan-Cx36-/- or rod-Cx36-/- mice, indicating that Cx36 is required for electrical coupling between rods. In addition, the rod/rod junctional conductance was dramatically reduced even in the cone-Cx36-/- retinas, suggesting that rods are primarily coupled by an indirect path (i.e. rod-to-cone-torod) due to rod/cone coupling. The rod/cone junctional conductance in the dark was very large, around 600 pS in WT mice and  0 pS in pan-Cx36-/-, cone-Cx36-/or rod-Cx36-/- mice. Application of the D2-like dopamine receptor antagonist 53

spiperone, known to increase photoreceptor coupling, increased rod/cone coupling  4-fold in the WT retina. Conclusions: The distribution of Cx36 expression in the conditional lines indicates that rod/cone gap junctions require Cx36 on both sides (rod and cone), as the absence of Cx36 on either side prevents the formation of a plaque. This implies that 1) Cx36 is expressed in rods and is required to form rod/cone gap junctions. 2) Most of the Cx36 plaques observed in the OPL are rod/cone gap junctions; we could not detect rod/rod gap junctions. 3) Rod/cone gap junctions are a key locus for the functional regulation of photoreceptors.

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31) Probing the retinal circuit by combining two photon holographic stimulation and multi electrode recordings G.L.B. Spampinato1, E.Esposito *,1, P.Yger 1, B.Lefebvre1, E. Ronzitti2, E. Papagiakoumou2, C. Robert1, W.Deschamps1, S.Picaud1, D.Dalkara1, V.Emiliani2, J.Duebel1,O.Marre1 1

Institut de la Vision, Paris, France Universite Paris Descartes, France

2

A major purpose of sensory neuroscience is to understand how a neural circuit generates complex activity patterns in response to sensory stimuli. While recent works have made possible to access the full connectome of a neural circuit, there is still a gap between its detailed anatomical reconstruction, and the functional characterization of its neural responses. To bridge this gap, we need to understand how the perturbation of each neuron will influence the activity of the circuit, and reconstruct the complete functional connectivity diagram. The retina transforms the visual scene in spikes and transfers them to the brain. Photoreceptors transduce light into electrical currents, bipolar cells process this signal and transmit it to ganglion cells, which send spikes to the brain A key component of retinal processing is the information transfer from the intermediate bipolar cell layer to the ganglion cell layer. Our understanding of this transfer is limited: while multi-electrode arrays allow recording large populations of ganglion cells, bipolar cells cannot be easily recorded or stimulated in the intact retinal circuit Here we present a novel method where we combined several techniques to record ganglion cells with multi-electrode arrays while perturbing individual bipolar cells using optical and optogenetic tools. We used an AAV and a specific promoter to express light sensitive proteins selectively in rod bipolar cells. We then used 2 photon computer generated holography, a technique to pattern light to stimulate individual neurons, while simultaneously recording ganglion cells with a multi-electrode array. Thanks to this combination of optical and electrophysiological tools, we could stimulate selectively rod bipolar cells and record the impact of this stimulation on the spiking activity of ganglion cells. Our method also allowed us to stimulate several bipolar cells simultaneously to measure the impact of complex stimulation patterns on the ganglion cell layer. We are currently using this technique to understand how each type of ganglion cell is modulated by rod bipolar cells. This method allows a precise probing of the retinal circuit and paves the way towards complete functional connectomics of the retina 55

TOOLS AGAINST RETINAL DISEASES SESSION 32) Tools for studying retinal circuits and disease Botond Roska Friedrich Miescher Institute for Biomedical research, Basel,Switzerland

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33) MANF as an immune modulatory intervention to improve retinal regenerative therapies in aging Joana Neves The Buck Institute for Research on Aging, Novato, CA, USA

Vision loss due to degenerative retinal diseases, such as age-related macular degeneration (AMD), is a common disability in the elderly. Although retinal degeneration is an irreversible process in humans, cell therapies, based on the transplant of retinal cells generated in vitro, hold promise as a therapeutic avenue, with a few clinical trials already underway. Nevertheless, the efficiency of such therapeutic approaches is compromised by the inefficient repair capacity of old, degenerating tissues. Age-associated inflammation, in particular, is likely an important roadblock for the success of regenerative therapies in aging. Thus, immune modulation aimed towards harnessing the anti-inflammatory potential of immune cells to promote endogenous repair mechanisms is a promising strategy to improve regenerative success in aging tissues. In our recent study, we discovered that mesencephalic astrocyte-derived neurotrophic factor (MANF), a previously described neurotrophic factor derived from neural glia, is also expressed in immune cells and has an autocrine immune modulatory function promoting anti-inflammatory activation. We further showed that the immune modulatory function of MANF could be used to protect the young retina from damage and to enhance the success of cell-replacement therapies in the models of retinal degeneration, improving cell integration and vision recovery following transplantation. We are currently expanding these studies to test the relevance of MANF therapy in the aging retina to protect against tissue damage and to improve the success of retinal regenerative therapies in old mice.

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LIGHT ADAPTATION SESSION 34) Adaptation of retinal processing in a dynamically changing environment Thomas Münch Institute for Ophthalmic Research (Department of Ophthalmology) and Centre for Integrative Neuroscience, University of Tübingen, Germany

The retina, as the front end of the visual system, is exposed to the full gamut of visual signals. It has to adapt to and compensate for the constant dynamic changes in ambient conditions. In our lab, we study the influence of such dynamics on the neural processing in the retina. I will discuss two such dynamic aspects: ambient luminance, and global image motion induced by eye movements. Ambient luminance has a strong qualitative influence on the output of the retina, to an extent that ganglion cells even change their response polarity (for example from OFF to ON-OFF) at different light levels separated by as little as one log unit. Similar effects are also found during natural movies, where a movie episode may elicit robust spiking at some, but not all, light levels. Such response changes occur at all light level transition, not only across the boundaries of major luminance regimes (scotopic, mesopic, photopic). No single mechanism can explain the observed phenomena, but different ganglion cells employ different mechanisms at different light level transitions. We find that rod photoreceptors can reliably signal at photopic light levels. When entering the photopic regime, rods initially have strongly reduced contrast sensitivity, but this recovers over time. Paradoxically, responses recover faster in brighter photopic light levels. This can be explained by enhanced rhodopsin bleaching, providing a form of bleaching adaptation previously known from cones, and by translocation of phototransduction elements between the inner and outer segments. Photopic rod responses are also robust during in-vitro experimental recordings from isolated retina. A large part of the dynamic image flow on our retina is self-inflicted, caused by self-motion of the observer. We characterized how ganglion cell responses to a “test stimulus” would be influence when it occurs in the context of global image shifts that resemble saccadic image flow. We see mostly response suppression that lasts up to 1 s, and some OFF ganglion cells show response enhancement. The strength of such modulation depends on the statistical properties of the image present during the “saccade”. Mechanistically, response modulation caused by the saccade, and responses to the saccade itself, employ different retinal pathways. 58

35) Light adaptation and correlated activity in the rodent retina Greg Field Neurobiology, Duke University School of Medicine, USA

Light adaptation influences nearly every aspect of retinal physiology. Changes in response gain induced by light adaptation have a clear purpose: to maintain sensitivity while avoiding saturation. However, other changes in retina function, such as light-dependent adaptations in receptive field structure and correlated activity have less obvious purposes. Furthermore, their dependence on light level presents a challenge to downstream brain circuits. For example, a change in receptive field structure indicates that spikes are signaling a different visual feature, suggesting that the decoding of these signals may need to change between low and high light levels. My lab is dissecting the impact of light adaptation on the receptive field structure and correlated spiking of diverse retinal ganglion cell (RGC) types to develop a more complete conceptual view that encompasses the ‘how’ and ‘why’ of light adaptation in the retina. I will draw from several examples of on-going research in my lab to illustrate some surprising effects of light adaptation on RGC function. I will describe the underlying mechanisms of these adaptations and identify the benefits they provide to the tuning of visual processing for low and high light levels.

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36) Is mouse vision more sensitive during the night? Sanna Koskela1, Tuomas Turunen2 & Petri Ala-Laurila1,2 1

Department of Biosciences, University of Helsinki, Helsinki, Finland Department of Neuroscience and Biomedical Engineering, Aalto University, Espoo, Finland 2

The retina is a rhythmic tissue with its own circadian clock and several circadian rhythms in its morphology, biochemistry and physiology. This has led to the hypothesis that visual functions are under circadian control. However, it has never been fully assessed whether visual sensitivity depends on the circadian clock. We determined if the sensitivity limit of mouse retinal ganglion cells (RGCs) and visually guided behavior depend on diurnal rhythm. We used both melatonin proficient (CBA/CaJ) and melatonin deficient (C57BL/6J) mouse strains to compare the visual sensitivity of mice in their subjective night with that in their subjective day. We used flat-mounted retinas and cell-attached patch clamp technique to measure the sensitivity limit of On and Off sustained alpha RGCs. We determined the visual threshold and detection strategies of mice in a watermaze test by using our fully-automated tracking system of mouse behavior. We show that both mouse strains have higher behaviorally measured visual sensitivity during the night than during the day. However, the sensitivity limit of RGCs does not show strong dependence on the diurnal rhythm. Instead, our results show that this improved visual performance during the night correlates with a more effective search strategy of visual cues by mice during the night.

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POSTERS POSTER SESSION I: RETINAL DISEASES AND THERAPIES 1) Short-term high-fat feeding accelerates retinal degeneration in an animal model of retinitis pigmentosa Pedro Lax1, Oksana Kutsyr1, Agustina Noailles1, Victoria Maneu2, Nicolás Cuenca1 1

Department of Physiology, Genetics and Microbiology, University of Alicante, Spain. Department of Optics, Pharmacology and Anatomy, University of Alicante, Spain.

2

Purpose: High-fat diet consumption has been associated with oxidative stress, metabolic damage and inflammation both in peripheral tissues and in the central nervous system. The aim of the current study was to investigate acute effects of high-fat feeding on retinal degenerative diseases. Methods: Rd10 mice 19 days old were fed either a normal chow (5,5 % kcal from fat) or a high-fat diet (HFD, 61,6% kcal) for 2 or 3 weeks (until P33 or P40, respectively). Age-matched C57BL/6 mice fed the same diets were used as control. After each of the two dietary periods, retinal function was assessed by electroretinography (ERG) and optomotor test. Results: HFD-fed mice significantly gained weight and developed glucose intolerance, regardless of strain or dietary period. In control C57BL/6 mice, shortterm high-fat feeding caused no significant effects on relative amplitudes of the a- and b-waves of the ERG or on visual acuity. However, the feeding of HFD for a period of 2 or 3 weeks deteriorated retinal function in rd10 mice. Concretely, HFD consumption produced rd10 mice with lower amplitudes in a- and b-waves and worse visual acuity when compared to age/strain-matched animals fed normal chow. The maximum scotopic a-wave (amax) and b-wave (bmax) recorded in rd10 mice fed HFD for 2 or 3 weeks were 42-55% lower than recorded in rd10 mice fed normal chow. Visual acuity of rd10 mice fell 23% two weeks after HFD introduction when compared to age/strain-matched animals fed normal chow. Conclusions: These results demonstrate that high-fat diets accelerate pathology progression in retinal degenerative diseases. The results suggest that in patients that suffer from ocular neurodegenerative diseases consumption of high-fat diets could accelerate the progress of the disease. Support: Ministerio de Economía y Competitividad, MINECO-FEDER-BFU2015-67139-R. Instituto Carlos III, RETICS-FEDER RD12/0034/0010. Generalitat Valenciana, Prometeo 2016/158.

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2) Systemic inflammation induced by LPS accelerates retinal degeneration in a mouse model of retinitis pigmentosa Agustina Noailles1, Laura Campello1, Oksana Kutsyr1, Victoria Maneu2, Pedro Lax1, Nicolás Cuenca1, 3 1

Department of Physiology, Genetics and Microbiology, University of Alicante, Spain. Department of Optics, Pharmacology and Anatomy, University of Alicante, Spain. 3 Institute Ramón Margalef, University of Alicante, Spain. 2

Purpose: Chronic microglial activation is associated with many neurodegenerative diseases and can contribute to retinal tissue damage during pathological processes. The main objective of this work was to study whether a damage caused by a chronic peripheral inflammatory process could be considered a risk factor that could worsen the progression of a degenerative disease of the retina. To do that, we studied the effect of a systemic administration of lipopolysaccharide (LPS) to an animal model of retinitis pigmentosa. Methods: Twelve homozygous P23H-3 and twelve wild-type Sprague-Dawley rats were injected twice weekly with LPS (60 μg/Kg, i.p.) or vehicle (PBS) from P20 to P60, six animals in each group. To evaluate the morphology and function of the retinal tissue, electroretinograms, flow cytometry and vertical retinal cryostat sections were used. Gene expression levels of inflammatory and apoptotic proteins were analyzed by quantitative RT-PCR. Results: LPS administration to P23H rats decreased the functional activity of the retina, as it reduced the a- and b-wave amplitudes in a significant way compared to the PBS-injected rats (34% reduction in both cases). This decrease was accompanied with a higher reduction in the number of photoreceptor cells and a worsening of synaptic connectivity, as compared to control P23H rats. In P23H rats, LPS administration increased the activation of microglia, evidenced by a statistically significant increase in the expression of MHCII and CD45 antigens in CD11b-positive cells detected by flow cytometry analysis. Immunohistochemical assays showed that in these P23H-LPS treated rats, microglial cells were located in all retinal layers, including inner and outer nuclear layers. In SD rats, the same LPS doses did not induce significant functional nor morphological changes. Gene expression analysis showed an increased level of apoptotic marker while a reduction in the autophagic activity in LPS-treated P23H rats was observed. Conclusions: Chronic administration of LPS accelerates photoreceptor cell death, increases microglial activation and accelerates retinal dysfunction in a retinitis pigmentosa rat model. Hence, in a similar way, chronic systemic inflammatory processes could worsen the progression of human retinal degenerative diseases. 62

Support: Ministerio de Economía y Competitividad, MINECO-FEDER-BFU201567139-R. Instituto Carlos III, RETICS-FEDER RD12/0034/0010. Generalitat Valenciana, Prometeo 2016/158. Support: Ministerio de Economía y Competitividad, MINECO-FEDER-BFU2015-67139-R. Instituto Carlos III, RETICS-FEDER RD12/0034/0010. Generalitat Valenciana, Prometeo 2016/158.

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3) Neuroprotective effect of glycyrrhizic acid on retinal degeneration in mice Gyu Hyun Kim, Sun-Sook Paik, Yong-Soo Park, In-Beom Kim Department of Anatomy, College of Medicine, The Catholic University of Korea.

The root and rhizomes of licorice (Glycyrrhiza) have been used as an herbal medicine, because they have various therapeutic effects, such as antiinflammatory and antioxidative activities. Glycyrrhizic acid (GA) is a major component in the root and rhizomes of licorice. In this study, we examined the effect of GA in an animal model for retinal degeneration (RD), which is the leading cause of blindness and characterized by the irreversible and progressive degeneration of photoreceptor cells in the retina. RD was induced in BALB/c mice by exposure to a blue light-emitting diode (LED) (460 nm) for 2 hours. To examine retinal functions, electroretinography (ERG) was performed. To assess histopathological changes, hematoxylin and eosin (H&E) staining were conducted. Apoptotic cell death was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. In addition, changes in proinflammatory cytokines were detected by real time RT-PCR and retinal stress and inflammation were evaluated by immunohistochemistry with anti-ionized calcium binding adaptor molecule 1 (Iba-1) and anti-glial fibrillary acidic protein (GFAP). Scotopic ERG showed that both a- and b-waves were significantly reduced in RD mice, while amplitudes of both waves were significantly increased in GA-treated RD mice, compared to those in non-treated RD animals. H&E and TUNEL assay showed that the outer nuclear layer where photoreceptors reside appeared to be more preserved and less apoptotic cells were observed in GA-treated RD retinas than in non-treated RD retinas. GA reduced expression of proinflammatory cytokines, such as TNF-α, IL-6, IL-1β, CCL2 and 6, iNOS, and Cox-2. In addition, GA reduced expression of Iba-1 and GFAP, indicating decreased glial response, retinal stress and inflammation. These results demonstrate that GA reduces retinal inflammation and prevents photoreceptor cell death from experimentally induced RD, suggesting that GA may have a potential for the treatment of RD as a medication.

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4) A fully organic polymer-based interface as potential retinal prosthesis in blind rats Mattia Di Paolo Department of Biotechnology and Applied Clinical Science, University of L’Aquila, Italy

Purpose: Degeneration of photoreceptors is the principal cause of irreversible loss of light perception in humans. Neuroprotective and substitutive strategies are main approaches to the retinal diseases. Neuroprotective effect it could be efficient in the chronicle disease or in the first stage of the pathology while substitution approach is required in the irreversible blindness phase. In particular, diseases affecting the retinal pigment epithelium and photoreceptors but preserve the inner retinal layers are preferential targets for implantation of visual prostheses that enable the electrical stimulation of neurons. Our partners have recently discovered that primary neurons can be successfully grown onto a photovoltaic organic polymer and electrically stimulated by light. Furthermore electronic device response was confirmed in implanted blind rats (Royal College of Surgeons, RCS) by recording an increase in the basal metabolic activity of the primary visual cortex. Here we show biocompatibility study by performing immunochemical and functional assays on the retina after prosthesis implantation in RCS and healthy rats. Methods: Experiments were performed on RCS rats and the non-dystrophic congenic animals (RCS-rdy). 2-3 months old animals were implanted with the prosthesis and let to recover for at least 3 weeks before experimentation. Electroretinographic recordings and immunofluorescence techniques were performed to verify the correct positioning of the prosthesis and its long-term tolerability. In particular the expression of inflammatory and trophic markers (FGF, GFAP), as well as microglia activation (Iba1), were monitored on retinas from RCS and RCS-rdy animals after implantation. Results: Our results describe a brief transient inflammation followed by a full recovery suggesting a long-term tolerability (up to 5 months) of the organic prosthesis. Moreover, electrophysiological techniques on RCS-rdy implanted rats confirm that the device presence does not compromise retinal function. Conclusions: In conclusion, our outcomes corroborate a good biocompatibility of the device confirming its potential application as substrate in developing of new generation of fully organic, highly biocompatible photovoltaic retinal prosthesis to treat degenerative blindness.

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5) Reconstruction of complex stimuli using spiking model of macaque retinal ganglion cells optogenetically restored using ChrimsonR Himanshu Akolkar1, Gregory Gauvain2, Romain Caplette2, Deniz Dalkara3, Celine Jaillard3, Jose A. Sahel3, Didier Pruneau4, Serge A. Picaud2, Ryad Benosman1 1

Vision and Natural Computation Lab, Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, Paris, France. 2 Visual Information Processing, Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, Paris, France. 3 Institut de la Vision, Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Paris, France. 4 Gensight Biologics, Paris, France

Introduction: Recently, we investigated overall effectiveness of AAV2.7m8 – ChrimsonR-tdTomato viral construct and its the clinical translation in peri-foveal retinal ganglion cells of non-human primates by performing full field stimulations to show response in large number of transfected retinal ganglion cells. Here, we build on this study using localized spot stimulations to model the firing response of the individual cells and use this model to reconstruct complex stimuli such as moving bar and letters. Methods: We performed ex-vivo multi-electrode array recordings (256 electrodes) from the peri-foveal retina of non-human primates (n=3) under a high resolution light stimulation setup using digital micromirror display (DMD). Pharmacological blockers were used to inhibit any remaining photoreceptors responses by blocking glutamatergic responses. The overall experiment was design in two stages. In the first stage, retina were stimulated with circular spots of varying diameters (25um, 50um and 100um) for duration of time ranging from 5ms to 40ms. Each condition was repeated over 20 trials. The cell responses to these local short-time stimulations were then fitted using a general integrate and fire forward model (Gerstner, 2001) to approximate the light input to spike-rate response relation of the cells. In the second part of the experiment, we presented more complex stimuli such as moving bar (width 25um and 50um) and letters (E,X,T,P) of different scales (55, 110, 220 and 330um) moving over the retina at speed of 11 degree/sec (equivalent to 2.23 mm/sec on retina). The forward-model of the cells was then used to convert the spike-rate of cells to estimates the presence of light stimulation at each cell location during these stimulations. Results: In the forward estimation, the response of the individual retinal ganglion cells was found to be consistent and reliable enough to achieve high fitting accuracy of the firing rate model. The residual errors of fitting was less than 1-10 with confidence p < 0.05. To test the performance of the model for bar and letter 66

stimuli, the cell response was converted into a circular spot of diameter varying from 25 to 100um at the location of cell and the response of the population was combined to create a reconstructed image. The reconstruction error was computed as the root mean squared error (RMSE) between the presented image on the retina and estimated image formed by the response of the cells. While, the reconstruction error of the model for the bar and letter stimuli was high, the reconstructed images were nonetheless successfully used to discriminate the different presented letters. To compute the discrimination power of the model and RGCs, the reconstructed images were passed through an image recognition algorithm (Lagorce, 2016). The algorithm was able to discriminate between a bar and the four presented letters with high accuracies. Conclusion: In this study we show that the retinal ganglion cell population restored using optogenetic therapy produce reliable response to local short-time stimulations that can be modeled and used to reconstruct complex patterns such as letters of different sizes and moving bars. These provide evidence that the visual restoration strategy may allow brain to use the retinal ganglion spikes and improve visual perception in treated patients. Financial support: This study was supported by the Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM), Pierre et Marie Curie University (UPMC) and was performed as part of the SightAgain project funded by BpiFrance.

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6) Neuroprotective effects of combined Nrf2 inducers and melatonin on retinal degeneration. Laura Fernández-Sánchez1, Laura Campello1, Oksana Kutsyr1, Agustina Noailles1, Isabel Ortuño-Lizaran1, Patricia Michalska2, Rafael Leon2, Antonio García García2, Victoria Maneu3, Pedro Lax1, Nicolás Cuenca1. 1 Department of Physiolgy, Genetics and Microbiology, University of Alicante, Alicante, Spain 2 Teófilo Hernando Institute of Drug R+D, University Autónoma de Madrid, Spain. 3 Department of Optics, Pharmacology and Anatomy, University of Alicante, Spain. Purpose: Oxidative stress, inflammation and apoptosis are common phenomena in all retinal neurodegenerative diseases. Nuclear transcription factor Nrf2 is a master regulator of the cellular antioxidant response. The aim of this work was to test the possible neuroprotective effect of Nrf2 inducers combined with the antioxidant melatonin for retinal degenerative diseases. Methods: Two retinal degeneration models were used: i) rd10 mice, a murine model of retinitis pigmentosa and ii) C57BL/6J mice with left eyes injured by ischemia/reperfusion, which triggers retinal ganglion cell death. Animals were administered intraperitoneally 1 or 10 mg/Kg of drug or vehicle twice a day, from P16 to P30 in rd10 mice or from 1 day before to 3 or 7 days after ischemia in C57BL/6J mice. Retinal function was assessed by electroretinography and optomotor test. The eyes were processed for morphological analysis. Results: Rd10 animals treated with the highest dose of drugs showed increased amplitudes in the a- and b-waves and a significantly augmented visual acuity and contrast sensibility, compared to control animals. In the retinal ischemia-reperfusion model no effects were found at either 3 or 7 days in the ERG tests, nor in the retinal ganglion cell number. By contrast, we detected reduced electroretinographic responses in the nonischemic eyes of injured animals. In these eyes both doses of treatment were able to improve retinal function, even restoring the ERG responses to normal values. Conclusions: These results indicate that combined Nrf2 inducers and melatonin exert neuroprotective effects in mild or moderate retinal injuries, while appear to be no effective in severe ischemic damage. Support: Ministerio de Economía y Competitividad, MINECO-FEDER-BFU2015-67139-R. Instituto Carlos III, RETICS-FEDER RD12/0034/0010. Generalitat Valenciana, Prometeo 2016/158.

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7) miR-204 is required for rod photoreceptor maturation and survival Intartaglia D., Naso F., Falanga D., Salierno F.G., Bhat R., Banfi S., Conte I. Telethon Institute of Genetics and Medicine, Via Campi Flegrei 34, Pozzuoli (Naples), 80078, Italy

Purpose: MicroRNAs (miRNAs), a class of small non-coding RNAs with a basic role in post-transcriptional regulation of gene expression, are emerging as key players in the control of fundamental biological processes during the differentiation of many tissues and organs. Their role in vertebrate eye patterning is just beginning to be elucidated. Recently, we identified members of miR-204/211 family as “master regulators” of eye development and function. We showed that miR-204 is required for dorsal-ventral patterning, lens differentiation and axon guidance of retinal ganglion cells. Most remarkably, we further identified a dominant mutation in miR-204 as the genetic cause of a unique phenotype of photoreceptor degeneration and coloboma, providing the first evidence, to our knowledge, of miRNA contribution to eye disease, likely through a gain-of-function mechanism. These findings also suggest an important role of miR-204 in the photoreceptor function. However, the relationship occurring between its function and the photoreceptor differentiation, maintenance and survival is still not analysed. Results: We found that deletion of miR-204 in Medaka fish resulted in specific eye phenotype, characterized by photoreceptor cell degeneration. Moreover, by taking advantage of stable miR-204 over-expression in photoreceptor cells, we show the key role of miR-204 in photoreceptor cell differentiation. Conclusions: Taken together, these studies allow us to gain a comprehensive understanding of miR-204 function and dysfunction, opening new perspective for the use of this

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8) miR-204/211 in RPE/photoreceptor crosstalk: an inticrate relationship Falanga D., Naso F., Intartaglia D., Barbato S., Marocco E., Pizzo M. Asteriti S., Cangiano L., Surace E.M., Banfi S., Conte I. Telethon Institute of Genetics and Medicine, Via Campi Flegrei 34, Pozzuoli (Naples), 80078, Italy

In recent years, microRNAs (miRNAs), a class of small non-coding RNAs, are emerging as key players in the control of fundamental biological processes in both physiological and pathological conditions. Their function in retinal cells is just beginning to be elucidated, and a few have been found to play a role in photoreceptors maintenance and function. We recently identified members of miR-204/211 miRNA family as new “key regulators” of vertebrate eye development. We demonstrated their role in eye morphogenesis including lens formation and dorso-ventral patterning of the retina. While their role in eye development has been characterized, the relationship occurring between their function and photoreceptor maintenance and function is still not completely characterized. To gain insights in this direction, we focus on the in vivo role of the miR-211. Interestingly, the functional analysis of miR-211 homozigous mutant mice revealed a progressive retinal degeneration characterized by a strong downregulation of photoreceptors markers together with an impaired electroretinogram response to light stimuli. MiR-211 knockout (KO) mice exhibited a progressive retinal dystrophy accompanied by significant alterations of visual function. The resulting phenotype mimics some of the pathological signs characteristic of retinal degenerative diseases, such as retinitis pigmentosa and age-related macular degeneration. These results not only allow us to gain a detailed understanding of miR-211 function and dysfunction, but also established the existence of precise network between miR-211 function and the RPE/photoreceptor crosstalk opening new perspectives for the use of this knowledge to develop novel therapeutic strategies to treat inherited retinal dystrophies.

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9) Sitagliptin slows retinal degeneration in a mouse model of retinitis pigmentosa Oksana Kutsyr1, Laura Fernández-Sánchez1, Victoria Maneu2, Pedro Lax1 António F. Ambrósio3, Nicolás Cuenca1 1

Department of Physiology, Genetics and Microbiology, University of Alicante, Spain. Department of Optics, Pharmacology and Anatomy, University of Alicante, Spain. 3 Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, Coimbra, Portugal. 2

Purpose: Retinitis pigmentosa (RP) is a retinal degenerative disease characterized by progressive loss of photoreceptors. Oxidative stress, inflammation and apoptosis are the main features in the progression of RP. Sitagliptin is a dipeptidyl peptidase-IV inhibitor that has demonstrated neuroprotective effects in mouse models of diabetes. The aim of this work was to evaluate the possible neuroprotective effect of sitagliptin in a mouse model of RP. Methods: Rd10 mice, carrying a mutation in the gene encoding the beta 6 subunit of phosphodiesterase in rods, were used as model of RP. The animals were treated orally with sitagliptin (10mg/Kg/day) from P18 to P25. Retinal responsiveness was determined by electroretinography (ERG) and visual acuity was evaluated by optomotor test. The hematoxilin staining was used to quantify the number of photoreceptor rows and immunohistochemistry techniques were performed to analyze retinal cells morphology and synaptic connectivity. Results: Sitagliptin administration ameliorated retinal function in rd10 mice, displaying higher amplitudes in a- and b-waves, as well as in oscillatory potentials. In addition, treated animals also showed better visual acuity. These functional findings correlated with a delay in photoreceptor degeneration revealed by raised number of photoreceptor rows founded in sitagliptin treated animals when compared to the control animals. Besides, better-preserved photoreceptor outer and inner segments as well as longer and well-defined photoreceptor axons terminals were observed in treated animals. Furthermore, presynaptic and postsynaptic elements, as well as synaptic contacts between photoreceptors and bipolar or horizontal cells were preserved. Conclusions: Sitagliptin treatment prevented retinal function loss, photoreceptor degeneration and preserved the morphology of rod bipolar cells and horizontal cells, as well as the synaptic connectivity between them. Our results support sitagliptin as a suitable option for treatment of RP. Support: Ministerio de Economía y Competitividad, MINECO-FEDER-BFU2015-67139-R. Instituto Carlos III, RETICS-FEDER RD12/0034/0010. Generalitat Valenciana, Prometeo 2016/158. 71

10) Time-course of anatomical and functional changes triggered by light exposure in the albino rat retina Serena Riccitelli1, Mattia Di Paolo1, Silvia Bisti1, Stefano Di Marco1 1

Department of Biotechnology and Applied Clinical Sciences, University of L’Aquila

PURPOSE: To investigate the time-course of functional and morphological modifications induced in the neuro-retina by exposure to high intensity light. METHODS: Sprague-Dawley adult rats, raised at 5 lux, were exposed to 1000 lux for 24 hrs (light damage, LD) and then placed back at dim light during recovery. At different survival times we performed scotopic flash-Electroretinogram to analyze a-wave, b-wave and oscillatory potentials. Flicker and photopic flash-ERG were performed to isolate cone-driven response. Retinas were subsequently collected and analyzed by sections and whole-mount immunohistostaining. Morphometric analysis were performed. They include measuring ONL and INL thickness, damaged extension area and opsin density. Tunel labeling was used to quantify photoreceptor death. Moreover, we evaluated immune localization of inflammation markers and selected neurotrophic factors. Experiments were performed at 7, 15, 30, 45, 60 and 90 days after LD. RESULTS: Visual function is significantly suppressed 7 days after LD, but shows partial slow recovery up to the 45th day. Analysis performed on retinal slices shows a progressive reduction of ONL thickness, due to photoreceptors loss, mainly in the dorsal retina. Thinning of the ONL is also observed in the ventral side, but retinal structure appears more preserved. Disorganization of retinal inner layers is observed by 30 days post-exposure. Whole-mount analysis shows that at increasing survival time “hot-spot” area becomes larger although remains locally confined in the dorsal retina. The number of TUNEL+ nuclei decreases over time and most are located in the hotspot or in the border of this expanding hot spot. Densitometric analysis shows a variation in both localization and expression of trophic factors and inflammatory markers. CONCLUSIONS: These data show that following an initial damaging stress neurodegeneration progressively extends. The initial strong reduction in retinal function is probably due to both electro-mechanical problems and activation of selfprotective mechanisms. These results support the hypothesis that characterization of relevant steps and mechanisms might be used to plan and develop protective treatments.

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11) The role of pro-apoptotic proteins BAK1 and BAX in rd1 rodent model of retinitis pigmentosa

Kiana Kakavand 1, Andrew I. Jobling1, Robb U. de longh1, Stephane Chappaz2, Benjamin T. Kile2, Erica L. Fletcher1 1

The University of Melbourne, Department of Anatomy and Neuroscience, Melbourne, Australia 2 The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia

Purpose: Photoreceptor cell death is the common outcome of mutations that give rise to retinitis pigmentosa (RP). However, the molecular mechanisms that lead to photoreceptor death are varied and not well understood. The mitochondrialmediated pathway is one of the two major apoptotic pathways regulated by the BCL-2 family members, BAK1 and BAX. The purpose of this study is to determine the role of BAK1 and BAX in the photoreceptor degeneration observed in the rd1 mouse model of RP. Methods: All strains of mice used in this study were on the C57Bl6/J background. Bak1-/-and Bax-/- mice were crossed with rd1 mice to generate rd1/Bak1-/-, rd1/Bak1-/-/Bax+/- and rd1/Bak1-/-/Bax-/- genotypes. Eyes were collected at post-natal day (P) 13, 14, 18 and 21 and the structure of the retina was assessed morphometrically using 5 μm hematoxylin and eosin stained paraffin sections. Retinae from rd1/Bax/Bak1 genotypes were compared to rd1 and C57Bl6/J controls. Results: Genetic ablation of Bak1 with (rd1/Bak1-/-/Bax+/+) or without (rd1/Bak1-/-/Bax+/-) a single Bax null allele had a slightly thicker outer nuclear layer (ONL) compared to age-matched rd1 retinae at P14 (25.6 μm and 27.5 μm compared to 20.9 μm; p<0.0001). However, total retinal thickness remained significantly less than that of in C57BL6/J mice (138.7 μm and 142.1 μm compared to 185.36 μm; p<0.0001). This increase in outer retinal thickness was transient, as no significant differences were found at P18 or P21, among any of the Bax;Bak1;rd1 mutants compared to the rd1 mice. While deletion of both Bak1 and Bax is associated with perinatal lethality on the C57Bl6/J background, we obtained viable offspring that survived to P13 (n=3). Retinae from these animals showed increased ONL and total retinal thicknesses when compared to rd1 and C57BL6/J mice. Conclusions: Genetic elimination of Bak1, with or without heterozygosity for Bax, resulted in a transient rescue of photoreceptor cells at early stages of retinal degeneration (P14) in the rd1 model of retinal degeneration. However, deletion of both Bak1 and Bax resulted in a dramatic rescue of rd1-induced cell death at 73

P13. These data suggest both Bak1 and Bax are required and act redundantly during photoreceptor degeneration in rd1 mice.

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12) Spontaneous oscillatory networks in the degenerated retina Prerna Srivastava1-3, Luke E. Rogerson1-4, Philipp Berens1,2,4, Thomas Euler1,2,4, Timm Schubert1,2 1

Werner Reichardt Centre for Integrative Neuroscience (CIN), Institute for Ophthalmic Research, 3 Graduate Training Centre of Neuroscience, 4 Bernstein Centre for Computational Neuroscience; All: University of Tübingen, Germany 2

Rod photoreceptor degeneration leads to severe anatomical and functional remodeling of synaptic connections between retinal neurons. In particular, the outer retinal network composed of remnant cone photoreceptors (cones), horizontal cells and bipolar cells (BCs) shows synaptic re-organization while generating spontaneous oscillatory activity. Morphological alterations in the inner retina are minor, nevertheless, the absence of light-driven input does affect inner retinal function, with ganglion cells, AII amacrine cells (ACs) and cone BCs showing spontaneous oscillatory activity bursts. It is still unclear, if the activity networks in the outer and the inner retina are linked. However, the involvement of BCs in both networks suggests a central role for this cell class. Here, we aim to understand if and how the outer retinal network drives/modulates the inner retinal network via BCs. To this end, we recorded Ca2+ signals from somata of neurons in the outer and inner rd10 retina by using 2P calcium imaging. To record glutamatergic synaptic activity, we expressed the glutamate biosensor iGluSnFR and imaged the glutamatergic input and output BCs receive at their dendrites and generate at their axon terminals, respectively. Consistent with earlier studies, we detected spontaneous oscillatory Ca2+ signals in both the outer and the inner retina; co-existing in the same tissue. Our glutamate biosensor data recorded in the outer plexiform layer suggest that outer retinal activity is generated in remnant cones, spreading to BC dendrites. Glutamate signals recorded in the inner plexiform layer at rod BC axon terminals suggest that BCs spontaneously release bursts of glutamate that could drive inner retinal neurons. To test if these signals reflect cone or AC input, we ‘clamped’ ONBC activity using the mGluR6 receptor agonist L-AP4. This reduced spontaneous activity in BCs and altered oscillations in the inner retina, consistent with a modulatory influence of the outer on the inner retina. Our approach allows us to study the two oscillatory networks in the rd10 retina in the same tissue. Based on earlier work and our preliminary data, it is tempting to speculate that the glutamatergic rod BC – AII AC synapse is crucial to connect outer and inner retinal activity. 75

13) CD47-/- mice accumulate highly pigmented subretinal macrophages agedependently similar to patients with degenerative retinal diseases. Sebastien Augustin1, Sophie Lavalette1, 2, Daniel Lewandowski3, Alexis Bemelmans1, 3 José-Alain Sahel 1, 4, Michel Paques1, 4, Xavier Guillonneau1, Florian Sennlaub1, 1

Institut de la Vision, 17 rue Moreau, Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, 75012 Paris, France. 2 Department of Microbiology and Immunology, Weill Cornell Medical College, New York, NY 10065, USA. 3 Commissariat à l'Energie Atomique et aux Energies Alternatives (CEA), Département des Sciences du Vivant (DSV), Institut d'Imagerie Biomédicale (I2BM), Molecular Imaging Research Center (MIRCen), F-92260 Fontenay-aux-Roses, France. 4 Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts, INSERM-DHOS CIC 503, Paris, France.

Age-Related Macular Degeneration (AMD) is characterized by the breakdown of subretinal immunosuppressivity and accumulation of Mononuclear Phagocytes (MP) that participate in photoreceptor degeneration, retinal pigment epithelium (RPE) dedifferentiation and pathological neovascularization and the appearance of hyperreflective foci (HF) in optical coherence tomography (OCT), a predictive progression marker. We have shown that Thrombospondin-1 (TSP-1) activation of CD47 on MPs is a key factor in the maintenance of subretinal immunosuppressivity and both TSP1-/-- and CD47-/--mice develop age-related subretinal MP accumulation. In parallel, the N-terminal domain of CD47 expressed on stromal cells serves as the ligand of Signal-Regulatory Protein alpha (SIRPα) expressed on MPs, which mediates a “don't eat me” signal and prevents stromal cell phagocytosis by MPs. We here show that most of the subretinal MPs in aged CD47 -/- mice are highly pigmented and are visible in optical coherence tomography (OCT) as hyperreflective foci (HF) contrary to un-pigmented subretinal MPs in TSP-1-/- mice. We also demonstrated that melanosomes in MPs are located within the phagosome in CD47-/- mice contrary to melanosomes in the retinal pigment epithelium. Adoptive transfer experiments of wildtype bone marrow to lethally irradiated wildtype- or CD47-/-- recipient mice showed that the age-dependent exaggerated subretinal MP accumulation is due to the lack of myeloid CD47 expression, but that increased pigmentation of subretinal MPs depends on the lack of CD47 in the host, likely in the RPE. Taken together our data confirms the importance of CD47 expression on MPs as a mediator of TSP-1 dependent subretinal immune-suppression and shows that 76

CD47 expression on the RPE inhibits the phagocytosis and accumulation of melanosomes in macrophages. Our results suggest that HFs in the retina are due to pigment-laden MPs. To our knowledge, this is the first rodent model of HFs that are an increasingly recognized marker for AMD progression and possibly other retinal degenerative diseases.

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14) Orally administration of melatonin and epigallocatechin gallate slows retinal degeneration in an animal model of Retinitis Pigmentosa, P23H rat Lorena Perdices Royo1, Elvira Orduna Hospital1, 2, Ana Isabel Sánchez Cano 1,3, Lorena Fuentes Broto1,4, Nicolás Cuenca5, Francisco Segura Calvo 1,2,3 Gema Insa Sánchez1, Isabel Pinilla Lozano1,3 1

Institute for Health Research of Aragón (IIS Aragón), Zaragoza, Spain Miguel Servet University Hospital, Ophthalmology, Zaragoza, Spain 3 Zaragoza University, Optics, Zaragoza, Spain. 4 Zaragoza University, Physiology, Zaragoza, Spain. 5 Department of Physiology Genetics and Microbiology, Alicante University, Spain 6 Hospital Universitario Lozano Blesa, Ophthalmology, Zaragoza, Spain 2

Purpose: to evaluate the efficacy of melatonin and epigallocatechin gallate (EGCG) for the treatment of retinal disease in an animal model of Retinitis Pigmentosa, the P23H rat. To evaluate the synergic effect of both drugs administered simultaneously. Methods: twenty P23H rats crossed with Long Evans (LE) rats, were used and compared to 20 Sprague Dawley (SD) (P23H background) crossed with LE rats. Vehicle, or 10 mg/kg/day of Melatonin and/or 10mg/kg/day of EGCG were administered for 6 months in drinking water. Visual function was evaluated by a monthly optomotor test and electroretinogram (ERG). The levels of lipid peroxidation, nitrites, catalase and superoxide dismutase in the liver tissue were determined. Results: P23HxLE rats showed lower values than SDxLE rats in all visual parameters studied. SDxLE rats treated with melatonin or EGCG increased, after 60 days of treatment, visual function parameters even higher than young animals. P23HxLE rats treated with melatonin or EGCG showed better visual acuity and b-wave amplitude than those treated with vehicle in all measurements done after 30 days of treatment, slowing the disease progression. In all animal groups, treatment with melatonin and EGCG simultaneously obtained better results than treatment with any of those compounds alone. We obtained higher levels of MDA + 4-HDA in the P23H rats, indicating a higher degree of lipid peroxidation compared to SDxLE; levels of activity of superoxide dismutase and catalase, two antioxidant enzymes, showed that they were lower in the P23H rats than in the SDxLE, control group, indicating that their defenses against oxidative damage were lower, and therefore, the oxidative stress was increased. Conclusion: oral treatment of melatonin or EGCG improved vision in wild type animals and delayed vision loss in P23H rats. Antioxidant treatments increased 78

antioxidant capacity and decreased lipid peroxidation. Furthermore, combination of both compounds had a better effect than any of those treatments alone, suggesting that they are protecting the retina by different mechanisms of action.

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15) Promoting the functional maturation of human iPS cell-derived retinal ganglion cells by a 3D/2D stepwise differentiation protocol Rabesandratana, Oriane; Chaffiol, Antoine J; Slembrouck, Amélie; Reichman, Sacha; Nanteau, Céline; Gagliardi, Giuliana; Duebel, Jens; Goureau, Olivier; Orieux, Gaël Institut de la Vision, Sorbonne Universités UPMC-Paris 06 ; INSERM U968 ; CNRS UMR7210, Paris, FRANCE

Purpose: Optic neuropathies have currently no effective treatment to stop the progressive loss of retinal ganglion cells (RGCs) and the degeneration of the optic nerve. Stem cell-based therapy using human induced pluripotent stem cells (hiPSCs) is one of the most promising strategy to replace lost RGCs. The present work displayed the optimized generation and characterization of purified hiPSCderived RGCs, based on our retinal differentiation method using adherent hiPSCs. Methods: 3D retinal organoids containing RGCs were generated according to our previous protocol (Reichman et al. PNAS 2014; 111:8518). To promote RGC axonal growth, retinal organoids were dissociated to further cultured retinal cells onto poly-D-lysin/laminin coated plates. At different time points, maturation of hiPSCderived RGCs was evaluated by immunohistochemistry with specific RGC markers and by electrophysiological recordings. For cell separation, cells were dissociated into single cells, immunostained with a CD90 antibody and Magnetic-Activated Cell Sorting (MACS) was performed by using anti-CD90 microbeads. Dissociated retinal cells (unsorted, CD90- and CD90+ fractions) were analyzed and characterized by cytofluorometry, immunohistochemistry and electrophysiological recordings. Results: Dissociated cells from retinal organoids cultured in 2D, expressed specific RGC markers (BRN3A, ISLET1, RBPMS, CD90). Axonal growth was highlighted by the expression of CD90 and βIII tubulin. Cytofluorometric analysis revealed an enrichment up to 87% of RGCs based on CD90 expression after MACS purification. On both unsorted and CD90+ cells, whole-cell patch-clamp recording revealed that hiPSC-derived RGCs displayed voltage dependent currents (up to 1500pA at +40mV). Furthermore, action potentials were triggered by depolarizing current injections (+40pA from the resting membrane potential), and in some cells spontaneous spiking was observed. Conclusions: Our results support promotion of axonal growth of hiPSC-derived RGCs in adherent culture conditions. MACS of CD90+ cells resulted in a significant enrichment of RGCs exhibiting specific RGC electrophysiological profile, which will be of a great utility for future clinical translation. For this purpose, the ability of 80

hiPSC-derived RGCs to survive, to fully differentiate and to establish new connections with the brain has to be demonstrated.

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16) Simultaneous imaging of calcium and calcium-dependent enzymatic activity in degenerating cone photoreceptors Michael J. Power1,2,3, Manoj Kulkarni1,2,3, Timm Schubert1,2, François PaquetDurand1, Thomas Euler1,2 1

Institute for Ophthalmic Research, University of Tübingen, Germany Centre for Integrative Neuroscience (CIN), University of Tübingen, Germany 3 Graduate Training Centre of Neuroscience, University of Tübingen, Germany 2

Calcium levels are strictly regulated within neurons and, thus, calcium dysregulation has been proposed to be linked to cell death in the nervous systems. Recent research has highlighted how various mechanisms of calcium dysregulation may be connected to both primary and secondary cone photoreceptor cell death in the mammalian retina (e.g. Arango-Gonzalez et al., PLoS One, 9:e112142, 2014). Here, we examine the activation patterns of the calcium-dependent cysteine protease calpain in comparison with cell death patterns seen in mouse models of photoreceptor degeneration. We determine the spatial activation of calpain at several time points in the secondary cone degeneration model rd1 (and later rd10), and the primary cone degeneration model cpfl1. Our preliminary data suggest that calpain activity is markedly increased in the rd1 and rd10 models (P30: rd1, 1.01 ± 0.113 calpain-positive cells in ONL/1,000 µm2, n=45 observations from 3 mice p < 0.0001 compared to wt; rd10, 0.869 ± 0.068, n=45 observations from 3 mice; wild-type, 0.086 ± 0.023 n=45 observations from 3 mice p = 0.0001 compared to wt). To link calpain activation to calcium dynamics within the cones, we use a transgenic HR2.1:TN-XL mouse line that expresses a calcium biosensor in cones to study calcium levels and, where applicable, light-evoked responses in an acute retinal slice preparation (Kulkarni et al., J Vis Exp. 6:e52588, 2015). We use twophoton microscopy to measure calcium dynamics in retinas of HR2.1:TN-XL mice as our wild-type control, as well as in the retinas of rd1 and cpfl1 which have been stably crossbred with HR2.1:TN-XL mice. By adapting an approach used commonly on thin, unfixed retinal sections, we have developed a protocol for the combination of detecting enzymatic activity of calpain and live-cell calcium imaging. In using this approach, we are able to simultaneously image two different parameters – here calcium and calpain activity – likely involved in cone cell death, in real-time. Future experimental manipulations (e.g. calpain inhibitors, Ca2+ channel blockers) will then allow us to establish if and how these factors are linked to each other and cell death. Data generated in this project may be used to 82

rationally design new therapeutic approaches for the treatment of cone degeneration.

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17) Characterization of monocyte derived macrophages in diabetic retinopathy Guillaume Blot1, Hugo Charles-Messance1, David Rivera2,3, Aude Couturier1, Sébastien Augustin1, Yonathan Garfias2,4, Florian Sennlaub*, Xavier Guillonneau1 1

Institut de la Vision, 17 rue Moreau, Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, 75012 Paris, France. 2 Institute of Ophthalmology, Conde de Valenciana Foundation, Mexico City 3 Centro de Atención Integral Del Paciente Con Diabetes, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico city, Mexico. 4 Department of Biochemistry, Universidad Nacional Autónoma de México,Faculty of Medicine, Mexico City.

Type 2 diabetes (T2DM) affects a growing part of the population. T2DM patients are characterized among other by hyperglycemia and dyslipidemia. This abnormal blood composition plays a critical role in the development of comorbidity. More than 60 % of people with T2DM will develop a diabetic retinopathy (DR) after 20 years of diabetes. T2DM patients demonstrate robustly elevated intravitreal levels of inflammatory cytokines. Recent evidences suggest that inflammatory macrophages (Mφ), mostly derived from infiltrating monocytes (Mo), account for most of the production of these cytokines. We hypothesize that pre-activated circulating inflammatory monocytes found in T2DM are an important pathogenic factor in DR. 72 T2DM and control patients were enrolled. Four groups were defined as follows: (i) controls, (ii) diabetic patients with no sign of retinopathy, (iii) patients with NPDR, and (iv) PDR patients. Patients with known history of nephropathy and periodontis were excluded. Fresh Mo were collected and some fresh Mo were differentiated into Mφ only by attachment to plastic for 18H. Mo and Mo-derived Mφ were then evaluated for their ability to produce cytokines known to be elevated in the vitreous of DR patients including IL-1ß, CCL2, VEGF. To assay the role of hyperglycemia in Mφ polarization, control Mo were exposed to high glucose condition (HG, 25 mM) for 2 days and compared to Mo kept in normal glucose condition (NG, 5mM). The addition of palmitic acid (50 mM) complexed with BSA (w/v 0,75 %) were also studied for Mφ polarization. The 3 diabetic groups are evenly distributed for age, BMI, glucose and Hba1c. Circulating Mo demonstrated low expression of cytokines with minor differences between groups. In contrast, upon differentiation Mφ increase their expression of all tested cytokines. After 18H expression of CCL2 and VEGF were different between groups. Similarly, control Mo-derived Mφ, were significantly upregulated for these genes in HG compared to NG. Interestingly palmitic acid highly upregulates CLL2 and IL-1ß but not VEGF. 84

Our preliminary data supports a model in which circulating Mo in DR patients have a greater propensity to produce inflammatory cytokines when they differentiate into Mφ. In vitro HG exposure increases inflammatory cytokines and VEGF mRNA expression, and palmitic acid exposure highly increases inflammatory cytokines mRNA expression. Together these conditions could mimic in part the Mφ polarization in diabetic environment.

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18) Photovoltaic stimulation of retinal ganglion cells with wide-field epiretinal prosthesis Naïg Chenais 1, Marta Airaghi Leccardi 1, Laura Ferlauto 1, Kevin Sivula 2 and Diego Ghezzi 1 1

Medtronic Chair in Neuroengineering, EPFL STI IBI-STI LNE, 1015 Lausanne, Switzerland Laboratory for Molecular Engineering of Optoelectronic Nanomaterials, EPFL SB ISIC LIMNO, 1015 Lausanne, Switzerland 2

Retinal prostheses have become in the past decade a promising and realistic technology to restore vision. Nonetheless, sight restoration with retinal prostheses in a clinically relevant perspective requires resolution and implantation challenges not yet achieved. Our goal is the development of a foldable and wide field-retinal prosthesis capable of achieving a wireless photovoltaic stimulation of retinal ganglion cells and restore at least 40° of visual field, though being injectable trough a minimal scleral incision. The 2345 organic photovoltaic stimulating pixels of our retina prosthesis are distributed into biomimetic pattern within an active area of 13 mm (44° of visual field) and the light-triggered current profile generated by those pixels shows a reproducible ability to elicit activity in explanted rodents’ retinas mimicking human retina dystrophies. Extracellular recordings of prosthetic-evoked spiking activity of retinal ganglion cells reveal both direct and network-mediated stimulation when the degenerating retina circuit is explanted on top of the stimulating device, while when retinas are layered on bare PDMS substrates, any lightevoked pattern of response can be detected among retina spontaneous activity. Screening of different conditions of illumination (pulse duration and intensity) with Rd10 retinas explanted on our prosthesis shows a direct activation from the minimal radiant exposure tested that is 7 times smaller than the maximum permissible retinal irradiance allowed for ophthalmic applications in this case- while network-mediated activation requires higher light exposure and can be supressed using synaptic blockers. The clinical compliance of the so-designed prosthesis and those preliminary results on explanted retinas represent a step forward in building advanced photovoltaic retinal prostheses.

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19) Modeling the behavior of microbial opsin ChrimsonR for optogeneticsbased Vision Restoration Quentin Sabatier1, 2, Gregory Gauvain2, Corentin Joffrois2, Pierre M. Daye2, Joël Chavas1, José-Alain Sahel2, Serge Picaud2, Ryad Benosman2 1

GenSight Biologics, Paris, France Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, UPMC, Paris, France 2

Purpose Optogenetics is increasingly used to externally drive neural behavior both in research and medical applications. Both fields need to control precisely the behavior of the neurons expressing the light-sensitive ion channels, and therefore need precise models describing the evolution of the conductance of the channels when submitted to an arbitrary light command. We developed a dual approach combining theoretical work and simulation-based parameter estimation based on patch-clamp recordings of HEK cells expressing the ChrimsonR channel to model the gating dynamics of the channel. Methods ChrimsonR channels were expressed into HEK cells which were then used in voltage-clamp experiments where the cells were submitted to light commands involving a wide range of light intensities and time scales for each cell. Markov kinetic models were used to describe the different conformations of the molecule and the transitions dynamics between the states. Using theoretical results on Markov kinetic models, we were able to set the lower bound to the number of states required to explain the data to five. The form of the Markov chain was a simplified version of the photocycles derived from spectroscopic studies on other channelrhodopsins. Results Theoretical considerations showed that at least five states, including two conductive states, were necessary to explain elementary observations and statistical analyzes. Using a five-state Markov chain, we were able to capture the main features of the recorded data. Among these features, we captured (i) the on and off dynamics of the response, (ii) the activation curve (i.e. the amplitude of the response on the whole range of light intensities) and (iii) a slow side reaction affecting the evolution of the response on long time scales (or the order of the minute). Additionally, our recordings reveal new observations on the behavior of channelrhodopsins that may be an important step towards building a complete model for their photocycle. Conclusion This preliminary study lays the groundwork for an integrated Markov kinetic model capturing the relationship between light stimulation and ChrimsonR conductance at multiple time scales. 87

20) CC2D2A mutations lead to variable phenotypes in a family with retinal dystrophy Cécile Méjécase1, Saddek Mohand-Saïd1,2, Camille Andrieux2, Aurélie Hummel3, Saïd El Shamieh1, Aline Antonio1,2, Fiona Boyard1, Christel Condroyer1, Christelle Michiels1, Steven Blanchard4, Mélanie Letexier5, Jean-Paul Saraiva6, José-Alain Sahel1,2,4,6,7, Christina Zeitz1, Isabelle Audo1,2,5 1

Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, 75012 Paris, France. 2 CHNO des Quinze-Vingts, DHU Sight Restore, INSERM-DHOS CIC1423, 28 rue de Charenton, 75012 Paris, France. 3 Department of Nephrology, Necker-Enfants Malades Hospital, Paris, France. 4 IntegraGen SA, Genopole, CAMPUS, 1 bat G8 FR-91030 EVRY, Paris, France. 5 Institute of Ophthalmology, University College of London, London, United Kingdom. 6 Fondation Ophtalmologique Adolphe de Rothschild, Paris, France. 7 Académie des Sciences, Institut de France, Paris, France.

Inherited retinal dystrophies (IRDs) are a heterogeneous group of disorders with a need of a better understanding of the underlying causes in absence of treatment. Our goal was to identify the underlying gene defects for IRD in three brothers from a consanguineous family. Ophthalmic examination of the eldest brothers revealed rod-cone dystrophy (RCD) whereas the youngest had earlyonset cone-rod dystrophy (eoCRD). Renal function and ultrasound was normal in the eldest brother whereas the youngest brothers had high blood pressure, hematuria and proteinuria in keeping with the diagnosis of glomerulopathy but not nephrophtisis, as confirmed by renal biopsy. Brain magnetic resonance imaging was normal for all. In addition, none had learning disabilities and all were otherwise fit and well. Initial targeted next-generation sequencing (NGS) covering exons of 120 genes mutated in IRDs did not identify any homozygous diseasecausing variant in the oldest brother. Subsequently, whole exome sequencing (WES) identified two compound heterozygous variants in CC2D2A (c.2774G>C p.(Arg925Pro) and c.4730_4731delinsTGTATA p.(Ala1577Valfs*5)) in all affected brothers, missed by targeted NGS due to the stringent filtering of only homozygous variants related to the reported consanguinity. In addition, WES analysis revealed a homozygous variant in CNGA3 (c.1235_1236del p.(Glu412Valfs*6)) in the eoCRD proband, heterozygously present in the two RCD brothers, which would account in him for the early onset phenotype in the form of achromatopsia, and the secondary rod involvement linked to the same variants in CC2D2A, shared with his brothers. In additional 1056 IRD cases screened by targeted NGS, two unrelated patients with CC2D2A mutations ([c.2774G>C 88

p.(Arg925Pro)]+[c.3182+355_3825del p.(?)]; [c.351T>G p.(Ser117Arg)]+[c.3556G>A p.(Val1186Met)]) were identified but co-segregation analysis is pending. CC2D2A mutations were previously reported in MeckelGruber (MS) and Joubert Syndrome (JS). Here we report that CC2D2A mutations can lead to non-syndromic RCD. The link between these mutations and the glomerulopathy present in the two youngest cases warrants further investigations. This study adds CC2D2A to the missing genes mutated in nonsyndromic IRDs with mutations accounting 0.1-0.3% of cases. Further studies are needed to understand the phenotype-genotype correlations in MS, JS and nonsyndromic IRD with or without renal defects.

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21) Towards a better understanding of ITM2B in retinal physiology and pathology. Juliette Wohlschlegel1, Christelle Michiels1, Sacha Reichman1, Amélie Slembrouck1, Valérie Forster1, Emmanuelle Clérin1, Manuela Argentini1, JoséAlain Sahel1,2,3, 4,5,6,7, Olivier Goureau1, Christina Zeitz1, Isabelle Audo1,2,3 1

Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, 75012 Paris, France. 2 CHNO des Quinze-Vingts, DHU Sight Restore, INSERM-DHOS CIC1423, 28 rue de Charenton, 75012 Paris, France. 3 Institute of Ophthalmology, University College of London, London, United Kingdom 4 IntegraGen SA, Genopole, CAMPUS, 1 bat G8 FR-91030 EVRY, Paris, France. 5 Fondation Ophtalmologique Adolphe de Rothschild, Paris, France. 6 Académie des Sciences, Institut de France, Paris, France. 7 Ophthalmology department, University of Pittsburg Medical College

Inherited retinal diseases are genetically and clinically heterogeneous disorders with limited therapeutic options to prevent blindness. Our group identified a missense mutation (c.782A>C, p.Glu261Ala) in ITM2B underlying a novel autosomal dominant retinal dystrophy (RD) with inner retinal dysfunction and progressive retinal degeneration. IT2MB is expressed in the brain, interacting with APP, and in the inner retina but its role remains unknown. However, mutations in ITM2B had previously been identified in two large autosomal dominant families with Alzheimer-like dementia (AD). Our project aims at better understanding the role of ITM2B in the retina, identify protein partners and decipher pathogenic mechanisms underlying ITM2B-related RD versus AD, including changes in protein interactions or retinal cell toxicity of ITM2B mutants. We used HEK 293 cells overexpressing the wild type or ITM2B mutants as a tool to establish immunostaining and western blot. In addition, through a collaboration with Dr. Goureau we developed iPS cells from a patient and a control for disease modeling. Conditioned media are tested for toxicity of the various ITM2B mutants on primary rat ganglion cell cultures. Immunoprecipitation experiments for ITM2B are underway to identify ITM2B interacting partners through mass spectrometry. Our project offers a unique model to better understand the role of ITM2B in neuronal physiology and validate therapeutic approaches also relevant for AD.

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22) Spectral reprogramming of photoreceptors Joseph C. Corbo, Jennifer M. Enright, and Matthew B. Toomey Washington University School of Medicine, St. Louis, MO USA

We are developing a 'red-shifted' optogenetic strategy for restoring vision to degenerated retinas. This therapeutic approach is based on a form spectral reprogramming used by migrating fish to enable better vision in turbid water. When salmon migrate from the open ocean (where blue-green light predominates) into inland streams (where incident light is red-shifted), they switch from using 11-cis retinal as their visual chromophore to 11-cis 3,4didehydroretinal which has red-shifted spectral properties. This chromophore switch causes a dramatic red-shift of the fish's opsin spectral sensitivity, thereby enhancing the animal’s ability to see infrared light. We have identified the enzyme that mediates this red-shift, and we plan to co-express it with optogenetic actuators in mammalian neurons in vivo, thereby red-shifting their action spectra. This spectral reprogramming strategy promises to enhance optogenetic therapies for blindness by permitting the use of near infrared light (>700 nm) for treatment purposes, thereby minimizing retinal photodamage.

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23) Live imaging of mitochondrial BAX recruitment in mouse retinal ganglion cells Maes, ME1,2, Schlamp, CL2, Nickells, RW2 1

Institute of Science and Technology Austria, Klosterneuberg, Austria Department of Ophthalmology and Visual Sciences, University of Wisconsin – Madison, Madison, WI, USA 2

Glaucoma is a group of optic neuropathies characterized by retinal ganglion cell (RGC) death and is one of the world’s leading causes for irreversible blindness. A major risk factor for this disease is elevated intraocular pressure (IOP), which is the target for all currently available treatments. Even with successful intervention to lower IOP, RGC loss often progresses, which highlights the need to develop neuroprotective strategies. RGC death is carried out through the intrinsic apoptotic program, which is controlled by the BCL2 gene family. BAX, a proapoptotic member of the BCL2 gene family, functions as the switch from cell life to cell death, or the point of no return in the apoptotic pathway. Genetic deletion of Bax is the only neuroprotective strategy that provides long term survival of RGCs after optic nerve damage in mouse models. Therefore, targeting BAX functionality could provide a successful neuroprotective strategy to complement current treatments. BAX is an inactive monomer predominantly located within the cytosol, but once activated, will recruit to the mitochondrial outer membrane (MOM) where it forms dimers, then oligomers, that permeabilize the MOM. In order to target BAX, it is critical to understand the timing to BAX activation after optic nerve injury and the rate at which BAX recruitment proceeds within RGCs. Through AAV2-GFP-BAX transduction of mouse RGCs, the percentage of RGCs exhibiting BAX activation was monitored after optic nerve injury in an acute model of optic nerve crush in two mouse strains (BALB/c and CB6F1) and in a chronic model of spontaneous IOP elevation (DBA/2J mouse). In the acute model, both mouse strains showed a significantly greater percentage of RGCs exhibiting BAX activation at 3, 5 and 7 days after optic nerve crush (BALB/c p < 0.05; CB6F1 p < 0.01) when compared to contralateral control. The chronic model showed a significant increase in percentage of RGCs exhibiting BAX activation between 8-12 months of age (p < 0.05). The rate of BAX recruitment can be quantified using time-lapse imaging and mitochondrial tracking. Live imaging of ex vivo mouse retina showed a significantly slower average rate of BAX recruitment (-5.1 ± 1.5 Log2RFU/min, p < 0.001) when compared to any of four tissue culture conditions. Additionally, a reversible BAX recruitment phenomenon was observed in a subset of RGCs after optic nerve 92

injury, which may be important when developing therapeutic strategies to target BAX.

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24) Functional evaluation of visual impairment in animal model of retinal exitotoxicity Pedro. de la Villa1, Santiago Milla-Navarro1, Estrella Calvo1, Violeta GomezVicente2, Laura Ramírez1, Nicolás Cuenca3; 1

Departamento de Biología de Sistemas. Universidad de Alcalá, Madrid. Spain Departamento de Optica, Farmacología y Anatomía 3 Departamento de Fisiología, Genética y Microbiología. Universidad de Alicante. Alicante 2

Background: In the previous years, Kainic acid (KA) and N-methyl-D-aspartic acid (NMDA) have been used to induce excitotoxic damage in neurons of the inner retina in animal models. Although structural characterization of retinal damage after the effect of KA/NMDA has been shown, no effective functional tests have been developed to characterize the degree of visual impairment. In the present work we demonstrate the utility of pattern Electroretinogram (pERG) and the optomotor test to fully characterize the degree of visual deficit in animal models of retinal excitotoxicity. Methods: Retinal cell damage was induced by injection of 1 μl of PBS containing different doses of KA/NMDA into the right eye. Same volume of PBS was injected in the left eye. Binocular recordings were performed by the pERG technique prior to, immediately after and one week after the induction of excitotoxic retinal damage. Different spatial frequencies were tested. In addition, we performed optomotor test in the same animals. Different spatial frequencies and degrade light contrasts was tested in clockwise and counterclockwise directions. Results: The pERG recordings from eyes injected with KA/NMDA showed a significant decrease in amplitudes of the P50 and N95 trace components one week after retinal damage. The pERG showed that at increasing concentrations of KA/NMDA, the amplitude of the P50 and N95 components were diminished, being almost null for highest excitotoxic concentrations. Results from the optomotor test corroborated all data obtained by pERG. At high concentrations of excitotoxic agents, the visual acuity of the animals showed a significant decrease when the optomotor stripes moved in counterclockwise direction, indicating the damage of the right eye. Conclusions: We demonstrate that the pERG and optomotor test are effective for evaluate the visual impairment induced in retinal cells. This work opens the possibility to test the effect of therapeutic agents on retinal damage in living animals.

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25) Long term visual restoration using optogenetic engineering of retinal ganglion cells with AAV2.7m8-ChrimsonR-td-Tomato Gauvain G, Akolkar H, Chaffiol A., Caplette R., Jaillard C., Brazhnikova E., Desrosiers M., Pruneau D.1, Duebel J., Benosman R., Dalkara D., Sahel J-A., Picaud S. Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision 1 Gensight Biologics (Paris)

Retinitis Pigmentosa (RP) is a collection of inherited dystrophies with similar clinical features resulting in the progressive loss of photoreceptors leading to blindness. Although gene therapy is currently in clinical trial for hereditary dystrophies, the considerable genetic heterogeneity in RP preclude the rapid development of treatments for patients. In this pre-clinical study we are considering an alternative to classic gene therapy and propose to use viral vector to deliver a microbial opsin, ChrimsonR, to optically control surviving retinal ganglion cells. We previously selected AAV2.7m8-ChrimsonR-tdTomato as the most potent of our viral constructs. We investigated here in nonhuman primates the treatment efficacy after long term (6 month) expression of our transgene, for three viral doses. None of the injected primates developed significant immune response or sign of morphological alteration based on the fundus images. We performed ex-vivo retinal ganglion cells (RGCs) recordings on 256 electrode multielectrode arrays (MEA). We isolated the response of RGCs expressing our construct from natural light response using pharmacological blockers (L-AP4, CNQX, CPP). We stimulated retinas using a digital micro mirror device (DMD) with a light source centered at 600nm (+/-20nm) allowing for: 1) full field stimuli of various durations, and 2) patterned stimulation (small spots, moving bar and objects). We also used a variable wavelength source in order to determine spectral sensitivity. Our result indicates the highest viral does as optimal for best efficacy in term of transduction efficiency (evaluated through overall induced activity on the MEA, and postfixation cell counting on confocal images) . We also demonstrate excellent spatiotemporal sensitivity of the reactivated retina (responses for spot as small as 50μm) as well as extremely short delay of responsiveness (~10msec for first evoked action potential). In combination of MEA recordings we did 2-photon guided single cell patch clamp experiments (whole cell and cell attached) to directly measure photocurrent induced by our opsin. These different experiments demonstrate stable expression of ChrimonR-tdTomato in non-human primates on a 6 month period post-infection, they also 95

provide evidence for functional efficacy at doses, which could represent the therapeutic dose range for the future clinical trial.

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26) Characterization of pathological mechanisms during aging of the Prpf31mutant mouse model of retinitis pigmentosa Abdallah Hamieh and Emeline F. Nandrot Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision

Mutations in the ubiquitously-expressed Pre-mRNA Processing Factors (PRPF) 3, 8, and 31 constitute the second most prominent cause of non-syndromic autosomic dominant retinitis pigmentosa (adRP) in humans. We previously determined that young Prpf-mutant mice exhibit abnormal RPE functions and develop a phenotype with age. Despite the difference in the pathological timing between patients and corresponding mouse models, human ARPE-19 cells downregulated for PRPF31 and Prpf31-mutant primary RPE cells display a similar defect in retinal phagocytosis, suggesting this mouse model can be used as a paradigm to identify related pathological processes. Thus, we set out to characterize RPErelated stress pathways occuring in Prpf31-mutant mice. Studies were conducted on 3 to 24-month-old animals in order to dissect the full series of molecular events. Gene and protein expression levels for the mitochondrial respiratory chain, the endoplasmic reticulum and detoxification pathways were assessed by qPCR and immunoblots. In the mitochondrial respiratory chain, in contrast to wildtype controls CoxIV (complex IV) expression increases between 6 and 18 months of age. Expression of the detoxifying enzyme SOD1 (CuZn-SOD, cytosol) varies at different ages in mutant mice. SOD2 (Mn-SOD, mitochondrial matrix) shows decreased gene and protein expressions at all ages. Accumulation of lipids was evaluated on histological sections by Oil-Red-O and Bodipy stainings. Lipid labelings were validated on control beta5 knockout integrin mouse sections that accumulate lipofuscin and lipids with age. Stainings on eye sections confirm our hypothesis that Prpf31-mutant RPE cells gradually accumulate more lipid droplets than wildtype littermates from 6 months of age. Taken together our results suggest that oxidative processes take place at different levels in Prpf31-mutant RPE cells. We believe our data will help us decipher the etiology of tissue-specific adRP cases linked to ubiquitously-expressed splicing factors and could contribute to define a new potential common therapeutic approach for all PRPF genes.

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27) Retinal prostheses with conjugated polymers: a new strategy to cure photoreceptor degeneration in the pig Maya-Vetencourt JF1, Colombo E1, Eleftheriou C1, Di Francesco M1, Desii A2, Mete M3, Zangoli M2, Pertile G3, Lanzani G2, Benfenati F1 1

Center for Synaptic Neuroscience and Technology, Istituto Italiano di Tecnologia, Genova, Italy 2 Center for NanoScience and Technology, Istituto Italiano di Tecnologia, Milan, Italy 3 Ophthalmology Department, Sacro Cuore Hospital, Negrar, Italy 4 Institute of Organic Synthesis and Photoreactivity, National Research Council, Bologna, Italy

In spite of the very high prevalence of age-related macular degeneration (dry or atrophic AMD), the therapeutic approaches to the vast majority of cases have been unsatisfactory so far and no approved treatment or cure exists. We recently introduced a new generation prosthetic device for neuronal photostimulation, which offers several advantages including photovoltaic functioning, high biocompatibility, mechanical compliance, and sensitivity close to daylight range. In particular, we developed a planar fully organic device composed of a flexible and highly conformable silk substrate covered with photoactive layers that, once implanted in the subretinal space of the eye of Royal College of Surgeons (RCS) rats, was able to rescue light sensitivity and visual acuity. In addition to their photovoltaic properties, conjugated polymers are highly biocompatible and entertain complex relationships with live cells. Although photovoltaic in nature, conjugated polymers are not classical current injectors and may involve several mechanisms of interaction with the excitable cells of the retina, including capacitive charging of the interface, local accumulation of protons and, at high light intensities, local temperature increases. With the aim of translating the rodent study to the pig that has an eye comparable to the human eye in terms of size and structure, we engineered a larger device using polyethylenterephthalate (PET) that is approved for clinical use and strictly adherent to the polymer layers. We tested the new device in the RCS rat and found a rescue of visual acuity as good as that obtained with the fibroin-based devices. We then setup a model of photoreceptor degeneration in the pig, based on the intravenous injection of iodoacetic acid (IAA) which causes the disappearance of the and b waves in the electroretinogram, both under scotopic and photopic conditions. Studies on the rescue of retinal activity after IAA lesion and prosthesis implantation in the pig eye are ongoing. Taken together, these results highlight the potential clinicalrelevance of this organic approach in retinal degenerative blindness.

POSTER SESSION II: RETINAL DISEASES AND THERAPIES 1) A new promoter allows optogenetic vision restoration with enhanced sensitivity in macaque retina Antoine Chaffiol1,2,3, Romain Caplette1,2,3, Céline Jaillard1,2,3, Elena Brazhnikova1,2,3, Mélissa Desrosiers1,2,3, Elisabeth Dubus1,2,3, Emilie Macé1,2,3, Olivier Marre1,2,3, Jens Duebel1,2,3, José-Alain Sahel1,2,3,4,5, Serge Picaud1,2,3, Deniz Dalkara1,2,3 1

INSERM, U968, Institut de la Vision, Paris, F-75012, France, Sorbonne Universités, UPMC Univ Paris 06, UMR_S968, Institut de la Vision, Paris, F75012, France, 3 CNRS UMR7210, Institut de la Vision, Paris, 75012, France , 4 CHNO des Quinze-Vingts, DHU Sight Restore, INSERM-DHOS CIC, 28 rue de Charenton, 75012 Paris, France. 5 Fondation Ophtalmologique Adolphe de Rothschild, Paris, France 2

The majority of inherited retinal degenerations converge on the phenotype of photoreceptor cell death. In these diseases second and third order neurons are spared 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 for the first time activation of macaque retinas, up to 6 months post-injection, using channelrhodopsin – CatCh at safe light intensities. High-level Catch expression was achieved thanks 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 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 (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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2) Unexpected prevalence of inflammatory response in a mouse model of Retinitis Pigmentosa: looking toward therapeutic potential Martina Biagioni, Viviana Guadagni, Elena Novelli1, Enrica Strettoi CNR Neuroscience Institute, Pisa, Italy

In Retinitis Pigmentosa (RP), a mutation causes the primary degeneration of rods, followed by the secondary death of cones, culminating into blindness. Due to their fundamental role in human daylight vision, preservation of even a fraction of cones would ensure relatively normal lives to RP patients. We previously showed that Environmental Enrichment (EE) slows down photoreceptor degeneration in rd10 mutant mice, a known RP model. Searching for molecular effectors of the protective retinal response triggered by EE, we demonstrated that at the peaks of rod and cone death, inflammation and immune responses constitute the prevalent biological processes. Here we aim at investigating whether a) EE beneficial effects are associated to a reduction of retinal inflammation; and b) rescue effects can be mimicked by steroid anti-inflammatory treatment. rd10 mice were reared from birth in EE with enhanced motor, social and sensorial stimuli. Controls were age-matched rd10 and WT C57Bl6/J mice kept in standard (ST) conditions. For molecular studies, retinas were harvested at 24 or 45 days and flash frozen in dry ice; the expression of cytokines and chemokines was determined by RT-PCR using 9 mice/experimental group. RNAs from retinas of mice raised in ST, EE and WT conditions and aged 24 and 45 days were processed for whole transcriptome analysis (Ion Torrent, Life Technologies, Grand Island, NY). For histology studies, retinas harvested at 24 or 45 days were fixed in 4% PFA, cryostat sectioned or processed as whole mount preparations and stained with cell-specific antibodies. Samples were examined with a Leica SP2 confocal microscope or with a Zeiss Axio Imager.Z2 ApoTome2 fluorescence microscope. Iba1 staining showed a strong pattern of microglial activation and infiltration in the outer retina of ST rd10 mice, a more moderate pattern in retinas of EE rd10 mice and virtually no activation in WT samples. RT-PCR followed by statistical analysis (Student t-test) showed widespread overexpression of inflammatory genes, including Ccl3 and Ccl5. In EE samples, this pattern was partially reverted toward the WT expression. Whole transcriptome analysis revealed a strong pattern of immune response in both rd10 ST and EE samples with respect to WTs, however more moderate in EE samples. Rd 10 mice administered oral steroids showed increased cone survival compared to controls. 100

In conclusion, environmental enrichment slows down inherited photoreceptor death concomitantly decreasing retinal inflammation. The effect of direct antiinflammatory administration on cones survival reveals a significant pathogenetic role of inflammation in RP. These data open the perspective of slowing down retinal decay in human RP targeting the retinal inflammatory response. Funding sources: Macula Vision Research Foundation, USA; Fondazione Roma, Italy; European Project H2020-MSCA-ITN-2014

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3) The contribution of microglia to early vascular dysfunction in diabetic retinopathy Mills, S.A.1 , Jobling, A.I.1 , Phipps, J.A.1 Bui, B.V.2, He, Z.2, Hui, F.2, Fletcher, E.L.1 1

Department of Anatomy and Neuroscience, The University of Melbourne Department of Optometry and Vision Sciences, The University of Melbourne

2

Background: Diabetic retinopathy remains the largest cause of blindness in working age adults in developed countries. Changes to the retinal vasculature occur early after diabetes onset, often preceding visual loss. Understanding the mechanism of early retinal dysfunction is therefore integral to halting the progression of the disease. Using a rat model of diabetes, retinal blood flow and the role that retinal microglia play in vascular control were assessed in the early diabetic retina. Method: Diabetes was induced in Dark Agouti rats by exposure to streptozotocin (STZ, 55mg/kg). After 4 weeks of hyperglycaemia, blood flow kinetics of arterioles, capillaries and venules were quantified by sodium fluorescein video angiography (n=21). Blood vessel diameters were measured from fundus images and optical coherence tomography angiography(OCTa) (n=10), while immunohistochemistry was used to quantify vessel density and the association of microglia (Iba-1) with vessels (n=13). Live cell imaging was performed on ex-vivo retinae to assess the dynamic effects of fractalkine on vessel diameter (n=5). Results: In STZ-diabetic rats, retinal blood vessels were slower to fill compared to control animals (arterioles: -23.1% [95% CI= -6.8% to -36.8%]; capillaries: -18.1% [-5.2% to -31.0%]; venules: -14.5% [-3.7% to -25.3%]) and had slower drain rates (arterioles: -52.6% [-26.6% to -78.5%]; capillaries: -48.1% [-18.1% to -78.0%; venules: -48.8% [-24.9% to -72.6%]). Arteriole to venous ratios remained unchanged in diabetes, with no loss of capillaries. However, the average width of capillaries was reduced in diabetes (control: 10.37μm ± 0.33; diabetes: 9.41μm ± 0.29, p<0.05). Retinal microglia showed an increased association with capillaries within the central retina (+319.8 cells/mm2 vessel [601.0 to 368.65]). Fractalkine, the sole ligand to the microglial receptor, Cx3cr1, caused vasoconstriction of vessels in healthy retina (-12.13% of baseline [0.77% to 23.49%]), while this was absent in diabetic animals (-5.19% [-16.55 to 6.172]). Summary: Early after the onset of diabetes, retinal blood flow is reduced and microglia increase their contact with retinal capillaries, which exhibit a reduced diameter. Microglia act as vasomodulators and this process is disturbed in early diabetic retinopathy. These results suggest that microglia are important players in the progression of vascular abnormalities in diabetic retinopathy. 102

4) Development of a gene therapy approach for cCSNB when mutations in GRM6 and LRIT3 are involved Juliette Varin1, Miguel Miranda de Sousa Dias1, Thomas Pugliese1, Marion Neuillé1, Christelle Michiels1, Melissa Desrosiers1, José-Alain Sahel1,2,3,4,5,6, Isabelle Audo1,2,3, Deniz Dalkara1, Christina Zeitz1 1

Sorbonne Universités, UPMC Univ Paris 06, INSERM U968, CNRS UMR 7210, Institut de la Vision, Paris, France. 2 CHNO des Quinze-Vingts, DHU Sight Restore, INSERM-DHOS CIC1423, Paris, France. 3 Institute of Ophthalmology, University College of London, London, United Kingdom. 4 Fondation Ophtalmologique Adolphe de Rothschild, Paris, France. 5 Académie des Sciences, Institut de France, Paris, France. 6 Department of Ophthalmology, The University of Pittsburgh School of Medicine, Pittsburgh, PA, United States

Complete Congenital Stationary Night Blindness (cCSNB) is a non-progressive heterogeneous retinal disease characterized by visual impairment and protein mislocalization. Affected individuals present a severe reduction in the amplitude of the b-wave on electroretinogram (ERG) recordings, an impairment of vision in dim-light conditions often associated with severe myopia, nystagmus and strabismus. Mutations in GRM6, LRIT3, NYX, TRPM1 and GPR179 lead to this subform of CSNB. These genes code for proteins expressed and localized at the dendritic tips of ON-bipolar cells. Mice lacking Grm6 and Lrit3 show a similar phenotype as patients, namely the severe amplitude reduction of the b-wave on the ERG. The respective proteins are absent in each model. In addition, other partners of the cascade show reduced or undetectable localization in the dendritic tips of ON-bipolar cells resulting in a signal transmission defect between photoreceptors and ON-bipolar cells. Although CSNB is responsible for visual handicap no efficient treatment is yet available. The aim of this work is to develop two gene therapy approaches to restore the wild-type phenotype in these two mouse models. Two different gene-specific AAV vectors have been developed for each model (AAV-Grm6 and AAV-Grm6-MBD or AAV-Lrit3-GFP and AAV-Lrit3), driven by a Grm6-promotor. These vectors have been injected intravitreally in KO mice (P300 or P30 or P15). Subsequently, the rescue has been followed by studying the visual function through ERG recordings and optokinetic tests but also gene expression and protein correct localization. Preliminary data showed encouraging results for both projects. For the Grm6 gene therapy approach, expression of the gene and correct localization of the protein and other partners of the cascade have been partially restored in one P300 mouse 5 months postinjection. ERG recordings and behavioral tests are currently ongoing for the newly 103

injected ones. Concerning the Lrit3 project, GFP fluorescence was detectable 15 days post-injection confirming the efficient transgene delivery for the AAV-Lrit3GFP construct. ERG recordings at 8 weeks post-injection or 24 weeks postinjection for the animals injected with AAV-Lrit3 were performed. Preliminary results revealed a small detectable b-wave recorded on 2 animals. This ongoing study has a real potential clinical value and hopefully will significantly contribute to treat patients with retinal disorders by a gene therapy based approach.

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5) Two convenient experimental models of photoreceptor degeneration for screening of molecular photoswitches Alexander Rotov1,2, Sitnikova Viktoriya1,2, Michael Firsov2, Mikhail Ryazantsev3 & Luba Astakhova2 1

I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry, RAS Peter the Great Saint-Petersburg Polytechnic University 3 Institution of the Russian Academy of Sciences Saint Petersburg Academic University 2

Many types of retinal disorders result in photoreceptor (PR) loss, while the inner retinal neurons survive. Those neurons can serve as a target of therapies, which restores the photosensitivity of retina devoid of PR. One of the strategies for restoring visual function is use of synthetic photoisomerizable small molecules, photoswitches. One of them, AAQ (acrylamide-azobenzene-quaternary ammonium), was shown to restore photosensitivity of retina of rd1 mice (model of PR degeneration) (Polosukhina et al., 2012). While perspective in some aspects, AAQ has too some disadvantages that makes need for search of new photoswitches with improved spectral and kinetic properties. Here we describe a new animal model that allows fast screening of candidate compounds. Method: The models are based on the frog R. ridibunda. Model I – mechanical removal of PR layer from isolated retina using filter paper. Model II – inducing PRspecific degeneration in the live frog by intraocular injection of antibiotic tunicamycin (1 μg/10 mg of body weight in DMSO, Fliesler et al., 1984, Fliesler, Basinger, 1985). To confirm the PR degeneration electroretinogram (ERG) from isolated retina (model I and II) and in vivo ERG (Model II – during development of the degeneration up to 3 weeks) were recorded. For model II extent of retinal degeneration was assessed by light microscopy. Retinal preparations with model PR degeneration were incubated with AAQ (1 mM) and then were tested by green and UV light (flashes and steps). Results: In the Model II degeneration developed within 3 weeks. Gradual decrease of in vivo ERG from injected eye and light microscopy suggests elimination of PR layer but surviving of inner retinal neurons. In the both models retinas in vitro ERG demonstrated no photoresponse to green/UV flashes and steps (up to 5 s). After incubation with AAQ both models acquired photosensivity for UV steps but not for green and UV flashes and green light steps. Conclusion: Both models of PR degeneration, mechanical removal of PR layer and tunicamycin-induced PR degeneration, are suitable for mass screening of the new candidate photoswitches. Study was supported by Russian Foundation for Basic Research (15-29-03872 ofi_m) and and Academic Program for Basic Research grant. 105

6) Dynamic interplay of innate and adaptive immunity during sterile retinal inflammation: Insights from the transcriptome Matt Rutar 1, Riccardo Natoli 2, Jan Provis 2 1

Anatomy and Neuroscience, University of Melbourne, Melbourne, VIC, Australia; The John Curtin School of Medical Research, The Australian National University, ACT, Australia. 2

Introduction: Inflammation is implicated in many retinal degenerations including AMD. Although microglia/macrophage responses are well-described in AMD and other modes of sterile retinal inflammation, the delineation of innate and adaptive immunity amongst the broad leukocyte infiltrate, and the gene networks which drive these responses, are largely unknown. To address this gap we mapped the global gene signature of the retinal leukocyte populations following sterile inflammation, using RNA-sequencing (RNA-seq) in the photo-oxidative damage model. Methods: Adult rats exposed to 1000 lux light damage for 24hrs were euthanized immediately (0 days), or at 3 and 7 days post-exposure. Retinas were collected and leukocytes retrieved by FACS using the pan-white blood cell marker CD45. Total RNA was extracted and cDNA libraries generated for RNA-seq. Functional enrichment of differentially regulated genes (ANOVA, adjusted p<0.05, FDR<0.05, n=3) were determined from the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, to predict dominant classes of immune cells recruited across the time course. Predicted subpopulations of retinal leukocytes were validated using flow cytometry Results: During the early phase following photo-oxidative damage (0 Days), the transcriptome was dominated by biological processes associated with innate immunity, including cytokine and chemokines, and this correlated with the acute infiltration of macrophages and neutrophils. By 3-7 days post-damage the transcriptome indicated high levels of antigen presentation and lymphocytedriven processes, which coincided with strong T-cell recruitment within the retinal environment and complete breakdown of the blood-retinal barrier. Conclusions: The transcriptional signature of the retinal CD45+ population reveals that sterile inflammation evokes dynamic changes in innate and adaptive immunity during retinal degeneration.

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7) Visual BOLD response in late-blind subjects with Argus II retinal prosthesis E. Castaldi1, G. M. Cicchini2, L. Cinelli3, L. Biagi4, S. Rizzo3, M. C. Morrone1,4 1

Department of Translational Research on New Technologies in Medicine and Surgery, University of Pisa, Pisa, Italy, 2 CNR Neuroscience Institute, Pisa, Italy, 3 Azienda Ospedaliero-Universitaria Careggi, SOD, 4 Stella Maris Scientific Institute, Pisa, Italy

The recent advance in retinal prosthesis technologies generates hope for partially restoring vision to blind people with retinal pathologies. However, these strategies require the visual system downstream of the retinal circuitry to be capable of transmitting and elaborating visual signals. We studied the capability of plastic remodeling in late blind subjects implanted with the Argus II Retinal Prosthesis with psychophysics and functional MRI (fMRI). After surgery, six out of seven retinitis pigmentosa (RP) blind subjects were able to detect high-contrast stimuli using the prosthetic implant and the performance correlated with the amount of training after the surgery. However, direction discrimination to contrast modulated stimuli remained at chance level in all of them. No subject showed any improvement of contrast sensitivity in either eye when not using the Argus II. Before the implant, the Blood Oxygenation Level Dependent (BOLD) activity in V1 and the lateral geniculate nucleus (LGN) was very weak. Surprisingly, after prolonged use of Argus II, BOLD responses to visual input were enhanced. This is, to our knowledge, the first study tracking the neural changes of visual areas in patients after retinal implant, revealing a capacity to respond to restored visual input even after years of deprivation.

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8) Two steps of photoreceptor degeneration in an animal model of Retinitis Pigmentosa Ulisse Bocchero1, Monica Mazzolini1, Beatrice M. Tam2, Colette N. Chiu2, Orson L. Moritz2 and Vincent Torre1 1

International School for Advanced Studies (SISSA), Trieste, Italy. Department of Ophthalmology & Visual Sciences, University of British Columbia, Vancouver, BC, Canada. 2

Retinitis pigmentosa (RP) leads to a progressive degeneration of rod OSs followed by the death of the entire rod. RP is a genetic disease caused by mutations in some dozens of different genes. Mutations in the gene coding for rhodopsin are responsible for a large portion of autosomal dominant RP (adRP). The mutation of proline23 in the N-terminal domain to histidine - mutation P23H - accounts for a significant fraction of all cases of RP. The exact mechanisms of cellular toxicity remain unclear. Previous studies suggest a mechanism involving the destabilization of P23H rhodopsin during light exposure, leading to a decreased endoplasmatic reticulum exit. In the present manuscript we probed phototransduction in Xenopus laevis tadpoles expressing the bovine form of P23H rhodopsin (bP23H) by measuring photoresponses with suction electrodes in animals bred and housed in continuous darkness. Under these circumstances bP23H rods do not degenerate, and have almost normal photoresponses, but following an exposure to light, sufficient to cause only a few photoisomerization of mutant bP23H rhodopsin per disc, phototransduction becomes impaired, although rods retained their morphological integrity. If the transgenic animals are exposed to light for at least 12 minutes, rods degenerate. Therefore in bP23H rods, light-induced degeneration has two steps: the first step occurring following a brief exposure to light causes an impairment of the phototransduction cascade while, under prolonged illumination, rods collapse and die.

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9) ON/OFF asymmetrical dysfunction of retinal mechanisms in Duchenne muscular dystrophy patients Mirella Telles Salgueiro Barboni1,2, Balázs Vince Nagy1,3, Cristiane Maria Gomes Martins1, Tina Tsai4,5, Marcelo Fernandes da Costa1, Francisco Max Damico1,6, Jan Kremers4,5, Dora Fix Ventura1 1

Department of Experimental Psychology, University of Sao Paulo, Brazil; Department of Ophthalmology, Semmelweis University, Budapest, Hungary; 3 Department of Mechatronics, Optics and Engineering Informatics, Budapest University of Technology and Economics, Budapest, Hungary, 4 Department of Ophthalmology, University Hospital Erlangen, Germany, 5 Department of Anatomy II, University of Erlangen-Nurnberg, Erlangen, Germany 6 Departament of Ophthalmology, University of Sao Paulo, Brazil 2

The b-wave attenuation of the dark-adapted full-field ERG is a characteristic visual clinical sign of Duchenne muscular dystrophy (DMD) patients. Recently we have reported that DMD patients show an asymmetric photopic ERG disturbance in which the ON-pathway is more affected than the OFF-pathway. On the other hand, in mesopic stimulation when there is rod contribution, this asymmetry is less clear, since in DMD patients both mesopic ON and OFF retinal responses are deeply altered. Here we compare mesopic and photopic ON/OFF asymmetry of DMD patients (N=9 males; 18.9 ± 4.6 years old) showing normal visual acuity and altered contrast sensitivity, with the ON/OFF asymmetry of an age-matched control group. The ERG recordings and the signal analysis have been previously reported (IOVS54:3195-3204;2013). Briefly, 4 Hz sawtooth (rapid-ON and rapidOFF) luminance modulation of white LEDs was used at mesopic (1 cd/m²) and photopic (60 cd/m²) conditions. Amplitudes of the b-wave (ON response) and of the d-wave (OFF response) were measured. The ON/OFF ratio was calculated by dividing the amplitude of the b-wave by the amplitude of the d-wave. The ON/OFF ratios were then compared. The ON/OFF mesopic ratios were similar between patients (0.91 ± 0.42) and controls (0.93 ± 0.60), meaning that both ON and OFF mesopic responses were similarly affected in DMD patients. The ON/OFF photopic ratios were significantly different (p < 0.01, t-test): patients = 0.92 ± 0.34 and controls = 2.00 ± 0.44 corroborating the asymmetrical alteration. Mesopic ON and OFF signals are similarly and deeply affected by DMD while photopic ON signals are more altered than the OFF signals. We have recently reported that ON/OFF photopic unbalanced signals in the human retina result in luminance signals in situations in which normally color-opponent signals would be found (IOVS 57:3581-3587; 2016). Now we add to this information that symmetric functioning 109

of the ON/OFF mechanisms is also necessary for the appropriate contrast sensitivity in perception. Financial support: BMBF (grant 01DN14009 to JK); CNPq (grant 470785/2014-4 and 404239/2016-1 to MTSB, and 490428/2013-4 to DFV); CAPES (grant 3263/2013 to DFV); DFG (grant KR1317/13-1 to JK); FAPESP (grant 2016/22007-5 and 2016/04538-3 to MTSB, and 2014/26818-2 to DFV).

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10) Optogenetically controlled photoreceptors derived from hiPSC respond to light and restore light mediated behavior in transplanted blind mice Marcela Garita‐Hernandez, Antoine Chaffiol, Laure Guibbal, Fiona Routet, Emilie Keomani, Hanen Khabou, Amelie Slembrouck, Giuliana Gagliardi, Marusa Lampic, Yannick Le Dantec, Sacha Reichman, Jose Alain Sahel, Olivier Goureau, Jens Duebel*, Deniz Dalkara* (*Co-last-authors) Institut de la Vision, 17 Rue Moreau, 75012 Paris, France

Retinitis Pigmentosa is a blinding retinal degenerative disease characterized by the loss of photoreceptors. The aim of our work is to provide functionally active photoreceptors for transplantation in patients suffering from photoreceptor degeneration. Human induced pluripotent stem cells (hiPSC) can be differentiated towards photoreceptors. Thus far, it has been impossible to obtain mature photoreceptors with a functional outer segment (OS) for light capture. In absence of an OS, a photoreceptor can be engineered to sense light by expression of optogenetic light sensors (Busskamp et al, 2010; Chuong et al, 2014). We have thus developed a protocol of differentiation to obtain photoreceptors from hiPSC, which we then modify genetically to carry Jaws. This hyperpolarizing chloride pump renders the photoreceptors responsive to light even in the absence of OS’s. The expression of Jaws is restricted to the membrane of the cones and is responsible for the light responses observed by patch clamp experiments. Jawsexpressing cones survive engraftment into retinas of rd1, rd10 and Cpfl/Rho mice; three animal models showing severe retinal dysfunction. The transplanted cells integrate into the outer nuclear layer forming functional synaptic connections with bipolar cells. Importantly the transplanted animals are able to perform lightguided tasks. RESULTS 1. We have efficiently generated retinal organoids for transplantation. We have generated retinal organoids using a 3-dimensional (3D) culture system. Based on a previously published protocol (Reichman et al. 2014) and starting from a highly-confluent 2D culture we observed the spontaneous generation of 3D structures growing out at around 30 days of differentiation. These retinal structures were isolated and further differentiated as retinal organoids in floating conditions expressing photoreceptor specific markers such as RCVN, CAR, OTX2 and CRX 2. We engineered cones in the organoids to express Jaws. Jaws was introduced into cones by AAV infection. AAV2-7m8 (Dalkara et al. 2013) 111

expressed Jaws in fusion with GFP under the control of CAR (cone arrestin) promoter. 3. Jaws is expressed in the membrane and responds to light. Immunofluorescence analysis showed Jaws is expressed mostly at the membrane of photoreceptor cells, immunoreactive for CRX, RCVN, OTX2 and for the cone specific marker CAR. More importantly, 2 photon guided clamp experiments demonstrated that Jaws-expressing cones respond to light stimulus. 4. Subretinal injections of dissociated retinal organoids in blind mice. Dissociated organoids were injected subretinally in rd1, rd10 and Cpfl/Rho mice. Fundus analysis revealed transplanted cells survive the engraftment. The Light/Dark box modified with a 590nm LED, showed significant difference between controls (GFP-only transplanted) and mice transplanted with Jaws photoreceptors demonstrating light avoidance behavior was achieved. 5. Photoreceptors from dissociated retinal organoids connect with the second order neurons. Histological analysis of transplanted animals showed Jaws+ cells have integrated only in the photoreceptor specific outer-nuclear layer, in direct contact with the second order neurons- the bipolar cells (PKCa and GOa). Integrated Jaws positive cells also expressed CRX, RCVN and CAR confirming their photoreceptor phenotype

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11) Transplantation of halorhodopsin expressing photoreceptor precursors restores visual responses in blind mice Maruša Lampič, Antoine Chaffiol, Laure Guibbal, Abhishek Sengupta, Marcela Garita-Hernandez, Fiona Routet, Jose Sahel, Marius Ader, Deniz Dalkara, Jens Duebel Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, Paris, France.

Transplantation of photoreceptor is a promising approach to substitute for the loss of photoreceptors in retinal degenerative diseases. While in mouse models of slow retinal degeneration, restoration of visual function after transplantation has been shown, cell replacement strategies have been more challenging in models of fast and severe degeneration. Lack of light sensitivity after photoreceptor transplantation is a major problem for future therapy in patients. To overcome this obstacle, we introduced a hyperpolarizing optogenetic tool (halorhodopsin: NpHR) in photoreceptor progenitors, derived from new-born mice, and transplanted them into blind mice with loss of rod/cone function (Rho−/−;Cpfl1[Pde6c−/−)]). Illumination of NpHR-expressing cells generates hyperpolarization, thus mimicking the function of healthy photoreceptors to light. Three weeks after transplantation of optogenetically transformed photoreceptors, retinas were dissected and functionality was tested by a set of electrophysiological experiments. Two-photon guided whole-cell patch-clamp from transplanted photoreceptors revealed robust light responses at 1016 photons cm-2s-1, matching the action spectrum and the temporal properties of halorhodopsin. Moreover, by using extracellular spike recording with a multielectrode-array (MEA), we could measure ON- an OFF-light responses in retinal ganglion cells from retinas of treated RD mice, demonstrating that transplanted photoreceptors can form synaptic connections to the inner retinal circuitry and that halorhodopsin induced signals are transmitted to the retinal output neurons. Subsequent immuno-histochemical analysis confirmed that the transplanted photoreceptors, equipped with NpHR, lack their outer segments, but integrated into the retina, forming contacts to bipolar cells. Finally, the light/dark box experiment showed significant light avoidance behavior in treated mice.

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12) Taurine deficiency induces retinal inflammation Hadj Said, Wahiba1; Dubus, Élisabeth1; Fouquet, Stéphane1; Manon Valet1; Garcia-Ayuso, Diego2; Villegas-Perez, Maria P.2; Sahel, Jose A.1, 3; Picaud, Serge A.1 1

Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, Paris, France. 2 Departamento de Oftalmología, Facultad de Medicina, Universidad de Murcia, Murcia, Spain and Instituto Murciano de Investigación Biosanitaria- Hospital Virgen de la Arrixaca (IMIB-Arrixaca), Murcia, Spain. 3 CHNO des Quinze-Vingts, DHU Sight Restore, INSERM-DHOS CIC, Paris, France.

BODY: Purpose: We recently showed that taurine deficiency induced the selective and concomitant degeneration of cone photoreceptors and retinal ganglion cells (RGC) (Hadj-Saïd et al., IOVS 2016; 57:4692-4703). We investigate here if the degenerative process may trigger retinal gliosis with microglial activation and their migration into the photoreceptor layers. Methods: We used the taurine transporter (Tau-T) inhibitor, guanidoethane sulfonate (GES), to induce taurine depletion. Retinas were dissected as wholemounts and immunolabelled for Iba1 (microglia) and PNA (cones). The retinal distribution of microglia was studied using 3D images from confocal microscopy. Retinal inflammation was also examined on retinal sections to define the relationship of microglial cells to apoptotic cells (Tunnel Assay). Results: In retinal sections, microglial cells were often observed in the outer layer (ONL) in GES-treated mice whereas they were not found in the same layer in non-treated mice. This abnormal distribution of microglial cells in the ONL was also clearly detectable on retinal flatmounts. This flatmount strategy enabled us to generate a systematic counting approach to quantify the drastic ongoing inflammation so that we can then assess therapeutic treatments. Furthermore, these observations provide a more integrated examination of the whole microglial populations and its relationship to blood vessel or degenerating cone photoreceptors. The complete cell morphology is also better appreciated showing the cell budging, which is another evidence of the microglial activation. Conclusion: This study demonstrates that taurine depletion causes microglial activation and migration and establishes a relation of cone loss and microglial migration to the ONL of retina. These results question if the retinal inflammation is consecutive to the photoreceptor degeneration from GES-treated mice.

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13) In vitro Modeling of the Circadian Rhythm of Phagocytosis of Photoreceptor Outer Segments Nemanja Milićević1,2, Ivanka de Bruin1, Esmée Wils1, Jacoline ten Brink1, Ghazaleh Hajmousa1, Anneloor ten Asbroek1, Marie-Paule Felder-Schmittbuhl2, Arthur Bergen1 1

Academic Medical Center Amsterdam (AMC), University of Amsterdam, Netherlands Institute of Cellular and Integrative Neurosciences (INCI), University of Strasbourg, France 2

Retinal photoreceptors undergo daily renewal of distal outer segment (POS) discs and their phagocytosis by a monolayer of retinal pigmental epithelial (RPE) cells. This process removes toxic metabolites and ensures proper functioning of photoreceptors which is essential for normal vision. POS phagocytosis is a cyclic process that takes place in rods and cones as a daily peak, occurring about one hour after light onset. Removal of shed POS is critical for maintaining photoreceptor viability as demonstrated in numerous retinal pathologies caused by defects in RPE phagocytosis. Surprisingly, little is known about the molecular machinery that drives this process. The daily rhythm of POS shedding and RPE phagocytosis is driven, in part, by circadian clocks, i.e. molecular pacemakers comprised of transcriptional-translational feedback loops with a period of roughly 24h. The development of an in vitro model of phagocytosis with circadian characteristics is an important step for investigating the link between circadian clock output and phagocytosis. In this study, we used a serum-shock procedure to synchronize ARPE-19 cells grown as a monolayer on a matrigel-coated surface. Semi-quantitative RT-PCR shows serum-shock induced rhythmic changes in clock (Per1, Per2, Bmal1, Clock) and phagocytosis (Itgb5) gene expression in ARPE-19 grown on a matrigel-coated surface and classical cell culture. The synchronized ARPE-19 monolayer will be challenged with bovine POS at different time-points to model the circadian rhythm in phagocytosis. In summary, this cellular model represents a novel tool for dissecting the molecular machinery driving POS phagocytosis.

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14) Selection of human iPS cell-derived photoreceptors by targeting of cell surface antigen CD73 G. Gagliardi, A. Chaffiol, K. Ben M’ Barek, J.B. Conart, A. Slembrouck, C. Nanteau, S. Reichman, O. Goureau Institut de la Vision, Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS UMR 7210, Paris, France

For retinal cell therapy based on human induced pluripotent stem (hiPSCs), one of the major challenges is to identify surface antigens as specific markers of hiPSCderived photoreceptors that could be used for the separation of transplantationcompetent cell population. Here, based on our retinal differentiation method using adherent hiPSCs, we focused our study on the cell surface antigen CD73. Analysis of CD73 expression in hiPSC-derived retinal organoids indicated that CD73 is specific of cells committed into the photoreceptor lineage, as all of the CD73+ cells co-localized with well-established photoreceptor markers (RECOVERIN, CONE ARRESTIN and OPSIN). Flow cytometry analysis indicated that the percentage of CD73+ cells in dissociated retinal organoids increased with maturation, with CD73+ cells representing more than 60% of cells at day 180 of differentiation. Dissociated retinal cells expressing CD73 could be sorted by Magnetic-Activated Cell Sorting (MACS), leading to enrichment to 90% of CD73+ cells in the positive sorted fraction. RT-qPCR analysis on sorted CD73+ cells showed over-expression of the most significant photoreceptor-specific genes compared to dissociated retinal cells before CD73 MACS. Re-plating of the CD73sorted cells demonstrated the cell viability, even several days after the separation process, and homogeneity (CRX and RECOVERIN expression) Freeze-thawing of both whole retina organoids and retinal cells obtained by previous dissociation of the retinal structures resulted in viable cells with unaffected expression of CD73 on the cell surface of photoreceptors. Functional analysis by Fura-2 calcium imaging showed cGMP-dependent calcium influx in CD73+ photoreceptors from 180 day-old retinal organoids. In vivo experiments in NUDE rats, suggested that both unsorted retinal cells and sorted CD73+ cells at D120 are safe as no malignant hyperproliferation of human cells was observed in the eye as far as two months after subretinal transplantation. As expected, injection of undifferentiated hiPSCs did result in teratoma development in half of the rat injected. To conclude, our results support the use of CD73 as a marker of hiPSC-derived photoreceptors. MACS of CD73+ cells resulted in a significant enrichment of photoreceptor cells. Importantly, this cell population do not present any tumorigenic risk as far as two months after transplantation. Hence, we think that CD73+ photoreceptor precursors hold great promise for future clinical translation. 116

15) Photoreceptor degeneration induced by mononuclear phagocytes relies on IL-1β-dependent glutamate disruption Hugo Charles-Messance1, Lourdes Siqueiros1,3, Valérie Forster1, Fanny Beguier1, Sébastien Augustin1, José-Alain Sahel1, Alvaro Rendon1, Ramin Tadayoni2, Florian Sennlaub1 & Xavier Guillonneau1 1

Institut de la Vision, Paris, France Hôpital Lariboisière, Paris, France 3 Cinvestav, Mexico DF, Mexico 2

Age-related Macular Degeneration (AMD) is the leading cause of irreversible blindness in the elderly. There are two clinical forms of late AMD : wet AMD, which is defined by choroidal neovascularization, and geographic atrophy, which is characterized by an extending lesion of both the retinal pigment epithelium and photoreceptors. Mononuclear Phagocytes (MPs) invariably accumulate in the subretinal space, in the vicinity of AMD atrophic lesions and contribute to rod and cone photoreceptor degeneration in an IL-1ß-dependent mechanism. We have previously shown in organotypic retinal explant model that IL-1ß induces rod apoptosis and cone segment reduction. Knowing that IL-1ß reduces glutamate uptake and also modifies the activity of glutamate transporters in astrocytes and macrophages, increasing glutamate release, we hypothesized that the accumulation of IL-1ß secreting MPs in AMD modifies glutamate homeostasis and leads to photoreceptor cell death. We thus cultured retinal explants in the presence of IL-1ß or monocytes. We here show that IL-1ß decreases the expression of EAATs transporters responsible for glutamate uptake by glial cells and increases the expression of xccystine/glutamate anti-porter in glial cells. We then show that CNQX, a glutamate receptor inhibitor protects rod photoreceptors against IL-1ß/monocytes mediated toxicity, but not cones, and that glutamate has a toxic effect towards rod photoreceptors only. We also demonstrate that Müller cells and microglial cells are responsive to IL-1ß and/or glutamate in our explant model. Taken together, our results strongly suggest that MP accumulation leads to a disruption of glutamate homeostasis that results in rod photoreceptor degeneration. We are now aiming to gain more insight into the cellular interactions that shape the overall injury response.

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16) Non-invasive and specific foveal cone targeting for vision restoration : A preclinical study, from rodents to primates Hanen Khabou1, Marcela Garita1, Antoine Chaffiol1, Sacha Reichman1, Céline Jaillard1, Elena Brazhnikova1, Stéphane Bertin1,2, Mélissa Desrosiers1, Valérie Fradot1, Céline Winckler1, Olivier Goureau1, Serge Picaud1, Jens Duebel1, JoséAlain Sahel1,2, and Deniz Dalkara1 1

Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, 75012 Paris, France 2 Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts, INSERM-DHOS CIC 503, 75012 Paris, France

Optogenetic gene therapy is a novel promising therapeutic approach for vision restoration. This strategy consists in using light sensitive proteins from microbial origin to restore light sensitivity in a degenerating retina that no longer responds to light. One of the target cells in optogenetic therapy are cone photoreceptors, they lose their outer segments -that are responsible for light capture- during degeneration. To express optogenetic proteins, such as Halorhodopsin or Jaws, adeno-associated viral (AAV) vectors are delivered locally into the eye. It is essential to design AAVs capable of targeting cones of the fovea without detaching this delicate region, as it is the cone rich region of the primate retina responsible for high acuity vision. Currently, subretinal AAV injections with retinal detachments that include the fovea are used to deliver therapeutic genes into cones. This administration route has been successful in several patients in clinical trials for Leber’s congenital amaurosis and choroideremia. But clinical experience also showed that subretinal injection is invasive and difficult to perform, particularly in a dystrophic retina. Furthermore, detachment of the fovea is more prone to complications such as holes or thinning that can severely compromise therapeutic benefits. In this study, we sought to develop AAV vectors capable of cone transduction without foveal detachment. Since mouse do not have a fovea, we used nonhuman primates, whose retina is similar to human retina. We first showed 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. Moreover, we used an engineered AAV2 variant to target 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. Lastly, we validated the efficacy of our gene therapy products on human cones. We obtained high level and specific cone transduction on post-mortem human retinas and retinal 118

organoids derived from human iPSCs. Collectively, our data support proof of concept for therapeutic potential of our vectors for the treatment of retinal diseases like retinitis pigmentosa or achromatopsia.

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17) HIF-1α and oxygen during the retinal degeneration of the RD10 mice, a model of retinitis pigmentosa Lorena Olivares-González1,2, Cristina Martínez-Fernández de la Cámara1, David Salom3, David Hervás4, José María Millán1,2,5, Regina Rodrigo1,2 1

Grupo de Biomedicina Molecular, Celular y Genómica, Instituto de Investigación Sanitaria La Fe, Valencia, Spain 2 CIBER de Enfermedades Raras (CIBERER), Madrid, Spain 3 Servicio de Oftalmología del Hospital de Manises, Valencia, Spain 4 Unidad de Bioestadística, Instituto de Investigación Sanitaria La Fe, Valencia, Spain 5 Unidad de Genética, Hospital Universitario y Politécnico La Fe, Valencia, Spain

Retinitis pigmentosa (RP) is a group of inherited retinal dystrophies characterized by progressive and irreversible loss of vision that in most models studied parallels photoreceptor cell death (rod and cones). RP present a high clinical and genetic heterogeneity with more than 90 genes involved. Photoreceptor cell death starts with rod degeneration by the genetic defect and, then, there is gradual degeneration of cones. Besides the genetic defect, evidence suggests that an unappropriated oxygen supply and consumption could contribute to the pathogenesis of RP. The transcription factor hypoxia-inducible factor 1 (HIF-1) is the primary hypoxic signalling protein that responds to low oxygen concentration regulating a large number of target genes. In the case of RP, the death of rods, the main consumers of oxygen in the retina, could cause an increase in the oxygen concentration, which could imply a reduction in HIF-1α content and also its target genes, contributing to oxidative damage. We analysed temporal profile of HIF-1α, their downstream effectors vascular endothelial growth factor (VEGF), endothelin 1 (ET-1), inducible nitric oxide synthase (iNOS) and glutamate transporter 1 (GLUT-1), the cytokines tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6) and microglial and glial activation in retinas of the mouse model, rd10 mice. We measured the content of pimonidazole adducts to detect tissue hypoxia. Finally, we assessed the effect of the stabilization of HIF-1α protein on photoreceptor loss, inflammation and antioxidant response. Our results suggest that during RP exist changes in oxygen availability that could affect photoreceptor degeneration, inflammation and HIF-1α and their downstream effectors.

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18) The origin of the RdCVF metabolic signaling Ait-Ali Najate, Blond Frédéric, Delelande Francois, Van Dorsselaer Alain, Leveillard Thierry Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, 75012 Paris, France

Purpose: The first ancestors of photoreceptors were cones. Agnathans such as lampreys are among the oldest living chordate relatives of the vertebrates and are the first animals to possess photoreceptors resembling rods. Hydra (Hydra vulgaris) belongs to the cnidarians family with feature of not having a visual eye, while being sensitive to light. The bifunctional gene NXNL1 encodes two proteins by alternative splicing, a trophic factor RdCVF that is secreted by rods and protects cones and the enzyme RdCVFL that protects photoreceptors against oxidative stress. Cones express only RdCVFL and not RdCVF. The alternative spliced product with intron retention occurs only in the rods. Our goal is to understand if this alternative splicing occurred in the ancestral gene or appeared with rods during evolution. On the other hand, the RdCVF receptor basigin1 (BSG1) is an alternative splicing product of the Bsg gene specific to photoreceptors with a third additional extracellular domain, necessary for RdCVF binding. What splicing event came first is a key to understand the emergence of RdCVF metabolic signaling. Methods: mRNA is extracted and quality controlled by lamprey retina and hydra tissues. An RT-PCR was performed by using specifics primers and an in situ hybridization (ISH) to discriminate the expression of the long and the short form of the Nxnl gene. The proteins were identified by mass spectrometry (MS/MS). Results: The short and the long form of Nxnl1 gene in the lamprey retina were detected by RT-PCR. In hydra, the Nxnl ancestor gene expression profile by ISH matches that of a thioredoxin but a short isoform with intron 4 retention was detected. Hydra genome does not contain a basigin gene with the third extracellular domain as lamprey does as shown by RT-PCR and proteomics. Conclusions: We demonstrated that inhibition of splicing of the ancestral gene Nxnl1 occurs before the emergence of the agnathans. The investigation of Nxnl in hydra shows that the intron retention occurs prior to the emergence of RdCVF metabolic signaling, a possible case of exaptation.

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19) HTRA1 blocks Thrombospondin-1 mediated immunosuppression Fanny BEGUIER1, Michael HOUSSET1, Sébastien AUGUSTIN1, Mustapha BENCHABOUNE2, Jean-François GIRMENS2, Philippe KAROYAN3, Zhiguo HE4, Thierry LEVEILLARD1, Michel PAQUES2, José-Alain SAHEL1, Xavier GUILLONNEAU1, Florian SENNLAUB1 1

Institut de la Vision, Paris, France, Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts, Paris, France, 3 Laboratoire des Biomolécules, Paris, France, 4Laboratoire BiiGC, Saint-Etienne, France 2

Physiologically, the Retinal Pigment Epithelium (RPE) expresses immunosuppressive signals such as FasL, TSP-1 or TGF-β to prevent the accumulation of Mononuclear Phagocytes (MP) in the subretinal space. Age Macular Degeneration (AMD) is associated with a breakdown of this immunosuppressivity and an accumulation of MP which cause photoreceptor degeneration, RPE dedifferentiation and pathological neovascularization. Genome association studies showed a strong link between AMD and a relatively common haplotype of 10q26 locus that contains the ARMS2 and HTRA1 gene. The disease associated haplotype is associated with increased HTRA1 transcription in some cell types such as lymphocytes and RPE cells. HTRA1 is a serine protease with a number of substrates, but the mechanism by which it might be involved in AMD pathogenesis remains unknown. We show here that HTRA1 expression is significantly increased in circulating monocytes from AMD patients. Mechanistically, we demonstrate that HTRA1 breaks down TSP-1 and and inhibits the physiological TSP-1/CD47-mediated MPs elimination in vitro in a coculture model and in vivo in a laser induced inflammation model. Pharmacological activation of CD47 efficiently reversed the pro-inflammatory effect of HTRA1 and might help control AMD progression in patients bearing the 10q26 risk haplotype.

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20) The stage of degeneration is critical for microglial manipulation in Crx mouse Yingdi Chen; Evelyne Sernagor; David Steel Institute of Neuroscience, University of Newcastle upon Tyne

Microglial cells constitute the innate immune population in the retina. The vital role they play in the maintenance of retinal homeostasis, and their function as the first-line defence against a variety of retinal insults have been widely demonstrated. However, recent studies has shown that pathways triggered by microglia activation can be either protective or degenerative. Indeed, microglia activation is not just a response to diseases, but also result in neural cell death. It is therefore crucial to understand the mechanisms underlying microglial activation to maximize the overall potential benefits of intervention. In this study, we aimed to pharmacologically regulate microglial activation in Crx mouse during retinal degeneration in order to determine the relationship between microglial activation and photoreceptor degeneration at different degeneration stages. Crx retinal slices between postnatal day (P) 15 and 5 months were counterstained with Iba1, TUNEL assay, and DAPI to investigate the time course of microglia activation and photoreceptor degeneration. The photoreceptors were quantified by measuring the thickness of the outer nuclear layer (ONL) and the numbers of TUNEL+ cells. The Iba1+ cells were categorized and counted according to their location in retinal layers. We then performed intraocular injections of the glial inhibitor Neurostatin. Neurostatin was injected at P25 in one group and at P60 in another, while plain saline was injected as a control. Retinas were harvested two weeks and a month after surgery and used for immunocytochemistry as described above. The decrease of ONL thickness could be detected from P10 and lasted for five months. Apoptosis was first observed at P30 and reached a peak at P70. Microglial activation was observed from as early as P25 and lasted until four months of age. Cellular depletion of ONL does not occur at a continuous rate, exhibiting slow progression except for two sharp drops at P30 and P60 respectively, matching the period of strong microglia activation. Neurostatin injections resulted in less pronounced microglia activation in both groups at two weeks post injection. Inactivating microglia at pre-apoptosis stages did not alleviate photoreceptor degeneration. In contrast, we observed a significant degree of ONL preservation within two weeks when Neurostatin was applied at P60. The effect became less conspicuous at a month post injection. Our results suggested that there may be different microglial activation pathways at the early and the later degeneration stages. Microglial activation may be 123

necessary for the maintenance of photoreceptors during early stage of retinal degeneration, but may aggravate photoreceptor degeneration at the later stages. Manipulating microglia activation during critical periods may therefore be therapeutically beneficial.

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21) Electrically stimulating retinal ganglion cells with smooth stimuli Larissa Höfling1², Florian Jetter1², Philipp Berens³, Günther Zeck1 1

Neurophysics Research Group, Natural and Medical Sciences Institute at the University of Tübingen, Germany 2 Graduate School of Neural Information Processing, University of Tübingen, Germany 3 Institute for Ophthalmic Research, University of Tübingen, Germany

Motivation: In degenerative diseases like Retinitis Pigmentosa photoreceptors degenerate, while the network of bipolar, horizontal, amacrine and ganglion cells initially remains largely intact. This remaining network can be stimulated electrically to evoke visual percepts, an approach that is already used in retinal implants. We use a smooth electrical stimulus to mimic physiological activity and to map the temporal electrical receptive field of mouse retinal ganglion cells (RGCs). The recovered temporal filters represent the RGCs’ preferred electrical stimulus and could be used to operate retinal prostheses more efficiently. Methods: We use a high-density CMOS-based microelectrode array to simultaneously stimulate and record from mouse retinal ganglion cells. The retina of adult rd10 mouse is prepared ex vivo and interfaced with the chip in flat-mount epiretinal configuration. Smoothed white Gaussian noise stimuli are delivered via 1024 capacitive stimulation electrodes, while electrical activity of the RGCs is recorded simultaneously on 4225 recording electrodes. Our approach allows recording and analysis of single neuron activity throughout the entire stimulation period without interference from stimulus artefacts. Custom-written data analysis software is used to recover the temporal electrical receptive fields of RGCs. Results: Using a physiologically plausible smoothed white Gaussian noise stimulus, we are able to activate retinal ganglion cells at low current densities (≤1.5 mA/cm²). Ganglion cell activity is evoked and modulated in a reliable manner for repetitive stimulus presentations. Electrical temporal filters with sub-millisecond precision could be recovered with this approach, demonstrating a direct RGC activation. The result has been confirmed after blocking the excitatory input of RGCs and recovering the same temporal filters. Conclusion: We present and discuss smooth electrical stimulation as an alternative to pulsatile stimuli for electrically activating and potentially controlling retinal ganglion cells in degenerating mouse retina.

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22) MERTK mutations account for 1.7% of cases with inherited retinal dystrophies Isabelle Audo1,2,3, Saddek Mohand-Said1,2, Elise Boulanger-Scemama1,4, Xavier Zanlonghi5,6, Vanessa Démontant1, Christel Condroyer1, Aline Antonio1, Fiona Boyard1, Said El Shamieh1, José-Alain Sahel1,2,3,4,7,8, Christina Zeitz1 1

Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, 75012 Paris, France. 2 CHNO des Quinze-Vingts, DHU Sight Restore, INSERM-DHOS CIC1423, 28 rue de Charenton, 75012 Paris, France 3 University College London Institute of Ophthalmology, 11-43 Bath Street, London EC1V 9EL, UK. 4 Fondation Ophtalmologique Adolphe de Rothschild, Paris, France. 5 Clinique Sourdille, 3 place Anatole France, 44000 Nantes, France. 6 Clinique Pluridisciplinaire Jules Verne, 2-4 route de Paris, 44300 Nantes, France. 7 Académie des Sciences-Institut de France, 75006 Paris, France. 8 Department of Ophthalmology, University of Pittsburgh Medical School, Pittsburgh, PA, USA

Purpose: MER tyrosine kinase (MERTK) encodes a surface receptor in the retinal pigment epithelium playing a critical role in photoreceptor outer segment internalization prior to phagocystosis. Mutations in this gene have been associated with severe autosomal recessive retinal dystrophies in the RCS rat model and in humans. The purpose of this study was to report genetic findings and phenotypic characteristics of patients affected with retinal dystrophies due to mutations in MERTK in a large cohort of patients applying state of the art techniques. Methods: A large French cohort of 1004 patients with inherited retinal diseases underwent a full ophthalmic examination. Informed consent was obtained from each patient and unaffected family member. The study protocol adhered to the tenets of the Declaration of Helsinki and was approved by the local ethics committee. The DNA of patients with a presumed diagnosis of autosomal recessive Rod-Cone Dystrophy and Cone-Rod Dystrophy was assessed. Microarray analysis, targeted Next Generation Sequencing (NGS) and Sanger sequencing were applied to 904 RCD and 100 Cone-Rod Dystrophies. NGS data was also analyzed to detect Copy Number Variation and cases with large deletion were documented applying Multiplex Ligation-dependent Probe Amplification. Familial cosegregation analysis was performed when possible. Results: A total of 23 patients from 18 unrelated families were found to carry predicted pathogenic mutations in MERTK including 19 cases of RCD and 4 cases of CRD. Twenty distinct mutations were identified among which 11 are novel. 126

Mutation spectrum in our cohort includes 3 small deletions, 2 small duplications, 1 large exonic deletion, 2 splice defects, 9 missense and 3 nonsense changes. The 23 patients carrying these mutations had severe inherited retinal disease with early onset macular involvement. Conclusion: Our study revealed that mutations in MERTK would account for ~1.7% of inherited retinal dystrophies in France. This new data are useful in the context of current and future therapeutic trials including gene replacement therapy or cell based therapy.

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23) The protective effect of OCX063 on reducing neovascularization and fibrosis in a mouse model of choroidal neovascularization Alice A. Brandli1, Fay Khong2, Roy Kong2, Darren J. Kelly2 and Erica L. Fletcher1 1

Department of Anatomy and Neuroscience, University of Melbourne, Parkville, VIC, Australia 2 OccuRx Pty Ltd., Level 9, 31 Queen Street, Melbourne, VIC, Australia

Vision loss in wet age-related macular degeneration (AMD) is caused by aberrant blood vessel growth and fibrosis in the choroid. Wet AMD is treated by vascular endothelial growth factor (VEGF) inhibitors that reduce neovascularization. Some wet AMD patients develop fibrosis in the retina in conjunction with neovascularisation. These anti-VEGFs don’t target the fibrosis processes and wet AMD patients can continue to lose vision. Hence, there is a need to test novel drugs that both reduce chorodial fibrosis and neovascularization. We investigated the effect of OCX063 in an experimental model of choroidal neovascularization (CNV). CNV lesions were induced in 8 week old female BL6 mice (n= 10 eyes/group) using laser photocoagulation (350 mW). Animals were intravitreal injected with either 0.5 uL of OCX063 (50 uM) or PBS immediately following lasering (acute) or at 7 days (long-term). Leakage was assessed using fluorescence angiography and total lesion size was quantified using image J at 7 days or 30 days and animals were euthanized at 7 days or at 30 days and the eyes were removed, fixed and stained using trichrome. The CNV lesion height/choroid height ratio was measured in image J. mRNA expression and gene ontology (GO) analysis study was also undertaken. RNA was extracted from the RPE, 7 days after laser. mRNA expression levels were compared using angiogenesis and fibrosis qPCR arrays. Significantly expressed genes relative to control (p< 0.05, fold change (FC) ± 1.5) underwent overrepresentation testing on the panther GO platform. CNV lesions outcomes were reduced in OCX063 treated mice relative to PBS treated mice in the acute and long-term experiments. The total leakage area (Pixels, 7 days; 5.7 x 104 vs. 2.8 x 104, 30 days; 2.3 x 104 vs. 1.4 x 104, p< 0.05) and the CNV lesion size (B/C ratio, 7 days; 5.5 vs. 3.5 (p< 0.01), 30 days; 3.6 vs. 2.6 (p< 0.05)) was significantly reduced. Gene expression changes showed that OCX063 significantly reduced (p< 0.05) the mRNA expression of CNV-promoting genes; Timp1 (-1.6 FC), Cxcl11 (-1.5 FC), and Thbs1 (-1.3 FC). Over-enrichment testing revealed TGF-beta pathways were being regulated by OCX063, indicating that OCX063 may be acting as an anti-fibrotic.

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OCX063 reduced CNV lesion size and leakage at 7 days and 30 days. OCX063 may provide an alternative treatment to wet AMD patients especially those that do not respond to anti-VEGF therapies.

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24) How the outer retina malfunctioning impacts on inner retinal circuitry Stefano Di Marco1, Davide Lonardoni2, Fabio Boi2, Alessandro Maccione2, Luca Berdondini2, Silvia Bisti1 1

Department of Biotechnology and Applied Clinical Science, University of L’Aquila Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia, Genova, Italy 2

Retina, within few cellular layers, transduce, analyze and organize visual information. The complexity and intensity of this encoding process is intrinsically susceptible to genetic defects or oxidative stress. Additionally, considering that transduction is an energetically demanding process, the more susceptible elements of this chain are the photoreceptors. In this talk I would like to highlight the strict relationship existing between photoreceptor failure and inner retina malfunctioning. I will show the long-term consequences that absence of light experience has during the critical period on the retinal ganglion cell’s ability to filter and decode space and time. I will also describe a similar paradigm transposed in an animal model for Retinitis Pigmentosa: the Royal College of Surgeon rat. Finally, I will introduce a new technological platform that allows the simultaneous recording of light-evoked responses from thousands of retinal ganglion cells in exvivo retina of mouse, rat and monkey, thus opening new perspectives in this field of research.

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25) Retinal Remodeling and Plasticity, Progressive Neurodegenerations Jones B.W., Marc R.E., Pfeiffer R.L.. Moran Eye Center, University of Utah

The challenges of vision loss in age-related macular degeneration (AMD), retinitis pigmentosa (RP) and other degenerations have motivated international efforts to develop late-stage vision restoring therapies including cell / tissue implants, optogenetics, photoswitches, and prosthetic implants, all requiring survival of the neural retina. It has often been asserted that the surviving neural retina is essentially unchanged in RP, but that is false. Remodeling includes neuronal death, rewiring, ectopic synapse formation, cell migration and emigration, glial transformations, and even vascular remodeling. Most of our knowledge of remodeling is based on analyses of short lifespan rodents using experimental tools that, with exceptions, are impossible with human autopsy tissue. A key difference between rodents and humans is severe retinal decimation (neuronal death + IPL morcellation) in human RP patients and the mild, albeit significant remodeling of many models of mouse and rat. Longitudinal analyses of the Tg P347L rabbit, a model with a lifespan 4x that of a mouse, shows that Phase 3 remodeling is relentlessly progressive, with continuing neuronal death until > 90% of neurons are gone, yielding an exponential death time constant of τND ≈ 2.5y. In parallel, Müller cells undergo metabolic collapse resembling the metabolic depression and hypometabolism of central nervous system neurodegenerative diseases. 
Based on analyses of mouse, rat, and short term 2y Tg P347L rabbit and Tg P23H pig models, we earlier concluded that remodeling reached stasis: retinas were aggressively remodeled but stable. That conclusion is wrong based our longitudinal analysis of 2-6y Tg P347L rabbit retinas and, importantly, advanced human RP. The long lifespan rabbit and human RP show that retinal ND never stops. 
These data demonstrate that retinal degenerations are binary diseases: acute photoreceptor degenerations convert to chronic neurodegenerations similar to progressive brain diseases. Targeting specific defects will be critical to achieving rescues vision rescue by molecular, cellular, or engineering approaches as the neurodegeneration is unremitting, decimating the neural retina on human time scales with > 90% loss of retinal ganglion cells, impeding rescue. We will demonstrate retinal plasticity from early to late stage, reveal networks affected, and discuss circuit topologies involved with metabolic chaos and collapse, the nature and scope of rewiring and neuronal loss.

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26) Preclinical validation of a novel tissue engineered product consisting in RPE derived from human embryonic stem cells disposed on human amniotic membrane. Karim Ben M’Barek,2,3, Walter Habeler1,2,3, Alexandra Plancheron1,2,3, Mohamed Jarraya4, Florian Regent1,2, Angélique Terray5, Ying Yang5,6, Laure Chatrousse1,2,3, Sophie Domingues1,2,3, Yolande Masson1,2,3, José-Alain Sahel5,6,7,8, Marc Peschanski1,2,3, Olivier Goureau5 and Christelle Monville1,2,3 1

INSERM U861, I-Stem, AFM, Institute for Stem cell Therapy and Exploration of Monogenic diseases, 2 rue Henri Desbruères, 91100 Corbeil-Essonnes, France; 2 UEVE U861, I-Stem, AFM, Institute for Stem cell Therapy and Exploration of Monogenic diseases, 2 rue Henri Desbruères, 91100 Corbeil-Essonnes, France; 3 CECS, I-Stem, AFM, Institute for Stem cell Therapy and Exploration of Monogenic diseases, 2 rue Henri Desbruères, 91100 Corbeil-Essonnes, France; 4 Banque de tissus humain, Hôpital Saint Louis, AP-HP Paris; 5 Institut de la Vision, Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, 75012 Paris, France; 6 CHNO des Quinze-Vingts, DHU Sight Restore, INSERM-DHOS CIC, 75012 Paris, France; 7 Fondation Ophtalmologique Adolphe de Rothschild, 75019, Paris, France; 8 Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, US.

Retinitis pigmentosa (RP) and age-related macular degeneration (AMD) are the main causes of blindness in the developed world. The Retinal Pigment Epithelium (RPE) is a continuous monolayer of cuboidal epithelial cells, localized between the photoreceptors and the fenestrated choroid capillaries. The RPE interact with the photoreceptors for the maintenance of visual function. Maintaining the epithelial morphology of RPE cells is a crucial parameter to consider in order to restore some visual function by cell therapy. Replacement of defective RPE by new RPE cells derived from human pluripotent stem cells provides a novel rational approach for treating forms of blindness that affect the RPE. First attempts in clinical trials demonstrated safety for the delivery of such cells as a suspension. Transplanting a more physiologically functional epithelium of RPE cells is the next challenge to effectively cure patients. We have developed, under clinically compatible conditions, a tissue-engineered product (TEP) consisting of RPE cells derived from human embryonic stem cells (hESCs) disposed on a biocompatible substrate: the human amniotic membrane. Through a new surgical approach to engraft the TEP into the subretinal space of Royal college of Surgeons (RCS) dystrophic rats, we demonstrated that TEP transplantation improved photoreceptor rescue and the visual function compared to RPE injected as a simple cell suspension. 132

These results are supportive for the initiation of our phase I/II clinical trial to treat Retinitis Pigmentosa patients.

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27) A fully organic retinal prosthesis restores vision in a rat model of degenerative blindness Maya-Vetencourt JF1, Ghezzi D1, Antognazza MR2, Colombo E1, Mete M3, Feyen P1, Desii A2, Buschiazzo A4, Di Paolo M5, Di Marco S5, Ticconi F4, Emionite L6, Shmal D1, Marini C7, Donelli I8, Freddi G8, Maccarone R5, Bisti S5, Sanbuceti G4, Pertile G3, Lanzani G2, Benfenati F1,9. 1Centre for Synaptic Neuroscience and Technology, IIT, Genova, IT. 2Centre for NanoScience and Technology, IIT, Milan, IT. 3Ophthalmology Department, Sacro Cuore Hospital, Negrar, IT. 4Department of health Science, Nuclear Medicine, University of Genova, IT. 5Department of Biotechnology and Applied Clinical Science, University of L'Aquilla, IT. 6Animal Facility, National Institute Cancer Research, IRCCS AOU San Martino, Genova, IT. 7Institute of Molecular Bioimaging and Physiology, CNR, Milan, IT. 8Innovhub-SSI, Silk Division, Milan, IT. 9Department of Experimental Medicine, University of Genova, Genova, IT.

The degeneration of photoreceptors in the retina is one of the major causes of adult blindness in humans. Unfortunately, no effective clinical treatments exist for the majority of retinal degenerative disorders. Here we report on the fabrication and functional validation of a fully organic prosthesis for long-term in vivo subretinal implantation in the eye of Royal College of Surgeons rats, a widely recognized model of retinitis pigmentosa. Electrophysiological and behavioural analyses reveal a prosthesis-dependent recovery of light sensitivity and visual acuity that persists up to 6–10 months after surgery. The rescue of the visual function is accompanied by an increase in the basal metabolic activity of the primary visual cortex, as demonstrated by positron emission tomography imaging. Our results highlight the possibility of developing a new generation of fully organic, highly biocompatible and functionally autonomous photovoltaic prostheses for subretinal implants to treat degenerative blindness.

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POSTER SESSION III: RETINAL CIRCUITS 1) Rewiring of bipolar cells in congenital stationary night blindness type 2 mouse models Kilicarslan I1, Seitter H1, Strettoi E2 and Koschak A1 1

Department of Pharmacology and Toxicology, University of Innsbruck, Innsbruck, Austria. 2 Neuroscience Institute, Italian National Research Council (CNR), Pisa, Italy.

Cav1.4 L-type voltage gated calcium channels are mainly expressed at photoreceptor (PR) terminals at the outer plexiform layer (OPL) and most likely also in bipolar cells in the inner plexiform layer (IPL). They are localized at specialized ribbon synapses where they allow sustained calcium influx and ensure neurotransmitter release. Mutations in the CACNA1F gene which encodes Cav1.4 channels are associated to congenital stationary night blindness type 2 (CSNB2) in humans. A mouse model carrying the Cav1.4 point mutation I745T (Cav1.4-IT) which results in a gain of Cav1.4 function - serves as a good model for the human CSNB2 phenotype. We compared Cav1.4-IT mice with Cav1.4 deficient mice (Cav1.4-KO) comprising a loss-of-function with respect to differential effects on the retinal morphology. Immunostaining with anti-PSD-95 showed that PR terminals were mislocated in the outer nuclear layer (ONL) in Cav1.4-IT mice (5weeks) in contrast to WT where they were located in the OPL and Cav1.4-KO retinas which lacked PSD-95 staining. Some rod bipolar cell dendrites of Cav1.4-IT mice approached to displaced PR terminals in the ONL and some invaginating contacts were also observed in the ONL of Cav1.4-IT mice in 11 to 14. Mislocated PR terminals of Cav1.4-IT retinas contained mostly immature, elongated synaptic ribbons. At the age of 28 weeks in Cav1.4-IT rod BC dendrites were completely lacking in the peripheral retina, likely due to the absence of PRs in the corresponding area. Immunoreactivity against to secretagogin elicited that some type of cone bipolar cells in Cav1.4-IT retinas also showed dendrites that were sprouting into the ONL (age group: 13-14 weeks) indicating that bipolar cells in the cone pathway are also affected in CSNB2. The synaptic organization in the OPL seemed more severely affected in KO than in IT mice confirming that different mutations in the CACNA1F gene can cause different types of morphological aberrations. On the contrary, there was no significant morphological variations observed in the inner plexiform layer of CSNB2 retinas. Taken together differences similar to those seen in two mouse models may also explain subtle variations in the clinical manifestation of CSNB2. ITN Switchboard 674901 to AK and ES, FWF P26881 to AK, University of Innsbruck and Center for Molecular Biosciences Innsbruck. 135

2) Parvalbumin-GFP mice can be utilized to perform type specific connexin36 dendritic arbor mapping of large field ganglion cells Gábor Debertin1,2,3, Orsolya Kántor1,4, György Sétáló Jr.5, Edina Szabó-Meleg6, Miklós Nyitrai6, Gábor Szabó7, Ferenc Erdélyi7, Béla Völgyi1,2,3 1

MTA-PTE NAP B Retinal Electrical Synapses Research Group, Pécs, Hungary Department of Experimental Zoology and Neurobiology, University of Pécs, Pécs, Hungary 3 János Szentágothai Research Center, Pécs, Hungary 4 Department of Neuroanatomy, Faculty of Medicine, Institute for Anatomy and Cell Biology, University of Freiburg, Freiburg, Germany 5 Department of Medical Biology, University of Pécs, Budapest, Hungary 6 Department of Biophysics, University of Pécs, Pécs, Hungary 7 Institute of Experimental Medicine, Budapest, Hungary 2

It has been established that many retinal ganglion cells (RGCs) of the mouse express connexin protein subunits that comprise both homologous and heterologous gap junctions with ganglion and amacrine cell neighbors, respectively. Most studies utilize tedious tracer injections of RGCs with a combination of immunocytochemistry to locate dendritic gap junction sites each RGC form. In this study we perform a high-throughput method to examine the distribution of connexin36 (Cx36) plaques that colocalize with dendrites of large field RGCs. To this end we use a parvalbumin-GFP (PV-GFP) mouse line, in which animal the PV expressing large field RGCs appear with their entire dendritic arbors thus allowing both the morphological identification of RGCs and the examination of Cx36 plaque distribution over dendritic branches of identified cells. In the retina of the PV-GFP mouse we repeatedly identify G1, G2 (ON alpha), G3 (OFF alpha) and G10 RGCs (Völgyi et al. 2009), whose entire dendritic arbors are revealed by the GFP marker. We took advantage of this phenomenon and created full-arbor Cx36 maps for examined cells with a particular emphasis on OFF alpha cells. We find that Cx36 plaques prefer middle dendritic areas and only a few plaques occur in the vicinity of dendritic tips. Besides Cx36 maps of individual cells we also checked dendritic crossings of neighbor OFF alpha cells in order to reveal potential homologous Cx36 gap junctions between OFF alpha cells. We found only a few example of such triple colocalizations, thus suggesting that homologous OFF alpha cell gap junctions are not comprised by the Cx36 subunit.

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3) Measuring glutamate neurotransmission in vivo at bipolar cell terminals using iGluSnFR Lea Darnet, Benjamin James, Sofie-Helene Seibel, Leon Lagnado School of Life Sciences, University of Sussex, Brighton BN1 9QG, UK

The fundamental unit of information transfer between cells in a neural circuit is the synaptic vesicle containing neurotransmitter. In the retina, the output from terminals of bipolar cells (BCs) not only exhibits quantized release, but also show coordinated multivesicular release (MVR), in which multiple vesicles are released simultaneously (Singer et al., 2004). Using zebrafish larvae expressing the intensity-based Glutamate Sensing Fluorescence Reporter (iGluSnFR) (Marvin et al., 2013) under the ribeyeA promoter, we have developed an approach for monitoring both uniquantal release and MVR in vivo using multiphoton microscopy of larval zebrafish. Full-field modulation of intensity at 5 Hz with different contrasts (1 to 100 %) as well as different frequencies from 0.5 to 30 Hz at 50 % contrast evoked fast localized transients that were sampled at 1 kHz using line-scans. These iGluSnFR signals recorded at an individual terminal were completely abolished by local photoablation of the soma of the same, but not neighbouring, bipolar cells demonstrating that the signals reflect release from the same terminal rather than “spillover” from neighbours. Next, we developed an algorithm that extracts, analyses and quantifies quanta release of glutamate from recorded iGluSnFR signals at different contrasts of visual stimulation. This method comprises 4 steps: spatial decomposition and deconvolution, Wiener filtering, event extraction, and amplitude clustering. Using this technique, 1) Glutamate quanta could be detected by examining the distribution of iGluSnFR signal deconvolved amplitudes upon light stimulation by analysing Gaussian Mixture Model (GMM) fit interpeak distances. The model accuracy of the GMM, as well as the nearly identical interpeak distances indicate that peaks within the GMM correspond to individual quanta, with a unitary quantal event amplitude equals to this interpeak distance, and 2) individual events separated by as little as 5 ms were distinguishable. We found that the distribution of inter-event times, even when accounting for our 5 ms discrimination window, does not follow an exponential distribution, indicating that vesicular release counts is not described by a Poisson process. Thus, our methods using iGluSnFR offer the spatial and temporal resolution to count glutamate vesicles as bipolar cell synapses transmit the visual signal to retinal ganglions cells. 137

4) What the fish’s eye tells the fish’s brain: The functional complement of zebrafish Retinal Ganglion Cells Mingyi Zhou1, Takeshi Yoshimatsu1 & Tom Baden1,2 1

Sussex Neuroscience, School of Life Sciences, University of Sussex, Brighton BN1 9QG, UK 2 Institute for Ophthalmic Research, University of Tübingen, 72076 Tübingen, Germany

In the vertebrate retina, incoming visual information is split into parallel information channels and sent to the brain by distinct types of retinal ganglion cells (RGCs) (Masland, 2012). In mouse, a minimum of 32 RGC types, and possibly as many as 50, have been charted both functionally (Baden et al., 2016) and anatomically (www.museum.eyewire.org) (see also Sanes and Masland, 2015). However, a similarly detailed understanding of the complement of zebrafish RGCs as well as their underlying feature-extracting microcircuits is lacking. In fact, there has yet to be a consensus on a definitive classification of RGCs in any species to date. Here, we performed two-photon population imaging of light-evoked calcium activity across different life stages of the zebrafish in vivo and in retinal explants to functionally chart what the fish’s eye tells the fish’s brain. While instructive in itself, in reference to existing data from mouse and other species, this will also allow us to study to what extent different species extract different aspects of visual scenes, and how this matches each species’ visual requirements. We used transgenic zebrafish lines HuC:GCaMP6f and Eno2:GCaMP3.5, both of which express an optical reporter for calcium under pan-neuronal promoters, which drives expression in RGCs under cytoplasmic localization. Preliminary data from adult retina indicates that only a low fraction of RGCs respond to full-field stimuli, and out of those that did, most were highly selective for a specific bandpass in the frequency domain. Further, all full-field driven RGCs displayed much stronger adaptation than observed previously in mice (Baden et al., 2016). Our preliminary results cautiously suggest that overall, the complement of zebrafish RGCs may be tuned to a more specific set of stimulus features than that of mice.

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5) Local signal processing in mouse horizontal cell dendrites Camille A. Chapot1-3, Luke E. Rogerson1-4, Tom Baden1,5, Sinziana Pop1-3, Philipp Berens1,2,4, Thomas Euler1,2,4 , Timm Schubert1,2 1

Institute for Ophthalmic Research, University of Tübingen, Germany Center for Integrative Neuroscience, University of Tübingen, Germany 3 Graduate Training Centre of Neuroscience, University of Tübingen, Germany 4 Bernstein Center for Computational Neuroscience, University of Tübingen, Germany 5 School of Life Sciences, University of Sussex, Brighton, United Kingdom 2

The mouse retina contains a single type of horizontal cell, a GABAergic interneuron that samples from all cone photoreceptors within reach and modulates their glutamatergic output via parallel feedback mechanisms. Because horizontal cells form an electrically-coupled network, they have been implicated in global signal processing, such as contrast enhancement. Recently, it has been proposed that horizontal cells can also act locally at the level of individual cone photoreceptor axon terminals. To test this possibility physiologically, we used two-photon microscopy to record light-evoked Ca2+ signals in cone axon terminals and horizontal cell dendrites as well as glutamate release in the outer plexiform layer. By selectively stimulating the two mouse cone opsins with green and UV light, we assessed whether signals from individual cones remain “isolated” within horizontal cell dendritic tips, or whether they spread across the dendritic arbour. Consistent with the mouse’s opsin expression gradient, we found that light responses recorded from dendrites of dorsal horizontal cells were dominated by M- and those of ventral horizontal cells by S-opsin activation. Light responses measured in neighbouring horizontal cell dendritic tips varied markedly in their chromatic preference, arguing against global processing. Rather, our data support the idea that horizontal cells can process cone input locally, extending the “classical” view of horizontal cells function. Pharmacologically removing horizontal cells from the circuitry reduced the sensitivity of the cone signal to low frequencies, suggesting that local horizontal cell feedback shapes the temporal properties of cone output.

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6) Effects of Noise on Contrast Gain Control Jacob Huth3, Emilie Mayer2, Timothée Masquelier1, Angelo Arleo3 1

CERCO UMR5549, CNRS, University Toulouse 3, France MINES ParisTech, Paris, France; Institut de la Vision, Paris, France 3 Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, Paris, France 2

Natural visual stimuli can occur at a large range of luminance and contrast levels. To be able to encode patterns under different lighting conditions with biological cells, which can only transmit a limited range of values, a linear gain between inand output has to be adapted dynamically. This can already be observed in the retina where response dynamics change under different luminace and contrast levels [1]. In addition, visual information is inherently noisy, as both the absorption of photons and the response of ganglion cells is a stochastic process [2]. We investigated the effect of noise on a simple contrast gain control mechanism model, first proposed by Shapley & Victor in 1978 [1]. We found that (1) noise effects can naturally be amplified or dampened by contrast gain control, depending on the stimulus and (2) that noise itself can counterintuitively enhance response reliability as it increases local contrast and thus engages contrast gain control locally, similar to the phenomenon of stochastic resonance. As a theoretic consideration, there exists a gain which optimizes the signal-tonoise ratio with respect to the noise level and the channel capacity, which we can show for the gain-control model. It remains to be determined whether there exist adaptive processes in actual visual systems that change gain control mechanisms in response to systematic changes in input noise, eg. In the natural aging process.

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7) Measuring the distance of neural morphologies using graph features S. Laturnus¹, D. Ghoshdastidar², U. von Luxburg², P. Berens¹ 1

Institute of Ophthalmic Research, University of Tübingen Department of Computer Science, University of Tübingen

2

The morphology of neurons is typically considered a defining feature of neural cell types. For example, 14 types of bipolar cells can be discriminated in the mouse retina based on their morphology (Helmstaedter et al. 2013, Kim et al. 2014, Greene et al. 2016), leading to a classification in good agreement with genetic and physiological data (Shekhar et al. 2016, Franke et al. 2017). Similarly, many retinal ganglion cells can be discriminated on morphological terms (Sumbul et al., 2014). Given recent advances in automatic reconstruction and crowd-based tracing techniques, the amount of available data is rapidly increasing (see e.g. www.neuromorpho.org). However, machine learning methods to automatically discriminate or cluster neurons rely on fairly simple representations of neural morphologies, discarding much of the richness of the three-dimensional morphology. Typically, these methods consider the neurite density in three dimensions or low dimensional projections thereof and measure the similarity between two neurons by the euclidean distance of these densities. For retinal neurons, this procedure has been used by analyzing the axon density of bipolar cells or the dendrite density of ganglion cells as a function of IPL depth. However, all fine details contained in the morphological reconstructions such as branching patterns are discarded. Here we propose to measure neuron similarity by retaining the original representation as a tree in the graph theoretical sense and comparing structural differences based on graph statistics. We investigate which similarity measures based on graph features allow reliable discrimination of neural types in the retina and how they can be combined with existing methods to improve discrimination and clustering of neural types.

141

8) Method for tracking the behavior of freely moving mice at visual threshold Tuomas Turunen1, Martta Viljanen2 & Petri Ala-Laurila1,2 1

Aalto University, Department of Neuroscience and Biomedical Engineering, Espoo, Finland 2 University of Helsinki, Department of Biosciences, Helsinki, Finland

The morphology of neurons is typically considered a defining feature of neural cell types. For example, 14 types of bipolar cells can be discriminated in the mouse retina based on their morphology (Helmstaedter et al. 2013, Kim et al. 2014, Greene et al. 2016), leading to a classification in good agreement with genetic and physiological data (Shekhar et al. 2016, Franke et al. 2017). Similarly, many retinal ganglion cells can be discriminated on morphological terms (Sumbul et al., 2014). Given recent advances in automatic reconstruction and crowd-based tracing techniques, the amount of available data is rapidly increasing (see e.g. www.neuromorpho.org). However, machine learning methods to automatically discriminate or cluster neurons rely on fairly simple representations of neural morphologies, discarding much of the richness of the three-dimensional morphology. Typically, these methods consider the neurite density in three dimensions or low dimensional projections thereof and measure the similarity between two neurons by the euclidean distance of these densities. For retinal neurons, this procedure has been used by analyzing the axon density of bipolar cells or the dendrite density of ganglion cells as a function of IPL depth. However, all fine details contained in the morphological reconstructions such as branching patterns are discarded. Here we propose to measure neuron similarity by retaining the original representation as a tree in the graph theoretical sense and comparing structural differences based on graph statistics. We investigate which similarity measures based on graph features allow reliable discrimination of neural types in the retina and how they can be combined with existing methods to improve discrimination and clustering of neural types.

142

9) Receptive field structure of sustained ganglion cells in the fish retina Alexey Aliper, Ilija Damjanovic, Pavel Maximov, Elena Maximova Institute for Information Transmission Problems of the Russian Academy of Sciences (Kharkevich Institute)

In vertebrates primary processing of visual information is carried out on retinal level. In lower vertebrates the structure that receives the majority of the retinal inputs is tectum opticum. The retinal ganglion cells send their axons to its outer layer. The ganglion cells projecting to tectum opticum can be divided into several types basing on the preferred stimuli they respond to. The axon terminals of sustained ganglion cells can be found in the deepest sublamina of retino-recepient layer of the tectum opticum. Their preferred stimuli are brightness shifts in the receptive field. Depending on the sign of contrast preferred sustained ganglion cells can be divided into light sustained and dark sustained subtypes. We recorded extracellular activity from the axon terminals of retinal GCs in the tectum opticum of Carassius gibelio, which is a close relative of goldfish and zebrafish. For the visual stimulation we used CRT monitor. The properties of stimuli and the experimental series were adjusted with unique software developed for our research. Responsive or excitatory central part of the receptive field (ERF) of the sustained ganglion cells appear to be of the same size as the other retinal GCs projecting to tectum opticum. However when we gradually increase the size of presented stimulus the number of spikes in response keeps growing even when the size of stimulus significantly exceeds the ERF. On the other hand total darkening or lightening of the visual scene (depending on a cell subtype) drastically inhibits the response from the cell. This means that the receptive fields of sustained GCs have large and complicated peripheral area. Supported by RFBR grant 13-04-00371

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10) Population omitted stimuli responses on retinal gangion cells J. Araya, R. Herzog, C. Miguel, MJ. Escobar, AG. Palacios Universidad de Valparaíso – Universidad de Santiago de Chile

The omitted stimulus response (OSR) is a phenomena that has been described in the retinal ganglion cells (RGC) when stimulated with repetitive light patterns. In brief, OSR is a response to a violation of the periodicity of the stimulus and its principal feature is the locking of the responses to a specific range of stimulus period. It has been reported a wide variety of OSR at the single cells level, finding that some cells spike both for the repetition and the violation of the pattern, joining different aspects of the stimulus on one single stream, making them inseparable for the brain. This suggests that we should focus at the population level: MJ Berry II & G Schwartz took this into account, and hypothesized that the retina separates the early visual information in two broad population responses: i) one associated with the predictable aspects of the stimuli, like the period of a repeated stimulus and ii) another related to surprising aspects of stimulus, as the periodicity violation. To test this hypothesis, we used multielectrode arrays to record hundreds of RGC (mean: 274, s.d: ± 50) from a patch of a diurnal rodent (Octodon degus, n=3) retina stimulated with a simple periodic flash sequence. The stimulus was 20 trials of 10 pulses of the same light-dark times, evaluated with different frequencies to evoke the OSR, finding it only on the 2.5 – 12.5 Hz range. For each trial, we took the population responses to every flash and its corresponding omitted stimulus response to build a similarity matrix between responses. Then, applying singular value decomposition to this matrix we extract two orthogonal population responses: i) the OSR which is correlated to the response at the beginning of each trial and ii) one response observed during the repetitions of the light pattern on each trial. These observations are valid only in the range where the OSR are observed. The second response becomes clearer after 2-3 repetitions of the pattern and not after a specific time. These results show that the retinal population responses under a repeated stimulus pattern are actually separated in two broad streams: one related to surprise (the pattern beginning and end) and another to the predictive aspects of stimulus (responses inside each trial). We propose as future direction to include this phenomena and corresponding results into the predictive coding framework, which could suggest that some predictions about the visual scene are actually generated in the early visual system. 144

11) Light induced ganglion cell responses in Cav1.4 mutant mouse retinas Zanetti L., Seitter H., Koschak A. University of Innsbruck, Institute of Pharmacy, Pharmacology and Toxicology, Innsbruck, Austria

Different mutations in the CACNA1F gene encoding for the alpha1 subunit of Cav1.4 channels are known to cause Congenital Stationary Night Blindness Type 2 (CSNB2) in humans. In this study we investigated the point mutation I745T (IT), which, in heterologous expression system, has been shown to result in gain of Cav1.4 channel function resulting in an abnormal calcium influx. To further examine the ganglion cell (GC) activity of IT mouse retinas, we differentiated dim light (scotopic) and bright light (photopic) conditions, and by the means of multiple light stimuli we aimed to detect specific GC response patterns. We confirmed a higher spontaneous firing rate in the absence of stimuli and a delayed response in both light conditions in IT whole-mount retinal preparations seen also in previous work . In addition, compared to controls, IT retinas showed a diminished firing frequency within the stimulus (WT= 16 Hz, IT= 6.7 Hz, mean of 5 and 4 experiments respectively). The higher spontaneous firing rate and the decreased light-driven firing response likely account for the inability of GC to transduce efficiently visual signals. Of note many GC previously did not respond to full-field stimulation undermesopic light conditions. In this study, the analysis of the same cell in two different light conditionsshowed that ON and OFF responses of IT GC are largely lost during bright light: while 275 GCs responded to dim light, only 80 GC (N= 4) responded using bright light stimuli. Gaussian white noise stimulus analysis instead, showed a loss of GCs response also at scotopic level. These preliminary data indicate that, although scotopic and photopic pathways show similarly impaired responses, in the IT CSNB2 model the cone pathway might be more severely affected. Together our findings reflect what is seen in electroretinographic analyses of CSNB2 patients.

145

12) Electrically imaging retinal neurons using high-density multi-electrode arrays Meng-Jung Lee1,2, Guenther Zeck1 1

Neurophysics Research Group, Natural and Medical Sciences Institute at the University Tübingen, Reutlingen, Germany 2 Multi Channel Systems GmbH, Reutlingen, Germany

Multielectrode arrays (MEAs) have been widely used in the study of neuronal physiologies. The advantages of high sampling rate, multiple recording sites and transparency make it an ideal tool to study not only single neuronal electrophysiology but also the networks of neural tissues. Here with the CMOSbased high-density microelectrode array (CMOS MEAs), we were able to further increase the spatial resolution of the MEAs to a level that enable us to electrically imaging retinal neurons, which will lead us to a better understanding of the networking signal transduction in the retina. In this study, we adapted isolated healthy C57BL/6 retinae in vertical slices onto poly-l-lysine coated CMOS MEAs with 1 mm2 sensor area and record with 20 k sampling rate. We provided light stimulus with LED setup and record the light response. Recordings from these retinae were further analyze and visualized in the customized python-based software SOMA and Matlab. In the vertical slices from healthy mice retinae, we were able to electrically image the vertical signal transduction of light response from photoreceptor side to retinal ganglion side. In the same preparation, we also observed the horizontal signal propagation within the vertical slices. With longer light stimulation and pharmacological treatment, we further revealed the ON and OFF pathways separately. Our results prove that with high-density CMOS MEAs, it is possible to electrically image retinal neurons in healthy retina to reveal the functional circuits. This technology will provide a fast and convenient way for neurological study in the future.

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13) The circuit mechanisms generating orientation-selectivity in the retina J. Johnston, Sofie-H. Seibel, Léa Darnet & Leon Lagnado Sussex Neuroscience, University of Sussex, Brighton, U.K

The detection and classification of moving objects is a fundamental task of the visual system. In the 1960s, Barlow and colleagues demonstrated that these more complex neuronal computations are not restricted to higher visual centers but can be encoded within the activity of retinal ganglion cells (RGC). Here we focus on one specific aspect of feature detection: the orientation of a moving edge. How is orientation selectivity (OS) generated within the retinal network and how is this signal propagated to the tectum? In order to address these questions, we performed 2-photon imaging of transgenic larval zebrafish expressing two different reporters; synaptically targeted GCaMP6f enabled us to measure presynaptic calcium influx into bipolar (BC) and amacrine cell (AC) synapses, while the glutamate sensor iGluSnFR allowed for evaluation of the excitatory drive from BCs to RGCs within the retina as well as of the output of RGCs within the tectum. We find that OS can already be detected at the output of BCs, with 25% of the terminals showing a strong preference for vertical orientation. We determined that BC receptive fields tend to be elliptical and oriented along the vertical axis in visual space. The integral of the response to orthogonally oriented bars in OS terminals was significantly different, indicating that despite their asymmetric shape, a single linear integration across the receptive field is not sufficient to generate OS. However pharmacological blocking of the inhibitory effects of ACs significantly decreased the OS of BCs. We propose that vertical OS of BCs is formed by lateral inhibition, provided by ACs distributed along the horizontal axis. Transiently expressing iGluSnFr in RGCs enabled us to first, simultaneously record the different glutamatergic inputs across the dendritic field of a single RGC and second, to look at its output in the tectum. Using this approach, we could show that differently tuned bipolar cells converge onto a single RGC. In the tectum, we find an overall increase of OS with terminals being tuned to vertical and horizontal orientations. The majority of the excitatory signals transmitted to the tectum were found to be rectified and high-pass filtered resulting in a stronger tuning to orientation. Furthermore, we show that the output of some RGCs can adapt to an ongoing stimulus but elicit a strong response upon alteration of the stimulus.

147

14) Population omitted stimuli responses on retinal gangion cells J. Araya, R. Herzog, C. Miguel, MJ. Escobar, AG. PalaciosLagnado Universidad de Valparaíso – Universidad de Santiago de Chile

The omitted stimulus response (OSR) is a phenomena that has been described in the retinal ganglion cells (RGC) when stimulated with repetitive light patterns. In brief, OSR is a response to a violation of the periodicity of the stimulus and its principal feature is the locking of the responses to a specific range of stimulus period. It has been reported a wide variety of OSR at the single cells level, finding that some cells spike both for the repetition and the violation of the pattern, joining different aspects of the stimulus on one single stream, making them inseparable for the brain. This suggests that we should focus at the population level: MJ Berry II & G Schwartz took this into account, and hypothesized that the retina separates the early visual information in two broad population responses: i) one associated with the predictable aspects of the stimuli, like the period of a repeated stimulus and ii) another related to surprising aspects of stimulus, as the periodicity violation. To test this hypothesis, we used multielectrode arrays to record hundreds of RGC (mean: 274, s.d: ± 50) from a patch of a diurnal rodent (Octodon degus, n=3) retina stimulated with a simple periodic flash sequence. The stimulus was 20 trials of 10 pulses of the same light-dark times, evaluated with different frequencies to evoke the OSR, finding it only on the 2.5 – 12.5 Hz range. For each trial, we took the population responses to every flash and its corresponding omitted stimulus response to build a similarity matrix between responses. Then, applying singular value decomposition to this matrix we extract two orthogonal population responses: i) the OSR which is correlated to the response at the beginning of each trial and ii) one response observed during the repetitions of the light pattern on each trial. These observations are valid only in the range where the OSR are observed. The second response becomes clearer after 2-3 repetitions of the pattern and not after a specific time. These results show that the retinal population responses under a repeated stimulus pattern are actually separated in two broad streams: one related to surprise (the pattern beginning and end) and another to the predictive aspects of stimulus (responses inside each trial). We propose as future direction to include this phenomena and corresponding results into the predictive coding framework, which could suggest that some predictions about the visual scene are actually generated in the early visual system.

148

15) Encoding of contrast using quanta in retinal bipolar cells José Moya-Díaz, Léa Darnet, Ben James & Leon Lagnado School of Life Sciences, University of Sussex, Brighton BN1 9QG, UK.

How is information transmitted across synaptic connections, where the fundamental signal is the quantum of neurotransmitter released from a vesicle? We are investigating this question in the retina using the intensity-based glutamate-sensing fluorescent reporter (iGluSnFR), which has the sensitivity to “count” vesicles in live zebrafish (abstract by Darnet et al.) Here we ask how bipolar cells encode changes in contrast. How many vesicles are used? How does this code vary between different types of bipolar cell?
 We find that: i) In most cases, at lower contrasts (between 1% to 20%) there is a predominance of events that represent the release of individual quanta from synaptic terminals, but at higher contrasts multiquantal events, likely reflecting multivesicular release, became more common. ii) The maximum release rate varies widely between active zones, ranging from 5 to 50 vesicles per second. iii) The contrast-response functions were often power relations: both linear and supra-linear relations were observed. iv) When stimulating at 5 Hz, individual terminals exhibit opposing forms of adaptation: some depressed while others sensitized. v) When we recorded from two or more active zones in a single terminal, we did not find significant differences in release rates, shape of contrast-response function or adaptive properties. These results help us understand how bipolar cell synapses transmit information about the contrast of a visual stimulus using the quantum of neurotransmitter as the “currency” of information.

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16) Plexin-A1 and Semaphorin-6D are involved in retinal axon targeting Delphine Prieur1, Cedric Francius1, Patricia Gaspar1, Carol A. Mason2 and Alexandra Rebsam1 1 2

INSERM U839, Institut du Fer à Moulin, Université Pierre et Marie Curie, Paris, France Columbia University, New York, USA

Retinal axons fasciculate together in the optic tract until they reach their retinal ganglion cell subtype-dependent target and exit the tract to arborize within this specific target. Furthermore, within the same target, such as the dorso-lateral geniculate nucleus (dLGN), retinal axons from the same (ipsilateral) and the opposite (contralateral) eyes innervate separate territories, forming an eyespecific map. We investigated the role of the guidance receptor Plexin-A1 and its ligand Semaphorin-6D (Sema6D) in these targeting processes. After anterograde tracing, Plexin-A1 -/- or Sema6D -/- mice present ectopic retinal projections in the dLGN, without alterations in topographic mapping. A 3DISCO clearing of Sema6D -/- mouse brains allowed 3D vizualisation and revealed that ipsilateral projections extend and form a new ipsilateral territory on the other side of the optic tract. A similar phenotype is observed in Sema6D +/- Plexin-A1 +/- mice, indicating that Sema6D and Plexin-A1 interact together for the appropriate target innervation by retinal axons. Both Plexin-A1 and Sema6D are expressed in retinal ganglion cells and dLGN during development. To determine where the expression of Plexin-A1 and Sema6D is required, we used in utero electroporation of Sema6D-shRNA or Plexin-A1-shRNA to knock-down their retinal expression before target innervation. Mice electroporated with Sema6D-shRNA or Plexin-A1-shRNA present similar targeting defect as Sema6D /- and Plexin-A1 -/- mice in the dLGN and we found non-cell autonomous effects for both of them. Thus, we showed that the expression of Sema6D and Plexin-A1 in the retina is essential for retinal axonal targeting.

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17) Processing of global motion images by local clusters of retinal ganglion cells Akihiro Matsumoto1, Masao Tachibana2 1

Global Innovation Research Organization, Ritsumeikan University Research Organization of Science & Technology, Ritsumeikan University

2

Our visual perception is unified and continuous, though our eyes repeatedly shift position (saccade) and alter fixation. The whole image projected on the retina (the retinal image) not only moves rapidly during saccade but also jitters even during steady gaze due to fixational eye movements. However, it is not yet fully understood how the retina processes the global motion images. Here, we show a novel coordinated processing of eye movement-like global motion images by retinal ganglion cells (RGCs). We recorded the firing of many RGCs in the goldfish isolated retina using a multi-electrode array, and classified each RGC into several groups based on the temporal profile of its receptive field (RF). We found that global jitter motion (simulated fixational eye movements) modulated the spatiotemporal RF properties in a group-specific manner. Fasttransient (Ft) RGCs showed significant changes in spatial and temporal RF properties. Whole-cell recordings revealed that global jitter motion which covered outside the RF estimated by the static background evoked excitatory postsynaptic currents with fast kinetics ("sharp EPSCs”) in addition to usual EPSCs in Ft RGCs. Subsequent global rapid motion (simulated saccades) evoked nonlinear and temporally coordinated responses in Ft RGCs. This fast and coordinated firing can transmit afferent retinal information to the brain during saccades and might help the brain respond to the visual scene during eye movements.

151

18) Establishing multi-electrode patch-clamp recordings to quantify population activity in the mouse retina Anna Stöckl, Sathish Narayanan, and Petri Ala-Laurila Department of Neuroscience and Biomedical Engineering, Aalto University, Espoo, Finland

Quantifying the synchronous activity of well-defined neural populations is often crucial for understanding neural circuit function. The retina is an ideal preparation for these studies, due to its accessibility, well-characterized structure and the possibility to use light as its natural stimulus. However, despite the great advances of multi-electrode spike recordings and optical imaging techniques, the current options to access neural populations in the retina across identified circuits, and at the level of input currents, are limited. Following the pioneering approaches in vertebrate brain slice recordings, we have built a state-of-the-art multi-electrode patch-clamp system to overcome these challenges. It allows simultaneous recordings of input currents and/or output voltages from four retinal neurons in flat-mounts and slice preparations, as well as access to their neuroanatomy and cell identity. The setup is designed to extend up to eight electrodes in the future. In addition to the system’s hardware, we are also developing custom-written open-source software to control data acquisition and visual stimulation, as well as online and offline data analysis which allows effective data sharing and reproducibility of results across laboratories, in accordance with Neurodata without Borders4. Here we give a detailed overview of our four-electrode patch-clamp setup and its software features - including preliminary test data – and provide an outlook on future projects using this novel approach to investigate population coding in the mouse retina.

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19) The visual system of the diurnal rodent, Rhabdomys Pumilio A.E. Allen1, A.A. Vugler2, R. H. Douglas3, G. Jeffery2, R.J. Lucas1 1

Faculty of Biology Medicine and Health, University of Manchester, UK Institute of Ophthalmology, University College London, UK 3 Department of Optometry and Visual Science, City University London, London, UK 2

The acuity and colour discrimination of the human visual system relies upon the high density of cone photoreceptors found within the central retina. Approximating this aspect of vision is challenging with conventional rodent models, owing to their low cone density (<3% in rats and mice). Diurnal rodents offer an alternative, with their ecological niche driving a shift in demands placed upon their visual system, optimising it for bright light and hence, cone vision. Rhabdomys pumilio, also known as the four-striped grass mouse, is one such species of diurnal murine rodent that has recently been domesticated. Rhabdomys share many characteristics with laboratory mice (approximate size, gross morphology, ecology, social structure, diet, predatory threats, etc.), making it an attractive experimental model. Here, we set out to make a primary characterisation of the Rhabdomys visual system, to assess its utility as a model for studying retinal circuitry and downstream visual processing in a visual system optimised for bright light. Our anatomical exploration of the Rhabdomys visual system has revealed a number of interesting hallmarks that are consistent with its diurnal phenotype. Histological examination of the Rhabdomys retina reveals a dichromatic, conedominated retina (>60% cones), an enlarged inner-nuclear and inner-plexiform layer, and an increased density of cone photoreceptors and retinal ganglion cells in the central retina. Rhabdomys has a pliable (accommodating) lens with low UVtransmission. Tracing of retinal ganglion cell axons shows dense innervation of areas including the dorsal lateral geniculate nucleus (dLGN), and superior colliculus (SC). We have also examined the functional capabilities of Rhabdomys vision using multi-channel recording electrodes to record from the visual thalamus of anaesthetised Rhabdomys. These recordings have uncovered high temporal and spatial acuity, colour opponency, and striking orientation selectivity. Put together, these findings highlight the potential utility of Rhabdomys as a rodent model that is experimentally tractable, and which represents an improved model for understanding cone vision compared with lab mice.

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20) A possible explanation for the difference in spatial integration between G9 and Off- alpha ganglion cells in the rabbit retina Iris Fahrenfort, Wei-Li Liu, Ming Tian, Steve Massey and Stephen Mills Ruiz Department of Ophthalmology and Visual Science

Background: Non-linear aspects of the receptive fields of GCs, are involved in interesting features such as sensitivity to fine spatial detail of the stimulus. Crossover inhibtion has been shown to play a significant role in spatial integration and has been found to rectify the output of bipolar cell terminals (Liang and Freed, 2010), which is associated with non-linear spatial integration. On the level of the GC dendrites, cross-over inhibition linearizes the output of the GC (Molnar et al., 2009). Methods: Both classical and extra-classical receptive fields of G9 and Off-alpha GCs were probed using both loose patch- and whole-cell voltage clamprecordings. Stimuli of different size, contrast, and spatial detail were used to characterize both their inhibitory and excitatory inputs. Possible anatomical correlates of cross-over inhibition onto the GC dendrites were measured by counting the amount of AII-associated α1-containing glycine receptors using triple-labeling imunocytochemistry. Results: Off-alpha GCs and G9 GCs have a pronounced difference in the way they spatially integrate stimuli. Off-alpha GCs show a characteristic second harmonic response to sinusoidally modulated gratings, indicating non-linear spatial integration. G9 GCs show a much more linear integration of stimuli that contain spatial detail. Off- alpha GCs also receive significantly more AII-associated glycine receptors compared to G9 GCs. In addition to On-pathway driven cross-over inhibition, G9 GCs receive Off-inhibition. Conclusion: The relatively higher cross-over inhibition onto the Off-alpha GC dendrites does not explain the relative non-linear behavior of the Off-alpha GC compared to the G9, given that cross-over inhibition onto GCs is often proposed to linearize the output of the GCs. G9s receive off pathway-driven glycinergic inhibition. Their response peak is always later than that of the excitation. It is tempting to conclude that G9 ganglion cells provide direct input to the AII lobules, although we cannot exclude input to a non-AII glycinergic amacrine cell type. We hypothesize that the “G9-off bipolar cell”-induced local hyperpolarization of the AII lobule reduces glycine release from the AIIs to the terminal of the same bipolar cell. This will depolarize the terminal, leading to less rectification of its glutamate release. This in turn, will linearize spatial integration. 154

21) Cone contributions to ipRGCs Josh Mouland, Tim Brown The University of Manchester

A subset of retinal ganglion cells (RGCs) within the mammalian retina express the photopigment melanopsin. These cells are therefore intrinsically photoreceptive (ipRGC) and also receive synaptic input from rods and cones. The contributions of inner and outer retinal photoreception to ipRGC sensory properties is still incompletely understood, however. Recent data indicate chromatic, conedependent, contributions to some ipRGC-driven physiological responses in mice and humans. However, whilst colour opponent ipRGCs have been reported within the primate retina, they have yet to be observed in any other species. Here then we set out to define the influence of long- and short-wavelengthsensitive (L-/S-) cone photoreception on ipRGCs responses across the murine retina. To this end we employed large-scale (256 channel) multi-electrode array recordings from the retina of human-cone knockin mice (Opn1mwR) alongside multispectral cone or melanopsin isolating stimuli. Although we find many mouse ipRGCs that exhibit robust L- and/or S-cone driven responses our data rule out the presence of large numbers of such cells that combine these inputs in an opponent manner, consistent with the generally low prevalence of colour opponency found here among conventional RGCS (~3% of all cells).

155

22) Characterization of VIP-1 amacrine cell coupling and its modulation by dopamine Luis Pérez de Sevilla Müller1, Janira de los Santos1 and Nicholas C. Brecha1,2,3,4 1

Department of Neurobiology, Department of Medicine, 3 Jules Stein Eye Institute, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095-1763, 4 Veterans Administration Greater Los Angeles Health System, Los Angeles, CA 900731003 2

A subset of retinal ganglion cells (RGCs) within the mammalian retina express the photopigment melanopsin. These cells are therefore intrinsically photoreceptive (ipRGC) and also receive synaptic input from rods and cones. The contributions of inner and outer retinal photoreception to ipRGC sensory properties is still incompletely understood, however. Recent data indicate chromatic, conedependent, contributions to some ipRGC-driven physiological responses in mice and humans. However, whilst colour opponent ipRGCs have been reported within the primate retina, they have yet to be observed in any other species. Here then we set out to define the influence of long- and short-wavelengthsensitive (L-/S-) cone photoreception on ipRGCs responses across the murine retina. To this end we employed large-scale (256 channel) multi-electrode array recordings from the retina of human-cone knockin mice (Opn1mwR) alongside multispectral cone or melanopsin isolating stimuli. Although we find many mouse ipRGCs that exhibit robust L- and/or S-cone driven responses our data rule out the presence of large numbers of such cells that combine these inputs in an opponent manner, consistent with the generally low prevalence of colour opponency found here among conventional RGCS (~3% of all cells).

156

23) Endocannabinoid modulation of retinal signalling Xiaohui Lin1,2, Andrea Yong1,2, Kevin Leung1,2, Jin Huang2,3, Charles Yates1,2 and Dario Protti1,2 1

Department of Physiology, The University of Sydney, Sydney, Australia Bosch Institute, The University of Sydney, Sydney, Australia 3 Discipline of Biomedical Science, The University of Sydney, Australia, 2

Signal processing in the retina involves complex neural computations carried out by different circuits and involving several distinct physiological mechanisms, including short and long-term plasticity phenomena. The endocannabinoid (eCB) system and all of its components have been identified in the retina. We have previously shown that an exogenous cannabinoid receptor agonist, WIN55212-2, reduced the strength of synaptic transmission, whilst the eCB receptor antagonist AM251 enhanced synaptic transmission onto retinal ganglion cells (RGCs). Whether or not eCBs are released in the retina and their potential physiological role, however, is still unknown. In this study we investigated whether eCBs are released in physiological conditions in the retina and, if so, how they affect retinal processing of visual stimuli and RGC excitability. We examined how eCBs modulate the response strength and receptive field organisation of RGCs by doing wholecell patch-clamp recordings from RGCs in current-clamp mode. In addition, we assessed the effects of modulating the eCB, as well as the potential involvement of TRPV1 channels as targets of the eCB system, on RGC excitability by monitoring the activity of sodium channels in RGCs using voltage-clamp conditions and by doing direct, optogenetic stimulation of channelrhodopsin-expressing RGCs. We recorded visual-evoked responses and voltage-gated sodium currents before and after bath application of URB597 (1 μM), an inhibitor of the enzyme that degrades Anandamide and co-application of URB597 and capsazepine, an antagonist of TRPV1 channels. Bath application of URB597 reduced the peak amplitude of visual-evoked postsynaptic potentials and the spatial tuning of ON and OFF RGCs. Paradoxically, URB597 increased RGC peak spike count of visual-evoked responses. URB597 also increased the strength of channelrhodopsin-mediated responses. In addition, URB597 shifted the current-voltage curve of the sodium current to the left. Capsazepine reversed the URB-induced effects on light-responses and RGC excitability. Our results indicate that eCBs are tonically released in the retina where they modulate synaptic transmission, consistent with their function in other central nervous system areas. These results also suggest that eCBs differentially target distinct receptors and channels presynaptic to RGCs and directly on RGCs and thus, their net effect will depend on the balance of these opposing actions. 157

24) Extrasynaptic GABAAR Gabra6 and Gabra4 subunit expression in mouse outer retina suggests horizontal cell signaling through tonic inhibition Arlene A. Hirano1,5, Alexander Solomon1, Steven Barnes1,4,5, Nicholas C. Brecha13,5 1

Neurobiology, Ophthalmology, 3 Medicine, Geffen School of Medicine at UCLA, Los Angeles, CA, 90095, USA; 4 Physiology & Biophysics, Ophthalmology & Visual Sciences, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4R2; 5 VAGLAHS, Los Angeles, CA, 90073, USA 2

Horizontal cells possess the molecular machinery for vesicular release, including VGAT/VIAAT, which accumulates GABA and inhibitory amino acids into synaptic vesicles, and horizontal cell VGAT/VIAAT knockout eliminated inhibitory feedback to photoreceptors (Hirano et al., 2016). To determine the possible targets of horizontal cell GABA release, we investigated the expression of GABA receptors in the mouse retina. Immunohistochemistry with Gabra6 (a6) antibodies produced hotspots of labeling deep within the outer plexiform layer (OPL) and faint outlines of bipolar cell bodies. Staining with peanut agglutinin to label cone pedicles demonstrated a6-like immunoreactivity was concentrated beneath cone pedicles. Double labeling with calbindin antibodies indicated that a6-like immunoreactivity is on horizontal cell processes and runs up the stalks of horizontal cell endings innervating rods. Rod bipolar cells, identified by protein kinase C immunostaining, showed a6-like immunoreactivity on the dendrites running along the same horizontal cell stalk-like regions. ON bipolar cell dendrites, immunolabeled for Galphao, showed a similar pattern of a6-like immunoreactivity, which did not seem to reach the very tips. RT-PCR using multiple sets of primers designed from a6 cDNA sequence(s) produced amplicons of the predicted sizes from retina, although the intensity was much less than that from cerebellum, indicating a low level of mRNA expression in retina. Immunolabeling with Gabra4 antibodies labeled putative cone photoreceptor and bipolar cell bodies, as well as the ChAT bands in the IPL. Light Gabrd (delta) subunit expression occured in the inner nuclear layer and outer plexiform layer. Extrasynaptic GABAA receptor expression in the outer retina suggests tonic inhibition by GABA/IAA of bipolar cells and photoreceptors and that these receptors may be targets of horizontal cell signaling.

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25) Functional diversity of mouse retinal ganglion cells in 4096-electrode CMOS array recordings Fernando Rozenblit, Vidhyasankar Krishnamoorthy, Tim Gollisch Department of Ophthalmology, University Medical Center Göttingen, Germany Bernstein Center for Computational Neuroscience Göttingen, Germany

Horizontal cells possess the molecular machinery for vesicular release, including VGAT/VIAAT, which accumulates GABA and inhibitory amino acids into synaptic vesicles, and horizontal cell VGAT/VIAAT knockout eliminated inhibitory feedback to photoreceptors (Hirano et al., 2016). To determine the possible targets of horizontal cell GABA release, we investigated the expression of GABA receptors in the mouse retina. Immunohistochemistry with Gabra6 (a6) antibodies produced hotspots of labeling deep within the outer plexiform layer (OPL) and faint outlines of bipolar cell bodies. Staining with peanut agglutinin to label cone pedicles demonstrated a6-like immunoreactivity was concentrated beneath cone pedicles. Double labeling with calbindin antibodies indicated that a6-like immunoreactivity is on horizontal cell processes and runs up the stalks of horizontal cell endings innervating rods. Rod bipolar cells, identified by protein kinase C immunostaining, showed a6-like immunoreactivity on the dendrites running along the same horizontal cell stalk-like regions. ON bipolar cell dendrites, immunolabeled for Galphao, showed a similar pattern of a6-like immunoreactivity, which did not seem to reach the very tips. RT-PCR using multiple sets of primers designed from a6 cDNA sequence(s) produced amplicons of the predicted sizes from retina, although the intensity was much less than that from cerebellum, indicating a low level of mRNA expression in retina. Immunolabeling with Gabra4 antibodies labeled putative cone photoreceptor and bipolar cell bodies, as well as the ChAT bands in the IPL. Light Gabrd (delta) subunit expression occured in the inner nuclear layer and outer plexiform layer. Extrasynaptic GABAA receptor expression in the outer retina suggests tonic inhibition by GABA/IAA of bipolar cells and photoreceptors and that these receptors may be targets of horizontal cell signaling.

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26) Probing the retinal circuit with two-photon computer generated holography and calcium imaging Ronzitti* E. 1, 2, Spampinato* G. L. B. 3, 4, Papagiakoumou E. 1,2,5, Khabou H. 3, 4, Robert C. 3, Dalkara D. 2, 3, Picaud S. 2, 3, Marre O. 3, 5, Emiliani V. 1, 2 1

Univ. Paris Descartes, Paris, France; CNRS, Paris, France; 3 The Vision Institute, Paris, France; 4 UPMC, Paris, France; 5 INSERM, Paris, France 2

A current major goal of neuroscience is to determine how neural circuits activity is related to sensory stimuli. In order to understand this link, it is necessary to elaborate specific strategies to reconstruct the complete functional connectivity diagram of a neural network. That demands appropriate toolkits including sensors and actuators to detect and trigger the activity of neurons, either individually or in groups. Here we have developed an all-optical approach to systematically investigate the functional connectivity between different neural layers, and applied it to the retina. A key component of retinal processing is the information transfer from the intermediate bipolar cell layer - which integrates the photoreceptor responses to the retinal ganglion cell layer - the output of the retina. In order to elucidate this process, we expressed opsin actuators in ON bipolar cells and GCaMP sensors in ganglion cells of wild type mice. We then performed two-photon excitation (2PE) imaging of ganglion cells, while using 2PE computer generated holography to selectively stimulate cells in the bipolar layer. Such a 3D probing strategy of the retinal circuit enables to measure the impact of complex stimulation patterns of bipolar cells on the ganglion cell activity and thus characterize the functional connectivity map between the two layers. This method paves the way towards complete functional connectomics of the retina, and could potentially be applied to any layered structure in the brain.

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27) Congenital stationary night blindness as a model to decipher retina signaling Christina Zeitz Sorbonne Universités, UPMC Univ Paris 06, INSERM U968, CNRS UMR 7210, Institut de la Vision, Paris, France

The first steps in vision occur when photoreceptors transform light into a signal, which then gets processed through the inner retina via the bipolar cells. The initial steps described by the phototransduction cascade are well understood, while the downstream transmission from photoreceptors to bipolar cells remains to be dissected in more details. Rods synapse with rod ON-bipolar cells and cones synapse with cone ON- and OFF-bipolar cells. Knowledge about the phototransduction cascade was gained by genetic studies on progressive retinal diseases, in which molecules of this cascade are mutated. Knowledge about the downstream signaling from photoreceptors to bipolar cells was gained by genetic studies on congenital stationary night blindness (CSNB), in which molecules of this cascade are mutated. Mutations in CACNA1F and CABP4 lead to incomplete CSNB, which represents ON- and OFF-bipolar cell dysfunction. This can be confirmed by rod and cone photoreceptor synapse immunolocalization of the respective proteins. Mutations in NYX, GRM6, TRPM1, GPR179 and LRIT3 lead to complete CSNB, which represents ON-bipolar cell dysfunction. This can be confirmed by rod and cone ON-bipolar cell immunolocalization of the respective proteins. Here we will present ongoing studies on CSNB gene defect identification, investigation of the pathogenic mechanism by in vitro and in vivo studies, which will help to better understand this disorder and retinal signaling in general. Our recent findings show that intronic or silent mutations in known genes may explain CSNB in several cases, previously excluded for mutations in the coding regions of these genes. In addition, although genotype-phenotype correlations in general overlap nicely with the immunolocalization of the respective proteins and distinguish two major forms, namely complete and incomplete CSNB, some gene defects cannot be classified as such. In addition, immunonolocalization studies, multi-electrode array and electron-microscopy studies in mouse models for CSNB point to small phenotypic differences most likely due to their slightly different role important for the signaling from the photoreceptors to bipolar cells. Together, although gene defect identification in CSNB patients helped to add molecules to this signaling cascade from photoreceptors to bipolar cells, other molecules need to be identified to elucidate this cascade in more detail.

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28) The role of fast noise correlations in encoding dynamic stimuli in the retina 1 Ulisse Ferrari, 2Stephane Deny, 3Gasper Tkacik, 4Thierry Mora, 1Olivier Marre 1

Institut de la Vision, INSERM & UPMC, Paris, France Neural Dynamics and Computation Lab, Stanford University, California 3 Institute of Science and Technology, Klosterneuburg, Austria 4 Laboratoire de physique statistique, CNRS, UPMC and Ecole Normale Superieure, Paris, France 2

A major challenge in sensory neuroscience is to understand how complex stimuli are encoded by neural circuits. In the retina, several layers of neurons process the visual stimulus, which is ultimately encoded by the spiking activity of ganglion cells. It is still unclear how the visual scene is encoded by the collective activity of ganglion cells. Here we recorded large populations of ganglion cells with multi-electrode array on rat retinas responding to complex stimuli, such as videos of moving objects. We found that neighboring cells exhibit fast correlations, that cannot be explained by the joint activation due to the stimulus. The time scale of these “noise correlations” is fast enough to be mediated by gap junctions, and they were present specifically for cells of the same type. In order to investigate the role of these noise correlations in the encoding of the visual scene we have constructed a model capable of predicting both the response of individual single cells (PSTH) and the observed noise correlations. This model is composed of a cascade of two layers of processing, and equipped with a recurrent interaction network among the ganglion cells to reproduce specifically noise correlations. We are currently using this model to measure how noise correlations impact the coding of these complex stimuli, and test if they are helpful or detrimental to the encoding capacity of the retinal network.

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29) Ganglion cell electrical synapses may serve as switches between independent and population coding operation modes in the mammalian retina Márton Balogh1,2,3,5, Gerrit Hilgen4, Evelyne Sernagor4, Béla Völgyi1,2,3 1

MTA-PTE NAP B Retinal Electrical Synapses Research Group, Pécs 7624, Hungary Department of Experimental Zoology and Neurobiology, University of Pécs, Pécs, H-7624, Hungary 3 János Szentágothai Research Center, University of Pécs, H-7624, Pécs, Hungary 4 Institute of Neuroscience, Newcastle University, Newcastle upon Tyne NE2 4HH, UK 5 Center for neuroscience, EFOP-3.6.1.-16-2016-00004 2

It has been demonstrated that neurons in the central nervous system interconnect via electrical synapses to synchronize their spiking output. Retinal ganglion cells utilize this strategy to create a synchronous spike based population code and inform the brain about certain aspects of the visual scene. Ganglion cell electrical synapses are thought to serve this spike synchronization. Therefore, ganglion cells that maintain gap junction contacts with their neighbor ganglion and/or amacrine cells have been hypothesized to partake in population coding rather than the individual receptive field based encoding mechanism. However, it has recently been shown that most ganglion cell types in the mouse retina display electrical and tracer coupling to their neighbors suggesting that most but a few ganglion cells are classic independent encoders. Here we provide evidence that gap junction coupled ganglion cells may serve both as independent encoders and also a population coders depending on the conductance of their corresponding gap junctions. We show that individual ganglion cell light responses are enhanced when gap junctions are blocked, whereas correlated ganglion cell spiking as a measure of population activity is augmented. This indicates that ganglion cell gap junctions likely serve as switches between encoding modalities thereby adapting to changes in ambient light conditions.

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30) Inactivation of microRNA183/96 leads to retinal dysfunctions in mice Jasmin Segelken1, Malin J. Lammers1, Hans Gerd Nothwang3 and Ulrike JanssenBienhold1,2 1

Visual Neuroscience, Dept. of Neuroscience, University of Oldenburg, Oldenburg, Germany 2 Neurobiology, Dept. of Neuroscience, University of Oldenburg, Oldenburg, Germany 3 Neurogenetics Group, Dept. of Neuroscience, University of Oldenburg, Oldenburg, Germany

MicroRNAs (miRNAs) are short, noncoding, highly conserved RNA molecules that regulate gene expression by targeting their downstream messenger RNAs (mRNAs) and inducing the breakdown and/or inhibition of protein translation. At least 78 miRNAs have been found to be expressed in the mouse retina (Xu et al., 2007). Several studies reported that certain miRNAs showed unique developmental stage-specific expression patterns and play an important role during differentiation and function of the retina. Here, we analyzed miR-183/96knockout mice to investigate the role of miR-183 and miR-96 in the retina. We performed in vivo electroretinography (ERG) recordings to evaluate light responses of the retina under scotopic and photopic conditions. To unravel potential morphological defects, we carried out histological analysis in vertical sections and wholemount retinas. We focused on photoreceptors and proteins of the photoreceptor synaptic complex. Additionally, apoptotic and degenerative processes have been observed via TUNEL assays and IBA-1 activated retinal microglia staining. The inactivation of miR-183/96 caused functional and structural changes of the outer retina. We observed abnormal ERG responses, photoreceptor degeneration and structural changes within the photoreceptor synaptic complex. In addition to that, prominent rosettes occurred in the miR183/96 knockout retinas, which decreased with age and disappeared in one year old mice. Inside the rosettes, microglia/macrophages were present, suggesting that they might contribute to the degradation process. In summary, our data indicate that miR-183/96 plays an important role in photoreceptor maintenance and structural integrity of the outer retina. The study was supported by the DFG (GRK 1885/1 to JS, UJB and HGN).

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31) Effects of saccade-like image shifts on retinal output: characterization and underlying mechanisms Saad Idrees1,2,3 and Thomas A. Münch1,2,4 1

Werner Reichardt Centre for Integrative Neuroscience, Tübingen, Germany; 2Bernstein Center for Computational Neuroscience; 3IMPRS for Cognitive and Systems Neuroscience, Tübingen, Germany; 4Institute for Ophthalmic Research, Tübingen, Germany

Purpose During natural viewing, image flow on the retina is determined primarily by the behavior of the observer. Eye movements during active visual exploration, such as saccades, are particularly dominating in this regard, especially because they incessantly occur 3-5 times per second. Despite this, retinal image processing has in the past often been studied with isolated stimuli, ignoring the highly dynamic aspects of natural viewing. We are therefore exploring the effects of eyemovement induced image motion on signal processing in the retina. We hypothesize that responses of ganglion cells, to a stimulus following a saccade, is modulated as a function of time between the saccade and that stimulus. Here we attempt to answer two broad questions: (A) How do saccades effect responses to subsequent stimuli within the retina? (B) What are the mechanisms behind these effects in the retina? Methods We recorded activity of ganglion cells from isolated mouse and pig retina using multi-electrode arrays (MEAs) while showing a visual stimulus paradigm mimicking saccades. The paradigm consisted of rapid saccade-like image shifts across the retina followed by a test stimulus at different intervals after the saccade. Results We found that the retinal responses to these "test stimuli", when presented in the context of saccades, are strongly modulated in comparison to responses to the same stimuli presented in isolation. Saccades exude their modulatory effects for up to 1 second, suggesting a highly relevant role of eye-movement induced effects under natural conditions with short inter-saccadic fixations. Such saccade-related retinal modulation critically depends on (1) the delay between saccade and test stimulus; (2) the statistical properties of the background scene present during the saccade; (3) the specific retinal cell type; (4) and the ambient luminance level. For example, the responses of OFF ganglion cells, but not ON ganglion cells, can be enhanced after saccades. Furthermore, our results indicate that these modulations originate from both the center and surround of a cell’s receptive field, but have different properties. Modulations originating from the surround 165

have rapid, short-lasting effects, whereas modulations originating from center have long lasting effects.

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32) The role of spikes for information transmission in bipolar cell models C. Schröder1, T. Baden2, J. Gjorgjieva3, T. Euler1, P. Berens1 1

Institute for Ophthalmic Research Tübingen / CIN Tübingen / Bernstein Center for Computational Neuroscience Tübingen 2 School of Life Sciences, University of Sussex / Institute for Ophthalmic Research Tübingen 3 Max Planck for Brain Research, Frankfurt

Traditionally, most inner retinal neurons are thought to respond with graded voltage changes to visual stimulation. These analog signals would be converted into spikes only at the level of the retinal ganglion cells, the output neurons of the retina. However, a growing body of evidence instead argues that this signal conversion from ’analog’ to ’digital’ can already take place at the level of the bipolar cell synaptic terminals (Protti et al., 2000; Baden et al., 2013; Puthussery et al., 2013). This conversion impacts information transmission and coding, for example by increasing the precision of the signal, but at the expense of decreased reliability and higher energy consumption (Baden et al., 2011; Sengupta et al., 2014). To understand the biophysical regimes in which spiking can occur and to study the effects of spiking on information coding, we built a simple two-compartment model of bipolar cells. The first compartment is a Hodgkin-Huxley-like channel system, which incorporates four types of ion channels (Na+, K+, L-type calcium and T-type calcium channels). It is driven by a light stimulus with different statistics, which is preprocessed by a simple linear filter aimed to model linear photoreceptor input. The second compartment models a ribbon synapse (Sikora et al., 2005). This ribbon synapse transforms the voltage signal into calcium concentration, which in turn drives the dynamics of three different vesicle pools (Burrone et al., 1997). The release rate of these neurotransmitters act as the output signal of our model and a “fair currency” for comparing information rates in different regimes. First, we investigated the impact of channel densities, channel dynamics and different sources of noise on the responses to light stimuli. In this regard we identified parameter regimes in which the first compartment switches from a graded signaling to a spiking mode. As expected, response waveforms were dominated by sodium and potassium channels, but were in addition strongly influenced by the density of calcium channels. Next, we evaluated the information that can be read-out linearly from the release rate, with high information rates indicating regimes that may be favorable for coding. 167

In summary, our analyses provide insights into the operating regimes of bipolar cells and their possible consequences on information transmission in vision.

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33) Neural system identification for large populations separating “what” and “where” David A. Klindt, Alexander S. Ecker, Thomas Euler & Matthias Bethge Centre for Integrative Neuroscience; Bernstein Centre for Computational Neuroscience; Institute for Ophthalmic Research; Graduate School of Neural & Behavioural Sciences, International Max Planck Research School, University of Tübingen, Tübingen, Germany

Neuroscientists classify neurons into different types that perform similar computations at different locations in the visual field. Traditional neural system identification methods do not capitalize on this separation of “what” and “where”. Learning deep convolutional feature spaces shared among many neurons provides an exciting path forward, but the architectural design needs to account for data limitations: While new experimental techniques enable recordings from thousands of neurons, experimental time is limited so that one can sample only a small fraction of each neuron’s response space. Here, we show that a major bottleneck for fitting convolutional neural networks (CNNs) to neural data is the estimation of the individual receptive field locations – a problem that has been scratched only at the surface thus far. We propose a CNN architecture with a sparse pooling layer factorizing the spatial (where) and feature (what) dimensions. Our network scales well to thousands of neurons and short recordings and can be trained end-to-end. We explore this architecture on ground-truth data to explore the challenges and limitations of CNN-based system identification. Moreover, we show that our network model outperforms current state-of-the art system identification models in the mouse visual system.

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34) From artificial to natural stimuli: encoding of visual signals in retinal bipolar cells Helene Marianne Schreyer1,2 and Tim Gollisch1,2 1

Department of Ophthalmology, University Medical Center Göttingen, 37073 Göttingen, Germany 2 Bernstein Center for Computational Neuroscience Göttingen, 37073 Göttingen, Germany

Current mathematical models in the retina fail to reliably predict responses of retinal ganglion cells to natural stimuli. Often, in these mathematical models, it is assumed that the input neurons of the retinal ganglion cells behave linearly. In this work, we focus on the excitatory input neuron: the bipolar cell and test how well standard models – like the linear-nonlinear (LN) model – can predict bipolar cell responses to artificial and natural stimuli. To understand and predict bipolar cell responses to light, we started with simple full-field contrast changes while recording the membrane potential of the cells intracellularly. Here, we found some cells with linear and others with nonlinear responses to contrast changes, and for both types the LN model successfully predicted responses to new contrast sequences (R2 of 82%-98%). We then continued with spatially structured artificial stimuli. For these stimuli, the performance of the LN model varied, for some cells the prediction was accurate (e.g. R2 of 82%) while for other cells the prediction failed (R2 of 34%). When investigating the reasons for the failure, we found a novel bipolar cell response property: nonlinear integration in space, similar to Y-type ganglion cells. Furthermore, this novel Y-type- like property observed in some bipolar cells caused the LN model to fail. To finalize, we showed natural movies to study how well we can apply the knowledge learned from artificial stimuli to predict responses to natural stimuli. Here, again, the model can predict the responses to natural movies well for linear cells, but failed for nonlinear Y-type-like bipolar cells. The findings suggest that nonlinear signal integration can start already at the level of bipolar cells and that nonlinear computations are crucial properties that mathematical models in the retina have to take into consideration for predicting responses to artificial and natural stimuli.

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POSTER SESSION IV: RETINAL CIRCUITS 1) Synergistic decoding of complex texture motion from populations of direction-selective ganglion cells Norma Kühn1, Tim Gollisch2 1

NERF, KU Leuven, VIB, IMEC; University Medical Center Göttingen, Bernstein Center for Computational Neuroscience Göttingen 2

Direction-selective (DS) ganglion cells are known to preferably respond to a certain direction of drifting motion but are suppressed by motion into the opposite direction. A subset of these cells is thought to report the direction of global image motion, as induced by body, head and eye movements, to downstream brain areas to guide compensatory eye movements. Although direction and velocity of the projected image are constantly changing during natural viewing, the directional preference of DS ganglion cells is usually probed with uniformly drifting bars or gratings. Here, we report from experiments in the isolated salamander retina that also during complex texture motion, the directional preference of DS cells is preserved. But in complex visual scenes the encoding of motion direction is ambiguous due to the cells’ simultaneous encoding of local contrast changes. These ambiguities in the encoding of motion direction of the individual cells can be resolved by reading from a population of DS cells with different preferred directions. Here, the joint population response provides more information about the global motion trajectory than would be expected by summing the individual contributions, resulting in a synergistic trajectory readout. Strong positive response correlations between DS cells enhance this synergy. This serves as an example of how population codes synergistically improve the extraction of single features from neurons encoding multiple features simultaneously in complex visual scenes.

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2) Specific inhibitory pathways mediate saccadic suppression in directionselective ganglion cells Benjamin Sivyer, Alexander Tomlinson, W. Rowland Taylor Benjamin Sivyer, Alexander Tomlinson: OHSU, Casey Eye Institute, Portland, Oregon, USA W. Rowland Taylor: UC Berkeley, Berkeley, California, USA

Two populations of direction-selective ganglion cells in the mammalian retina likely have different roles in mediating behavioural responses to motion. Our analysis of ON and ON-OFF direction-selective ganglion cells (DSGCs) reveal different responses to simulated saccades. ON-DSGCs are strongly suppressed, while ON-OFF DSGCs are activated. We show that these differences are present during stimulation with natural images, and demonstrate that the selective activation of glycinergic inhibitory pathways can explain much of the difference between the two cell types.

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3) Functional characterisation of parvalbumin-expressing cells in the mouse retina Gerrit Hilgen, Evelyne Sernagor Institute of Neuroscience, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK

It is well established that the Ca2+ binding protein parvalbumin (PV) is expressed in many retinal ganglion cells (RGCs) and amacrine cells (ACs). However, virtually nothing is known about their functional correlates. We have investigated the functional properties of PV-expressing cells in the mouse retina using pharmacogenetics, based on silencing (hyperpolarizing) PV neurons with Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) in the presence of the DREADD agonist clozapine N-oxide (CNO). A high-density large-scale MEA (Biocam, 3Brain) featuring 4096 electrodes covering most of the mouse retina was used to study light-evoked responses from 100s1,000s RGCs simultaneously. Various stimuli were used to characterize RGC receptive field organization and direction/orientation selectivity (DS, OS) in control conditions and in the presence of CNO (2 μM). RGCs with >50% decrease in activity in CNO were classified as PV-RGCs and were compared to all other RGCs (non-PV RGCs). The role of PV-expressing ACs was established by comparing how non-PV RGCs respond to light without and with CNO. Overall, we found a high incidence of OFF transient cells (17.5%) amongst PV-RGCs, whereas non-PV RGCs tend to have a higher incidence of ON transient responses (19.8%). We used the bias index (BI) to classify RGCs into ON (0.33
establishing DS/OS, presumably by providing a robust direct or indirect asymmetric inhibitory input specifically onto DS/OS cells. Finally, they appear to control oscillatory activity between RGCs during specific visual tasks.

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4) Photoreceptor inputs for light-induced dopamine release in the mouse retina. Morven A Cameron, Victor Perez-Fernandez and John W Morley School of Medicine, Western Sydney University

Adaptation of the retina to the presenting light conditions relies considerably on modulation of retinal pathways by dopamine (DA). DA is released by a unique set of neurons known as dopaminergic amacrine cells (DACs). Inputs to DACs from all three photoreceptor classes, cones, rods and intrinsically photoresponsive retinal ganglion cells (ipRGCs), have been identified, however, measurement of dopamine release in the retina in response to light does not align with electrophysiological and/or immunohistochemical reports. The purpose of this study was to describe the photoreceptive origin of light-induced dopamine release using pharmacology and transgenic/mutant mouse models. We used UHPLC-MS/MS analysis to quantify DA released from the mouse retina in an ex vivo preparation in response to 4 light intensities (max 1.6 log W/m2; ~10,000 lux). Wild-type, Gnat2A517G (cone-functionless), and rd/rd (lacking rods and cones) animals were light pulsed and pharmacological blockers applied. We found that light caused a significant increase in dopamine release in the retinae of both wild-type and Gnat2 A517G mice but only at very bright light intensities (> 0.65 log W/m2; ~1000 lux). In agreement with previous studies, no significant increase was obtained in response to light in rd/rd mice at any light intensity. The gap junction blocker MFA (200 μM) caused a significant reduction in light induced DA release in both wild-type and Gnat2A517G animals, but did not completely abolish the light-induced increase in comparison to dark conditions. While the glycine receptor blocker strychnine (10μM) did not affect dopamine release in either light or dark, the GABAA/C receptor blocker picrotoxin (100μM) caused a significant DA increase in both light and dark in wild-type mice. Interestingly, all the glutamatergic receptor class blockers reduced the light induced DA release in the wild-type mouse: L-AP4 (50 μM; blocks mGluR) and CNQX (10 μM; blocks AMPA/kainite Rs) both individually abolished light-induced DA release, while it was significantly reduced by AP-5 (50 μM; blocks NMDARs). Blockage by L-AP4 suggests that light-induced DA release depends on an ON bipolar cell mediated input from classical photoreceptors. Furthermore, our data implicates an important co-incident role for AMPA/kainite and NMDA receptors in this response. While all our data thus far support the theory that rods are driving the majority of this light-induced DA release, the threshold for this response is far (6 log units) above the rod threshold. 175

We hypothesize that the NMDA mediated input originates from ipRGC photoreceptors that is only effective when paired with inputs from the outer retina, allowing this input to be “gated” to such a high intensity

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5) Response properties of retinal ganglion cells and their underlying circuits vary with retinal location Rebekah Warwick and Michal Rivlin Weizmann Institute of Science

Retinal ganglion cells (RGCs) are divided into subtypes. Cells belonging to a single subtype share similar receptive field response properties. However, using a transgenic mouse line in which the transient Off-alpha RGC (tOff-αRGC) expresses GFP, we demonstrate that cells belonging to a single RGC subtype may reveal different response properties depending on their retinal location. While tOff-αRGCs in the ventral retina displayed transient responses to a dark spot, tOff-αRGCs in the dorsal retina displayed significantly more sustained responses. Voltage clamp recordings suggested the difference originates from a sustained disinhibitory current at light offset, which was significantly larger in dorsal-tOff-αRGCs. The mouse retina displays a non-uniform cone opsin distribution, dominated by mid-wavelength opsins in the dorsal retina and short-wavelength opsins in the ventral retina. Yet, the difference between dorsal- and ventral-tOff-αRGCs did not merely result from greater cone activation in the dorsal retina. First, dorsal-tOffαRGCs were still more sustained in response to UV illumination, which preferentially activated cones in the ventral retina. Second, the responses of dorsal-tOff-αRGCs in cone knockout mice (gnat2-/-) were even more sustained than in WT mice, whereas the responses of ventral-tOff-αRGCs were significantly diminished in gnat2-/- mice. Third, strychnine – a glycine receptor antagonist which blocks tOff-αRGCs inputs that are derived from the primary rod pathway – shortened the responses of dorsal-tOff-αRGCs so that they appeared similar to ventral-tOff-αRGCs. These experiments suggest that the differences between dorsal- and ventral-tOff-αRGCs are not merely mediated by distinct cone inputs. Instead, the rod input via the glycinergic AII amacrines differs between dorsal and ventral-tOff-αRGCs, underlying the different response durations. These data suggest that retinal circuits are subject to location dependent variations, possibly optimizing representation of the different stimulus statistics encountered in the upper and lower visual fields.

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6) Chromatic processing in the zebrafish outer retina. Takeshi Yoshimatsu1, Philipp Bartel1 and Tom Baden1,2 1

School of Life Sciences, University of Sussex, UK; Institute of Ophthalmic Research, University of Tuebingen, Germany.

2

Zebrafish colour vision is based on four spectrally distinct cone-photoreceptors. In the outer retina, signals from different cones are combined and modulated by horizontal cells to compute contrasts in wavelength. The result is then conveyed to the inner retina by bipolar cells. However, little is known about how chromatic information from each cone type is processed in vivo in the outer plexiform layer, with long range connections preserved. Here, we used two-photon calcium and glutamate imaging to study chromatic response properties of cones and horizontal cells in live larval zebrafish. We first examined the anatomical distribution of cones across the eye using transgenic lines and immunostaining to label specific cone types. This revealed a series of striking anisotropies across cone types with retinal position. The sum of all cones peaked temporally, indicating an increased spatial acuity in front of the animal. However, different cone distributions peaked in different regions within this overall trend. For example, blue cones were elevated at the visual horizon in either direction, while UV cones peaked temporo-ventrally, thus looking forward and upwards. We next expressed SyGCaMP6f in all cones or iGluSnFR in horizontal cells to investigate chromatic response properties of cones. Stimulation was performed with one of two custom Arduino-based tetrachromatic or hyperspectral full-field stimulators (see poster by Bartel et al.) that were synchronised with the scan retrace to avoid interference with the imaging. Chromatic stimulation revealed that cone action rarely matched predicted single opsin absorption spectra, but instead exhibited broader tuning and/or, in many cases, colour opponent responses. Further, the number of functional cone types far exceeded the four genetic cone types in zebrafish, suggesting functional diversity amongst single cone types which appear to be related to differential processing in different parts of the eye.

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7) Chromatic Processing in the Zebrafish Inner Retina Maxime Zimmermann1, Takeshi Yoshimatsu1, Tom Baden1,2 1

School of Life Sciences, University of Sussex, UK; Institute of Ophthalmic Research, University of Tuebingen, Germany.

2

Colour vision is a crucial capability of many visual systems that allows organisms to differentiate objects wavelength independent of intensity. Zebrafish are an interesting model to study colour vision as it possesses four principal conephotoreceptor types: red-, green-, blue- and UV- sensitive. Bipolar cells are the first projection neurons of the retina, receiving input from the photoreceptors and sending their axon in the inner plexiform layer (IPL). We imaged light-driven calcium signals from bipolar cell synaptic terminals in larval zebrafish and show that the IPL is highly organised into chromatic and achromatic layers across both traditional ON and OFF sublaminae. Chromatic responses were found in at least half of bipolar cell terminals, and included both spectral preferences as well as full antagonism. Furthermore, depending on their relative position in the eye, bipolar cell terminals systematically shift their chromatic and achromatic response properties to match natural spectral input statistics (cf. abstract by Nevala et al.). Taken together, chromatic processing is clearly a dominant feature of the zebrafish retina, with bipolar cells already encoding complex chromatic responses before driving retinal ganglion cells.

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8) Mapping the natural visual world of the zebrafish (Danio rerio) Nevala NE1, Nilsson DE2, Osorio D1 and Baden T1,3 1

School of Life Sciences, University of Sussex, UK; Lund Vision Group, Lund University, Sweden; 3 Institute of Ophthalmic Research, University of Tuebingen, Germany 2

The zebrafish (Danio rerio) is a popular model in vision research, but we know little about its natural visual world. A detailed understanding how their visual system is adapted to the natural environment, and what is important for the fish to see in their habitats in shallow freshwaters of the Indian subcontinent, is still missing. Mapping the visual environment of the animal and comparing this to the architecture and function of the eye across different life stages can help us to understand the evolution of vision and provide new perspectives when designing species-specific visual stimuli. Here, we surveyed what spectral content the environment holds for the zebrafish to see. We find that chromatic information systematically varies along the vertical axis with increasing depth, resulting in 3 separate zones with different chromatic features. Zebrafish are tetrachromats with 4 cone types sensitive to UV, blue, green and red light. To examine their natural visual world and to understand which wavelengths of light provide meaningful information for them, we used a custom waterproofed hyperspectral scanner to measure full spectrum images under water. Altogether 31 scenes were scanned in 6 different habitats in shallow ponds and slowly moving streams of West Bengal, India. As expected from previous work, the water itself and dissolved particles absorb most of the shorter wavelengths of light thereby red-shifting the spectrum available for underwater vision. The resultant peak wavelength aligns near perfectly with the sensitivity of red cones, leaving the UV cones at the extreme edge with minimal number of photons available. Next, we separated achromatic information from the chromatic content by principal component analysis (PCA). This revealed a chromatically rich horizon separating two more achromatic zones above and below. In the upper visual field shorter wavelengths are still present, while the areas close to the bottom are dominated with longer wavelengths. These results provide a quantitative and testable series of key predictions for chromatic processing in the zebrafish retina (see posters by Yoshimatsu et al. and Zimmermann et al.).

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9) Spatial Integration in Mouse Retinal Ganglion Cell Dendrites Yanli Ran1-3, Katrin Franke1,2,4, Ziwei Huang2,3, Tom Baden2,5, Philipp Berens1,2,4, Thomas Euler1,2,4 1

Centre for Integrative Neuroscience, Tübingen, Germany; Institute for Ophthalmic Research, Tübingen, Germany; 3 Graduate Training Centre of Neuroscience, Tübingen, Germany; 4Bernstein Centre for Computational Neuroscience, Tübingen, Germany; 5 School of Life Sciences, University of Sussex, Brighton, UK 2

Retinal ganglion cells (RGCs) are the output neurons of the eye. They send the visual information extracted by the retinal network to the brain. Recent functional and anatomical work suggests that there are more than 40 RGC types in the mouse retina, each selective for specific visual features like contrast, motion or edges. Much of this functional diversity is generated in the inner plexiform layer (IPL). Here, RGCs receive excitatory inputs from bipolar cells (BCs) and inhibitory inputs from amacrine cells (ACs). However, how specific RGC types integrate these inputs in their dendritic arbours to generate their characteristic responses is still largely unknown. To study how RGCs pool information across their dendrites, we inject the calcium indicator dye Oregon Green BAPTA-1 into individual RGCs in the ex-vivo intact retina. We then use two-photon calcium imaging to record local responses to various light stimuli in dendritic segments. After functional recordings, a high resolution stack with the cell’s dendritic morphology is acquired. Using semiautomatic neurite tracing, we then reconstruct the cell’s 3D structure to extract detailed morphological properties, including stratification and branching patterns, and the exact locations of the recorded segments. This allows us to relate functional features of dendritic segments like receptive field (RF) size to their morphological characteristics such as branch order. Our preliminary data indicates that different RGC types exhibit distinct forms of spatial integration: For instance, in transient Off alpha RGCs (and likely their On counterparts), dendritic segments closer to the soma systematically exhibit large, overlapping RFs, whereas segments closer to the dendritic tips are small and more spatially independent. In contrast, a small Off RGC type that co-stratifies with the transient Off alpha type exhibits no systematic change in RF size with distance from the soma.

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10) Linear and nonlinear integration of chromatic stimuli in retinal circuitry Mohammad Khani1, 2, 3, Tim Gollisch1, 2 1

Department of Ophthalmology, University Medical Center Göttingen, 37073 Göttingen, Germany 2 Bernstein Center for Computational Neuroscience Göttingen, 37073 Göttingen, Germany 3 International Max Planck Research School for Neuroscience, Göttingen, Germany

In the retina, the photoreceptors divide the light into separate chromatic channels. The ganglion cells receive the photoreceptors’ chromatic inputs through bipolar cells and integrate these inputs to generate their output. However, little is known about how retinal ganglion cells integrate and encode different chromatic inputs. Here, we established a new stimulation approach to investigate chromatic integration in the mouse retina. We asked whether retinal ganglion cells integrate s-cone (UV sensitive) and m-cone (green sensitive) inputs linearly or nonlinearly. We stimulated the retina with cone-isolating UV-green stimuli and recorded the responses of many ganglion cells with multielectrode arrays. We used a stimulus that has combinations of UV and green colors with opposite contrasts. We found that ganglion cells integrate their chromatic inputs either linearly or nonlinearly with the majority of ganglion cells showing linear responses. For these linear cells, the responses induced by the UV stimulus is cancelled by the opposite green stimulus. Furthermore, we found two types of responses for the linear cells: some do not respond to stimuli with non-preferred polarity (on or off) while others reduce their activity below the spontaneous firing rate. Surprisingly, in addition to linear cells, we found populations of ganglion cells that integrate chromatic stimuli nonlinearly. Nonlinear ganglion cells show clear responses to every applied combination of UV-green contrasts. Therefore, these cells encode chromatic features of the stimulus. Furthermore, our data shows differences between on, off and on-off nonlinear cells. For chromatically nonlinear cells, we identified the interactions between center and surround of the receptive field as a putative mechanism of the observed nonlinearity. Together, we defined a new experimental approach to study signal integration in neurons. Beyond that, our data reveal nonlinear retinal computations involved in the integration of chromatic stimuli. Our framework can assist existing functional classification schemes of the ganglion cells that usually ignore chromatic processing.

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11) Comparing AII and A8 amacrine cells in the mouse retina Shubhash Chandra Yadav, Karin Dedek Animal Navigation/Neurosensorics, Institute for Biology and Environmental 26111 Oldenburg, Germany

Both AII and A8 amacrine cells are narrow-field, bi-stratified glycinergic amacrine cells in the mammalian retina [1,3]. The function of A8 is poorly understood; however, AII cells were shown to be a crucial node in the primary rod pathway and are thought to mediate fast switching between rod and cone vision [2]. Previous studies suggest that A8 cells, like AII cells, form homocellular A8-A8 [3] and heterocellular A8-ON cone bipolar cell gap junctions [1,3]. However, in contrast to AII, A8 cells are primarily cone-driven [3]. Recent connectomics data indicate that AII and A8 cells may couple to a common bipolar cell type [1,2] and receive input from dopaminergic amacrine cells [4,5]. Further, both AII and A8 receive glutamatergic input from the same bipolar cell they make gap junctions with [1,2,4]. Thus, it is likely that A8 gap junctions are also dopamine-/lightdependent and work in concert with AII cells to amplify or suppress rod signals or even facilitate cone signals. To test this, we compared the light dependence of AII and A8 coupling. Tracers were injected in whole-mount retinas of wild-type and Ier5-EGFP mice under lightadapted conditions. Also, we used various somatic and synaptic markers to determine the type of connexin expressed by A8 cells and to discern whether AII and A8 cells share a common gap junction partner. The results thus far show that A8 cells abundantly express Cx36 in the ON IPL layer although Cx36 expression is considerably weaker than in AII cells [6]. Unlike AII, A8 cells also sparsely express Cx36 in the OFF IPL layer. AII cells are coupled to more ON bipolar cells than other AII cells under light-adapted conditions. Unexpectedly, A8 are coupled to less bipolar cells than AII cells in the same conditions, which might mean that AII-ON bipolar cell gap junction is dominant in photopic conditions, whereas AII-AII and A8-ON bipolar cell coupling might be more evident in scotopic and/or mesopic conditions, which we aim to investigate in future. Supported by the DFG (DE1154/5-1, JA854/3-1) and the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 674901 and the European Commission (to KD).

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12) How does multivesicular release contribute to the transmission of the visual signal? Ben James, José Moya-Díaz, Léa Darnet, & Leon Lagnado University of Sussex, School of Life Sciences

Within the inner retina, Bipolar Cells (BCs) transmit information to post-synaptic cells by graded releases of the neurotransmitter glutamate through ribbon synapses specialized to transmit early sensory information. These synapses, unlike many other synapses, release vesicles in a quantized manner; rather than occurring in an all-or-none or Poisson fashion, where a maximum of one vesicle can be released at any given time, glutamatergic events from BCs can be composed of several vesicles being released simultaneously in a mechanism known as coordinated or multi-vesicular release (MVR). Thus, in MVR, the resultant glutamatergic event is quantized, with each quantum representing a single glutamate vesicle. The effects of MVR on retinal processing are, however; little understood. To investigate this question, we use the intensity-based Glutamate Sensing Fluorescence Reporter (iGluSnFR) to decompose glutamatergic events into units of individual vesicles in order to quantify the effects MVR has on stimulus encoding and decoding. We use four main methods for investigating the role of MVR: 1) mutual information to quantify how much information can be learned about the stimulus after observing the BC response; 2) Bayesian alternative forced choice paradigms to test how well we can predict the stimulus that elicited an observed response; 3) leaky integrate-and-fire neuron simulations to observe the effects MVR has on down-stream retinal ganglion cell activity; and 4) signal detection theory to calculate the maximum number of discriminable stimuli based on BC responses. In all of these analyses, we use either the extracted BC time series (including the quantized vesicle-release time series), or an altered time series where the quanta released at each event is set to the average number of quanta per event. We thus use the same event timing and total number of quanta in each condition, allowing us to tease out any additional information transmitted through MVR. Based on these analyses, we show that the use of MVR and variable numbers of vesicles per event increases the encoding ability of BCs, thus increasing the information capacity of the ribbon synapse.

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13) Chromatic processing in the zebrafish brain Philipp Bartel1, Tom Baden1,2 1

University of Sussex, School of Life Sciences Institute for Ophthalmic Research, University of Tübingen, Germany

2

The zebrafish is a small freshwater species with an impressive visual chromatic processing apparatus. Its tetrachromatic retina comprises short-, medium-, long, and UV-sensitive cones, extending the borders of its spectral sensitivity beyond those of humans. We set out to map chromatic representations in zebrafish central visual circuits. Retinal ganglion cells (RGCs) are the retina’s only output neurons and project to one or multiple contralateral pretectal and tectal axonal arborisation fields (AFs). We therefore systematically surveyed the chromatic response properties of these AFs in the larval zebrafish brain in vivo using 2-photon calcium-imaging of GCaMP6f expressed throughout the nervous system. To achieve high chromatic resolution we adapted a custom-built “Light Synthesiser” (Belusic et al, 2016) to estimate spectral tuning curves for each region of interest. Subsequent fitting of each curve with the linear sum of the four opsin absorption spectra yielded a four-dimensional vector in opsin space for comparison of chromatic representation across the visual areas of the brain. Our results indicate that most Regions of Interest exhibited some form of chromatic bias, including many putatively colour-opponent units throughout the tectum and pretectum. Our growing dataset indicates the existence of a complex and highly specific organisation of central chromatic circuits.

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14) Response transiency of retinal ganglion cells are maintained when the input dominance switches between parallel signaling streams Alma Ganczer, Ádám Tengölics, Béla Völgyi Department of Experimental Zoology and Neurobiology, University of Pécs, Pécs, Hungary MTA-PTE NAP B Retinal Electrical Synapses Research Group, Pécs, Hungary János Szentágothai Research Center, Pécs, Hungary

It has been known that retinal ganglion cells (RGCs) produce either a singular burst of action potentials or keep spiking as a response to prolonged stimulation and thus can be categorized either as transient or sustained, respectively. Although various mechanisms have been proposed to determine RGC response transiency, it remains largely unknown whether the original RGC response characteristics are maintained when input dominance switches between parallel retinal pathways. In this work we recorded action potential trains from a cohort of mouse RGCs while an input dominance switch was initiated either by pharmacological manipulations or by changing certain parameters of the light stimulation. We found that RGC transiency values did not change considerably when responses were recorded at different levels of light intensity, whose signalling is associated with primary-, secondary rod pathways and cone pathways. In addition, OFF ganglion cell responses retained their original transiency values following dl-2amino-4-phosphonobutyric acid (APB; mGluR6 agonist) incubation when a pharmacological rerouting of signal flow occurs from the primary rod pathway to the secondary rod pathway and/or to the cone signalling stream. This suggests that an input dominance switch from rod- to cone pathway does not alter RGC response transiency considerably. By altering the spectral feature of light stimulation we show that RGC response transiency is also retained when a putative input switch occurs between short-wavelength and medium-wavelength signalling cone-patways. Contrary to the above observations, however, response transiency was altered more prominently when horizontal signaling in the inner retina was perturbed by a pharmacological blockade of GABAergic synapses and/or RGC gap junctions. Therefore, the above data indicates that changes in the RGC light response temporal characteristics are largely dependent on signalling through horizontal but not parallel pathways. This further suggests that changes in response transiency likely carry information about the horizontal pathway encoded spatial characteristics of the visual scene but not its intensity or spectral features.

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15) Spatial integration profiles of mouse retinal ganglion cells Dimokratis Karamanlis1,2,3, Tim Gollisch1,2 1

Department of Ophthalmology, University Medical Center Göttingen, Germany Bernstein Center for Computational Neuroscience Göttingen, Göttingen, Germany 3 International Max Planck Research School for Neurosciences, Göttingen, Germany 2

The bipolar cells form the spatial subunits of a retinal ganglion cell’s receptive field center. It has long been suggested that signal integration over bipolar cells can be either linear or nonlinear, and different types of nonlinearities have been identified. By precisely stimulating single ganglion cells, one can infer the different types of spatial nonlinearities on a cell-by-cell basis. However, a method that allows the measurement of such nonlinearities in population recordings is still missing. We characterize ganglion cells based on their spatial integration properties using multielectrode array recordings of spiking activity from isolated mouse retinas. We use a spatially-structured artificial stimulus to study how ganglion cells integrate visual contrast over space. The stimulus consists of a checkerboard arrangement with two contrast values shown alternatingly over the tiles. The size of the tiles is chosen so that individual ganglion cells typically have multiple tiles in their receptive field centers. For each stimulus presentation, the contrast values of the tiles are chosen randomly from a set that covers the entire contrast space. By relating the stimulus pairs to the ganglion cell responses, we develop two different methods to determine nonlinearities in spatial integration. One is based on the iso-response framework: we identify stimulus patterns that elicit the same response in ganglion cells and quantify the relationship of those patterns. The other method includes fitting complete input-output models to large sets of stimulus-response pairs, and extracting the nonlinearities as parameters of the models. Utilizing both methods, we show that spatially nonlinear ganglion cells have a variety of integration profiles, with rectification of non-preferred contrasts and supra- or sublinear integration of preferred contrasts. Furthermore, we relate those profiles to major cell classifications, based on contrast preference (ON, OFF or ON-OFF) and direction selectivity. By showing that the nonlinear cells in the mouse can be further subdivided, our approach can assist existing functional classification schemes. Additionally, estimating nonlinearities can be of use for the construction of nonlinear encoding models for different ganglion cell types. 187

16) Differential localization of murine CaMKII isoforms indicates CaMKII- and - as specific elements of retinal gap junctions made of connexin36 Stephan Tetenborg1, Shubhash C. Yadav1, Ype Elgersma2, Ulrike JanssenBienhold3,4 and Karin Dedek1,4 1

Animal Navigation/Neurosensorics, Institute for Biology and Environmental Sciences, University of Oldenburg, 26111 Oldenburg, Germany 2 Erasmus Medical Center, 3015 Rotterdam, Netherlands 3 Visual Neuroscience, Dept. of Neuroscience, University of Oldenburg, 26111 Oldenburg, Germany 4 Research Center Neurosensory Science, University of Oldenburg, 26111 Oldenburg

AII amacrine cells are essential interneurons of the primary rod pathway and transmit rod-driven signals to cone bipolar cells to enable vision under scotopic conditions (Bloomfield and Völgyi, 2009). Gap junctions made of connexin36 (Cx36) mediate electrical coupling among AII cells and between AII cells and ON cone bipolar cells. These gap junctions were shown to underlie a remarkable degree of plasticity as they are modulated by different signaling cascades (Kothmann et al., 2009, 2012). In particular Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been characterized as an important regulator of Cx36, capable of potentiating electrical coupling (del Corsso et al., 2012). However, which CamKII isoform is mediating this effect is not known so far. To obtain a more detailed understanding of the subunit composition of CaMKII at retinal gap junctions, we analyzed the retinal distribution of all four CaMKII isoforms using confocal microscopy. These experiments revealed a differential distribution for all CaMKII isoforms: Strong CaMKII-α expression was detected in starburst amacrine cells which are known to lack electrical coupling. CamKII-β was strongly expressed in OFF bipolar cells which form electrical synapses in the outer and inner retina. CaMKII-γ displayed no cell type-specific expression and was diffusely distributed across the entire retina. CaMKII-δ labeling was evident in bipolar and AII amacrine cells, which contain the majority of Cx36-immunoreactive puncta in the inner retina. We double-labeled retinas for Cx36 and all four CamKII isoforms and revealed that the composition of the CamKII enzyme differs between gap junctions in the outer and inner retina: in the outer retina, we only found CamKIIβ at Cx36-containing gap junctions, whereas in the inner retina, CamKII- β and -δ colocalized with Cx36. This finding suggests that gap junctions in the inner and outer retina are regulated differently although they both contain the same connexin. Moreover, we found that the association between CaMKII and Cx36 was connexin-specific since none of the tested CamKII isoforms colocalized with Cx45. 188

Taken together, our study identifies CaMKII-β and -δ as Cx36-specific regulators in the mouse retina. This work was supported by the Deutsche Forschungsgemeinschaft (DE1154/5-1, JA854/31) and the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 674901 and the European Commission.

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17) Melanopsin immunoreactive neurons in the goldfish retina Tareq Yousef1,2, William H. Baldridge1,2,3,4 1

Retina and Optic Nerve Research Laboratory, Departments of Medical Neuroscience 3 Ophthalmology & Visual Sciences 4 Dalhousie University, Halifax, Nova Scotia, Canada 2

Background: Intrinsically photosensitive retinal ganglion cells (ipRGCs) express the photopigment melanopsin, making them capable of responding to light independent of photoreceptors. In mammals, melanopsin expression is limited to ipRGCs. Interestingly, melanopsin has been shown to be ubiquitously expressed in a teleost (zebrafish, D. rerio) retina, in every class of retinal neuron (Davies et al., Cell Mol. Life Sci., 68:4115-4132, 2011). Here, we investigate the expression of melanopsin in the retina of another teleost, the goldfish (C. auratus) retina. Methods: Goldfish eyes were fixed using a modified 4% paraformaldehyde solution containing 134 mM HEPES and 200 mM sucrose (Stradleigh et al., J.Comp. Neurol., 523:545-564, 2015). Cryostat sections (20 µm) of retina were cut and labelled using a rabbit polyclonal antibody (pas350 at 1:400) that was raised against a 13 amino acid synthetic peptide (CVPFPTVDVPDHA) corresponding to a highly conserved region of mammalian-like melanopsins (opn4m). A goat antirabbit Cy3-conjugated secondary antibody (Life Technologies, Carlsbad, CA) was used to visualize pas350 labelling. In some cases, sections were double labelled with an antibody against tyrosine hydroxylase (TOH) preconjugated to Alexa 488 (MAB318-AF488, at 1:500; EMD Millipore , Billerica, MA). Confocal microscopy was used to acquire images of labelled neurons. Results: In the goldfish retina, the pas350 antibody labelled all cell types in the INL, a subpopulation of ganglion cells, and produced intense, often punctate, labelling in the OPL. Weaker punctate labelling was also found in the IPL. To characterize further the type(s) of amacrine cells that are pas350-immunoreactive (-IR), we first looked for co-localization with TOH. We examined 131 TOH-IR neurons and in no case were these neurons pas350-IR. Conclusions: Melanopsin is ubiquitously expressed in the teleost retina, including by amacrine cells. Which specific types of amacrine cells express melanopsin remains to be determined, but we have established that TOH-IR neurons (dopaminergic I1 interplexiform cells) do not express melanopsin. Acknowledgements: We thank Drs. Mark Hankins and Steven Hughes (Oxford University) for the generous supply of the pas350 antibody. We also thank Dr. Andrew Ishida from the University of California, Davis for advice concerning optimal fixation. This work was 190

supported by a grant from NSERC Canada (to WHB) and a Scotia Scholar NSHRF Award (to TY).

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18) Oscillating eye movement in nob mice is explained by oscillating retinal ganglion cell activity Beerend Winkelman1,2; Coen Joling1; Kathryn Fransen3; Gobinda Pangeni3; Marcus Howlett1; Maj-Britt Holzel1; Maureen McCall3; Chris De Zeeuw1,2; Maarten Kamermans1,4 1

Netherlands Institute for Neuroscience, Amsterdam, The Netherlands; Erasmus Medical Center, Rotterdam, The Netherlands; 3 University of Louisville, Louisville, KY, United States; 4 Academic Medical Center, Amsterdam, The Netherlands 2

Retinal motion detection is elemental to image stabilizing eye movement reflexes and is mediated by ON-direction selective ganglion cells. Here we report on eye movement control in mice without functional ON-bipolar cells (nob mice). Nob mice have a mutation in the X-chromosomal gene encoding for nyctalopin, causing defective signaling within the mGluR6 cascade in retinal ON-bipolar cells. We studied optokinetic reflex eye movements and single cell responses of retinal ganglion cells in wildtype and nob mice under various pharmacological conditions. Stimulation with moving sine-wave gratings revealed severe impairment of the optokinetic reflex and defective gaze holding in the nob mice. Even more, eye movements of nob mice exhibited a prominent horizontal oscillation with an average frequency of 5 Hz. Electrophysiological recordings in the retina confirmed that ON-direction selective GCs of nob mice are unresponsive to light stimulation, but their membrane potential showed robust sub-threshold oscillations with a frequency of 5 Hz on average. Oscillatory modulation at this frequency was generally observed in the spiking output of a large fraction of RGCs. Oscillations could be eliminated or significantly reduced by simultaneously blocking the AMPA and NMDA receptors, using DNQX and D-AP5. Intravitreal injection of these drugs in both eyes of nob mice abolished the 5 Hz oscillation in spontaneous eye movements, recorded while the animal was awake. These results strongly suggest that synchronous oscillation of RGCs is the direct cause of the horizontal eye movement oscillation. This mechanism could explain the small amplitude horizontal nystagmus observed in CSNB patients (Simonsz et al., 2009).

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19) Sensitivity of Retinal Ganglion Cells to Fine Spatial Details in their Receptive Field is Determined By Contrast Sensitivity of Nonlinear Subunits 1 Adam Mani, 1,2Gregory W. Schwartz 1

Department of Ophthalmology, Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611 2

For more than 50 years, we have known that the receptive fields of retinal ganglion cells (RGCs) contain nonlinear subunits, enabling them to respond to fine spatial structure on a scale much smaller than that of the classical receptive field center. As we approach a complete classification of RGC types in mouse, it is worth exploring the diversity of nonlinear subunits that allow each RGC to respond to coarse or fine spatial structure with varying sensitivity. While bipolar cells are the main substrate of receptive field subunits, which bipolar cells parameters determine spatial sensitivity is not well understood. Using measurements of spatial sensitivity and contrast sensitivity across a wide range of RGC types, we have created a model that links the contrast response function of a RGC to its nonlinear spatial sensitivity, via the contrast sensitivity of its subunits. We considered interactions between contrast and spatial sensitivity both in the receptive field center and surround. Overall, our work unifies several ideas about receptive field structure to reveal a more general framework to account for the responses of different RGC types in different conditions.

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20) Horizontal cells regulate photoreceptor synaptic output via an integrated GABA and pH dependent mechanism Steven Barnes, James C.R. Grove, Arlene A. Hirano and Nicholas C. Brecha Physiology & Biophysics, Ophthalmology & Visual Sciences, Dalhousie University, Halifax, Nova Scotia, Canada; Neurobiology, Ophthalmology, Medicine, Geffen School of Medicine at UCLA, Los Angeles, CA, USA; VAGLAHS, Los Angeles, CA, USA

Horizontal cells send inhibitory feedback to photoreceptors, regulating the gain of the photoreceptor output synapse and contributing to the formation of receptive field properties of all downstream neurons in the retina and visual pathway. Previous studies make clear that horizontal cell membrane potential drives actions that modulate the activity of presynaptic Ca channels in photoreceptors, which are responsible for modulating glutamate release. We proposed that horizontal cells release GABA, which in turn acts back on the horizontal cells to modify cleft bicarbonate (HCO3ˉ) levels to indirectly modulate photoreceptor transmitter release via cleft pH changes (Liu et al., 2013). Here, we tested key steps underlying this feedback pathway by patch clamping horizontal cells and photoreceptors in mouse, rat and guinea pig retinal slices. We patch clamped cone photoreceptors and affirmed that block of GABARs produces pH-dependent disinhibition of cone Ca channels. The requirement of GABA release by horizontal cells was confirmed by its absence in the Cx57-VGAT KO mouse. We reproduced the autaptic site-specific actions of GABA on cone Ca channels with targeted expression in horizontal cells of engineered anion channels (PSAM-GlyRs), which, activated by their orthogonal ligand, mimic the role of GABARs in modulating cone Ca channels. The present investigations substantiate the model, in which 1) horizontal cells release GABA, 2) autoreception via GABARs increases horizontal cell HCO3ˉ conductance, 3) HCO3ˉ efflux, driven by the horizontal cell membrane potential, buffers the pH of the synaptic cleft, and 4) cone Ca channels are modulated by pHinduced shifts of the voltage- dependence of their activation. Since cone Ca channels determine the release of glutamate onto second order bipolar cells, these results suggest that a GABA and pH dependent multistep mechanism acts on photoreceptor synaptic output to regulate synaptic gain and lateral inhibition in visual processing in the retina.

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21) Horizontal cells regulate photoreceptor synaptic output via an integrated GABA and pH dependent mechanism Steven Barnes, James C.R. Grove, Arlene A. Hirano and Nicholas C. Brecha Physiology & Biophysics, Ophthalmology & Visual Sciences, Dalhousie University, Halifax, Nova Scotia, Canada; Neurobiology, Ophthalmology, Medicine, Geffen School of Medicine at UCLA, Los Angeles, CA, USA; VAGLAHS, Los Angeles, CA, USA

Horizontal cells send inhibitory feedback to photoreceptors, regulating the gain of the photoreceptor output synapse and contributing to the formation of receptive field properties of all downstream neurons in the retina and visual pathway. Previous studies make clear that horizontal cell membrane potential drives actions that modulate the activity of presynaptic Ca channels in photoreceptors, which are responsible for modulating glutamate release. We proposed that horizontal cells release GABA, which in turn acts back on the horizontal cells to modify cleft bicarbonate (HCO3ˉ) levels to indirectly modulate photoreceptor transmitter release via cleft pH changes (Liu et al., 2013). Here, we tested key steps underlying this feedback pathway by patch clamping horizontal cells and photoreceptors in mouse, rat and guinea pig retinal slices. We patch clamped cone photoreceptors and affirmed that block of GABARs produces pH-dependent disinhibition of cone Ca channels. The requirement of GABA release by horizontal cells was confirmed by its absence in the Cx57-VGAT KO mouse. We reproduced the autaptic site-specific actions of GABA on cone Ca channels with targeted expression in horizontal cells of engineered anion channels (PSAM-GlyRs), which, activated by their orthogonal ligand, mimic the role of GABARs in modulating cone Ca channels. The present investigations substantiate the model, in which 1) horizontal cells release GABA, 2) autoreception via GABARs increases horizontal cell HCO3ˉ conductance, 3) HCO3ˉ efflux, driven by the horizontal cell membrane potential, buffers the pH of the synaptic cleft, and 4) cone Ca channels are modulated by pHinduced shifts of the voltage- dependence of their activation. Since cone Ca channels determine the release of glutamate onto second order bipolar cells, these results suggest that a GABA and pH dependent multistep mechanism acts on photoreceptor synaptic output to regulate synaptic gain and lateral inhibition in visual processing in the retina.

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22) Activity-Dependent modulation of gap junction circuits in developing retina Marla B. Feller. and Franklin Caval-Holme Department of Molecular and Cell Biology & Helen Wills Neuroscience Institute University of California, Berkeley

How does the same neural circuit mediate two distinct functions? We explore this question in the developing retina, where at stages of development before vision is possible, immature neurons fire bursts of action potentials that propagate across the tissue in a spontaneous activity pattern termed “retinal waves”. Our previous studies have contributed to understanding the generation of retinal waves and to elucidating the critical role they play in properly wiring retinal projections to the brain. Recent findings have revealed light-guided behaviors prior to the maturation of conventional photoreceptors and during the same period of development as robust retinal waves. I will present recent data from the lab that explores how retinal waves interact with emerging light-responsive circuits.

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23) Outer retinal information transfer Marcus Howlett, Rozan Vroman, Maarten Kamermans Netherlands Institute for Neuroscience

Light input entering the visual system has already undergone several processing stages by the time it reaches the retinal bipolar cells. Cone outer segments detect photons and convert them into the photocurrent, which then drives the cone membrane potential to produce the photovoltage. Voltage activated processes of the membrane react to the changes in voltage shaping the activation of the voltage gated Ca-channels in the cones. Ultimately the resulting fluctuating calcium concentration in the synaptic terminal regulates the release of glutamate from the ribbon synapses. Horizontal cells detect this glutamate and feed a signal back to the cones than in turn modulates the cone Ca-channels. Here we detail the information transfer and capacity of these various sites via the “effective noise” (cell noise dividing with the stimulus-response transfer function) and “noise whitening” procedures. The photocurrent and photovoltage had closely matching tuning curves peaking at approximately 3 Hz, and similar information capacities (48.6 ± 2.9 vs 49.5 ± 6.2 bits/s), indicating the high fidelity of the conversion process. TEA and CsCl sensitive processes are essential to the fidelity of this conversation as their blockage reduced the photovoltage information capacities by 53% and 34%, respectively. On the other hand, calcium dependent processes play little role in maintaining the fidelity as CoCl2 had little effect on the photovoltage information capacity. Horizontal cell had a tuning curve that peaked at around 5 Hz and an information capacity of 113 ±10.9 bits/s. Additionally, compared to cones the HC tuning curve was more bandpass in nature with a “preferred” frequencies band extending out into much higher temporal frequencies. The tuning curve of the feedback signal (FB) closely matched the shape of the tuning curve of the HCs, but as the effective noise level was higher the information capacity of FB (66.5 ± 4.4 bits/s) was less than that of HCs, but similar to that of cones (65.2 ± 2.7 4.4 bits/s) under the same stimulus conditions. However, as the FB tuning curve matched that of the HC, and not the cones, the FB signal received by the cones for frequencies above approx. 7Hz had a higher information rate than it was for the cones themselves. Finally, we calculated the information capacity of each cone ribbon synapse to be 21.8 bits/s. The results presented here represent the first description of information transmission at the first stages of vision in a vertebrate retina. One unexpected 197

outcome was the performance of FB in that it is delivering a negative feedback signal at higher frequencies to cones with more information than the cone response. This suggests that at higher frequencies the FB signal induces a temporal “inhibitory surround” in the cone output. Finally, any description of outer retinal processing that cannot account for the levels of performance we show here will be incomplete

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24) Multiplexed computations in retinal ganglion cells of a single type Stephane Deny, Ulisse Ferrari, Emilie Mace, Pierre Yger, Romain Caplette, Serge Picaud, Gasper Tkacik, Olivier Marre Institut de la Vision, Paris

In the early visual system, cells of the same type perform the same computation in different places of the visual field. How these cells code together a complex visual scene is unclear. A common assumption is that cells of the same type will extract a single stimulus feature to form a feature map, but this has rarely been observed directly. Using large-scale recordings in the rat retina, we show that a homogeneous population of fast OFF ganglion cells simultaneously encodes two radically different features of a visual scene. Cells close to a moving object code linearly for its position, while distant cells remain largely invariant to the object's position and, instead, respond non-linearly to changes in the object's speed. Cells switch between these two computations depending on the stimulus. We developed a quantitative model that accounts for this effect and identified a likely disinhibitory circuit that mediates it. Ganglion cells of a single type thus do not code for one, but two features simultaneously. This richer, flexible neural map might also be present in other sensory systems.

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25) Transcriptional control of an optic pathway driving unique features of the pupillary light reflex Onkar S. Dhande1, Tania A. Seabrook1, Ann H. Phan1, Phong L. Nguyen1, Jack T. Wang1, Sylvia Evans2, Andrew D. Huberman1, 3, 4, 5 1

Department of Neurobiology, Stanford University School of Medicine, Stanford, CA 94305, USA 2 Department of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA 3 Department of Ophthalmology 4 Stanford Neurosciences Institute, and 5 BioX, Stanford University School of Medicine, Stanford, CA 94305, USA

The eye is tasked with extracting salient information from the visual scene and relaying it to appropriate targets in the brain to create percepts and necessary behaviors. The differential distribution of cell types across the retinal surface allows for biased sampling of relevant visual features from different portions of visual space. Little is known about the genetic control of non-uniform cell topographies in the retina and how they contribute to visual percepts or behaviors. Here we discovered an unique optic pathway that preferentially encodes dorsal visual space. We used a comparative microarray-based approach to identify the T-box family transcription factor Tbx20 as a molecular marker for this ‘sky viewing’ pathway. To study the role of Tbx20, whose function in the nervous system is relatively unknown, we generated Tbx20 mutant mice using a Cre/loxP system. Using a combination of transgenic mice and modern anatomical techniques we show that Tbx20 function is critically required for the development of this ‘sky viewing’ pathway. Furthermore, Tbx20 expressing retinal ganglion cells (RGCs) project to a distinct domain within the olivary pretectal nucleus (OPN), a principal station in the neural pathway for generating the pupillary light reflex (PLR). PLR is critical for optimizing visual acuity and sensitivity over a wide range of lighting conditions. Conditional ablation of Tbx20-RGCs revealed that unique visual information is indeed relayed to distinct OPN domains and that these parallel retina-to-OPN pathways cooperatively act to drive different phases of the PLR. These data offer new understanding of the mechanisms that establish functionally specialized parallel optic pathways and the role of such pathways in vision.

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26) Integrating anatomy, connectivity and biophysics to understand horizontal cell computations in the mouse retina Christian Behrens 1-4*, Timm Schubert 1,2,*, Yue Zhang1,2,4, Camille A. Chapot1,2,4, Thomas Euler 1,2,3,+, Philipp Berens 1,2,3,+ 1

Werner Reichardt Centre for Integrative Neuroscience (CIN); Institute for Ophthalmic Research; 3 Bernstein Centre for Computational Neuroscience (BCCN); 4 Graduate Training Centre of Neuroscience, all University of Tübingen, Germany 2

In the outer plexiform layer of the mouse retina, two types of cone photoreceptors (cones) provide input to 13 types of cone bipolar cells (CBC). At this first synapse of the visual system, one type of horizontal cells (HC) provides feedback and feedforward input to cones and BCs, respectively. The full computational role of HCs is still unclear: First, recent studies suggest that - in addition to providing lateral (global) feedback to cones - HCs may also provide local, cone-specific feedback (Jackman et al., 2012, Chapot et al., 2017). However, it is unclear how the signal is kept locally isolated in the large HC dendritic tree. In addition, if and how HCs provide direct feedforward input to the different BC types is unknown. To understand the connectivity of HCs with cones and bipolar cells, we reconstructed the cone axon terminals as well as the dendritic trees of three HCs in a serial block-face electron microscopy volume (Helmstaedter et al., 2013), and quantified the contacts (Behrens et al, 2016). Horizontal cells contacted cones and ON-CBCs with their distal varicosities in the invaginating cleft of the cone axon terminal. In addition, HCs contacted ON- and OFF-CBCs with dendritic “thickenings”, short segments of increased dendritic diameter (‘’bulbs’’) on their main dendrites, suggesting that horizontal cells contact CBCs in a separate synaptic strata in the outer plexiform layer. To better understand the conditions under which local signaling is possible in HCs, we built a biophysically realistic model of a HC dendritic branch based on the detailed morphology and connectivity. The model contains AMPA-type glutamate receptors, voltage-gated calcium and potassium channels, as well as intracellular calcium buffers and pumps. Preliminary simulations suggest that distinct features of the morphological structure of HCs support local processing as inputs from different cones remain well isolated in the very fine dendritic tips just as in physiological measurements. As a next step, we will use the model to explore the computational role of the “bulbs” and explore the effect of active conductances commonly found in HCs for switching between local and global information processing. 201

27) Electrical Synapses Convey Orientation Selectivity in the Mouse Retina Amurta Nath, Gregory W. Schwartz Interdepartmental Neuroscience Program, Northwestern University Department of Ophthalmology, Department of Physiology, Northwestern University, Chicago, Illinois 60611, USA

Sensory neurons downstream of primary receptors are selective for specific stimulus features, and they are described as deriving their selectivity from (1) excitatory and inhibitory inputs from other neurons and (2) their own intrinsic properties. Retinal ganglion cells (RGCs), the diverse feature detectors carrying visual information to the brain, receive excitatory input from bipolar cells and inhibitory input from amacrine cells. Here we describe a RGC, which lacks chemical excitatory input, and relies on gap junctions to convey its selectivity for the orientation of a visual stimulus. Morphological studies reveal that these RGCs are coupled to inner nuclear layer (INL) amacrine cells which possess highly asymmetric oriented dendrites. Selective blockade of gap junctions in these RGCs leads to a loss of orientation selectivity. This represents both a new functional role of electrical synapses as the primary drivers of feature selectivity and a new mechanism for orientation selectivity in the retina.

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28) Probabilistic Analysis of Two-photon Microscopy Data Using Gaussian Processes Luke Edward Rogerson 1, 2, 3, 4, Zhijian Zhao 1, 2, Katrin Franke 1, 3, Philipp Berens 1, 2, 3 , Thomas Euler 1, 2, 3 1

Institute for Ophthalmic Research, University of Tübingen, Germany Centre for Integrative Neuroscience, University of Tübingen, Germany 3 Bernstein Centre for Computational Neuroscience, University of Tübingen, Germany 4 Graduate Training Center for Neuroscience, University of Tübingen, Germany 2

Two-photon imaging allows light-evoked retinal activity to be measured with a relatively high spatiotemporal precision, and has been used to characterize functional diversity in the mouse retina. It is, nonetheless, constrained: signals are contaminated by optical distortion, photon-shot noise and nonlinear indicator kinetics, and recording sessions are time-limited to avoid bleaching and phototoxicity, preventing an exhaustive exploration of the stimulus space. The key to advanced downstream analysis or to making predictions to unobserved stimuli is modeling the uncertainty about the signal correctly. We propose to recover the fluorescence signal using a Bayesian non-parametric method called Gaussian Process (GP) regression, providing a probabilistic estimate of the underlying signal including a model of the uncertainty. Our GP models are fit to calcium and glutamate imaging data in retinal tissue driven by oscillatory stimuli, using a Poisson likelihood function to model photon-shot noise explicitly. We use sparse approximation methods and variational inference to overcome challenges with fitting the GP model parameters to a large number of observations. The GP models can then be used as the foundation for further statistical analysis: For detecting periods of response differences, one can use GP equality tests which extend inferential statistics from points to functions; effects of stimulus parameters can be tested for in a non-linear ANOVA-like framework; for clustering, the Bhattacharyya distance can be used to describe the similarity of two responses, taking the uncertainty into account. Additional processing steps can be included, accounting for the point spread function of the optical system or the kinetics of the fluorescent indicator. We wish to use this framework to explore response diversity in the IPL of the mouse retina. In particular, to identify the conditions under which co-stratifying bipolar cell terminals, both on the same and different cells, decorrelate to form distinct functional pathways. Our imaging configuration includes an electricallytunable lens which allows fast vertical scanning, allowing us to image across 203

multiple layers in the IPL almost simultaneously. The use of parametric stimuli (such as harmonic stimuli with frequency and contrast parameters) allows us to optimize our stimuli to maximize decorrelation between neurites.

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POSTER SESSION V: HUMAN PRIMATE RETINA, MYOPIA, LIGHT ADAPTATION, OTHERS 1) Establishment of an ex vivo retinitis pigmentosa model by efficient delivery of PRPF31 siRNA to human organotypic retinal culture Daftarian, Narsis2, 1; Azizzadeh Pormehr, Leila3, 2; Ahmadian, Shahin3; Rezaei Kanavi, Mozhgan2, 1; Ahmadieh, Hamid2, 1 1

Ophthalmology, Ophthalmic Research Center, Shahid Beheshti University o Medical Sciences, Tehran, Iran (the Islamic Republic of). 2 Ophthalmology, Ocular Tissue Engineering Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Tehran, Iran (the Islamic Republic of). 3 Biochemistry and Biophysics, Dept. of Biochemistry, Institute ofBiochemistry and Biophysics, University of Tehran, Tehran, Tehran, Iran (the Islamic Republic of).

Purpose: The splicing factor PRPF31 is the most commonly mutated general splicing factor in the autosomal dominant retinitis pigmentosa. There is a high demand for safe and efficient delivery of siRNA into Human Organotypic Retinal Culture (HORC). We used a rapid, convenient and safe transfection method with an efficient PRPF31 knockdown in HORC in order to study the PRPF31 silencing effect on retinal gene expressions in this ex vivo model. Methods: Donor human eyes were obtained within the 24 hours post mortem from Iranian eye bank in compliance with the Declaration of Helsinki. The retinas were isolated and cultured in DMEM serum free medium. HORC transfection using PRPF31 siRNA and scramble siRNA as negative control were done with modified calcium phosphate method. PRPF31 down regulation was assessed by quantitative real time PCR (qPCR) within 63 hours. The tissue viability of HORC was measured using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The PRPF31 gene down regulation effects on retinal gene expression such as RP1, GNAT and ROM were analyzed by qPCR. Results: After 63 hours of calcium phosphate transfection method in HORC, the qPCR results showed that the levels of PRPF31 mRNA was reduced significantly in the PRPF31 siRNA treated samples in comparison to controls (over 90%). The PRPF31 mRNA silencing with calcium phosphate had no effect on cell viability in the time period of experiment. PRPF31 reduced expression in the siRNA treated samples led to reduction of retinal specific gene expression such as RP1 (over 100 times), GNAT (10 times) and ROM (10 times) compared to controls, based on qPCR results. Conclusions: We represent an efficient and safe delivery method of PRPF31 siRNA to HORC that could be a useful tool for ex vivo modeling of RP disease. Other than 205

significant effect of PRPF31 down regulation on reduction of RP1, GNAT and ROM gene expression detected by qPCR in this study, further examinations for understanding of PRPF31 down regulation effects in HORC are in progress.

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2) Is the McCollough effect related to the red-green or yellow-blue color signal processing? Paul V. Maximov Institute for Information Transmission Problems of the Russian Academy of Sciences (Kharkevich Institute)

In the primate retina, there are three pathways for transmission of color-specific information, according to the type of cones which determine the response of the central part of their receptive fields: short-wave (S), middlewave (M) or long-wave (L) ones. In the evolutionarily ancient short-wave pathway, connections between cones, bipolar and ganglion cells of certain types are established with the use of special labels that allow cells to recognize each other during the development. An assumption that M and L cones also carry some specific labels that link them during development to rightful bipolar cells meets with a series of theoretical difficulties. Moreover, the experiments with genetically engineered mice (Jacobs et al., 2007) and with males of Saimiri (Mancuso et al., 2009) demonstrated that gene-driven changes in receptor organization can lead to the appearance of a new capacity for chromatic discrimination. Since no special labels were introduced, such colour vision modification could occur only due to an appropriate plastic reorganization of neural wiring in the visual system. Earlier we proposed two computational models of neural networks with modifiable synapses, providing selective wiring in chromatic neural pathways. Experiments with the models showed that secondary neurons were able to establish colour-specific connections with unlabelled input neurons after adaptation to visual stimulation with random sequence of patched images, where colours of nearby pixels were correlated. The longterm illusions like contingent colour aftereffects occur in the models as by-products. In the present work we determine whether the McCollough effect is related only to the red-green or to the yellow-blue opponent color signal pathway as well. To do this we use specially designed pairs of monitor colours specified in terms of human cone spectral sensitivities. A yellow-blue pair is designed so that the signal of S cones would change in opposite with the signals of L and M cones whereas the ratio of the signals of L and M cones would remain constant. A red-green pair is designed so that the signal of L cones would change in opposite with the signal of M cones and the S cone signal would remain constant. Our experiments show that the McCollough effect can be induced by both yellow-blue and red-green grids. The result suggests that the corresponding neural mechanism is not restricted on finding synaptic connections for unlabelled neurons in L and M pathways, but performs some global functions. 207

3) Cellular Identification of the outer retinal bands in optical coherence tomography in human fovea using immnucytochemical markers Nicolás Cuenca1; Isabel Ortuño-Lizarán1; Isabel Pinilla2 1

Physiology, Genetics and Microbiology, University of Alicante, Alicante, Spain. Lozano Blesa University Hospital, Aragon Institute for Health Research, Zaragoza, Spain.

2

Purpose: The emergence of optical coherence tomography (OCT) has meant a technological breakthrough in the diagnosis, treatment and follow-up of many ocular diseases, especially retinal and neuro-ophthalmological pathologies. Several controversies have arisen over the specific cell types that the bands observed in the OCT represent, especially over the four outer retinal bands. The aim of this study was to correlate the hyperreflective bands observed in the OCT at the fovea level with the retinal histological structures using immunocytochemistry. Methods: Eyes from human donnors were used in this study. Vertical cryosections were immunostained with antibodies specific for cones photoreceptors, bipolar cells, mithocondria, Müller and RPE cells, and visualized using confocal microscopy. Triple immmunolabeling allowed distinguishing between cells types and different cell compartments. Results : Triple immmunolabeling allowed distinguishing between cells types and different cell compartments. Immunostaining with GNB3 and CRALBP antibodies showed all retinal layers at the foveola, especially the separation between the outer nuclear layer and the Henle fiber layer. CRALBP and cytochrome C immunolabeling revealed that the hyperreflective bands 1 and 2, observed in the OCT, correspond to the outer limiting membrane and the cone ellipsoids respectively, separated by the cone myoids. CRALBP, cytochrome C and GNB3 showed that the RPE interdigitations extend along the entire external segment of the cones, what discard them as the responsible structure that form the 3rd band. However, the identification of small fragments of cone outer segments within the RPE led us to characterize the 3rd band as the cone phagosomes located in the top of the RPE. Finally, we propose that the 4th band corresponds to the accumulation of mitochondria at the basal portion of the RPE as identified by cytochrome C immunoreactivity, and that the hyporeflective band between band 3 and 4 corresponds to the RPE nuclei and melanosomes zone. Conclusions: This study proposes a new interpretation of the outer retinal bands that leads to a more accurate interpretation of OCT images providing information about the health of cones and its relationship with the retinal pigment epithelium and could help to a better understanding of retinal diseases diagnosis and prognosis. 208

Support: Ministerio de Economía y Competitividad, MINECO-FEDER-BFU2015-67139-R. Instituto Carlos III, RETICS-FEDER RD12/0034/0010. Generalitat Valenciana, Prometeo 2016/158

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4) Morphological and neurochemical characterization of narrow and broad thorny ganglion cells in marmoset retina Ulrike Grünert, Ashleigh Chandra, Sammy C.S. Lee Save Sight Institute and Department of Clinical Ophthalmology; The University of Sydney; ARC centre of Excellence for Integrative Brain Function, Sydney Node

Different types of retinal ganglion cell play specific roles in visual processing and project to different regions in the brain. In primates, at least 17 types of ganglion cell can be identified based on morphological criteria. Here we identified the ganglion cell types in the marmoset retina, Callithrix jacchus, expressing the calcium binding protein calretinin. Retinal whole mount preparations were double labeled with antibodies against RNA binding protein with multiple splicing (RBPMS, a ganglion cell marker) and with antibodies to calretinin in order to distinguish calretinin positive ganglion cells from calretinin positive displaced amacrine cells. Prelabeling with antibodies against calretinin and intracellular injections with the lipophilic dye DiI was used to identify the calretinin positive cells in the ganglion cell layer. The double labelling experiments demonstrated that at eccentricities between 2 and 8 mm, the large majority (80%) of the calretinin positive cells in the ganglion cell layer were retinal ganglion cells, the remainder were displaced amacrine cells. The calretinin positive ganglion cells on average made up 12% of the total ganglion cell population with the proportion of calretinin positive ganglion cells increasing with distance from the fovea. The large majority (76/88) of the intracellularly injected cells were ganglion cells, 14% were displaced amacrine cells. The population of calretinin positive ganglion cells mainly comprised broad thorny (41%) and inner and outer stratifying narrow thorny (30%) cells. In addition, some weakly labeled cells with a large soma were identified as parasol cells (20%). Our results show that thorny ganglion cells in marmoset retina can be distinguished based on their neurochemical properties. The findings suggest that the proportion of thorny ganglion cells increases in peripheral retina.

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5) Contrast summation of opponent cone inputs to blue-on (S-cone) cells in marmoset monkeys Martin PR1,2,3, Eiber CD1,2,3, Pietersen ANJ1,2,3, Zeater N1,2,3, Solomon SG4 1

Save Sight Institute, The University of Sydney, Sydney, NSW, Australia. ARC Centre of Excellence for Integrative Brain Function, The University of Sydney. 3 School of Medical Sciences, The University of Sydney 4 Department of Experimental Psychology, University College London, London, UK. 2

Purpose: In retina of diurnal primates, a small bistratified ganglion cell class shows "blue-on/yellow-off" receptive field properties. This property arises from combination of on-type input from short-wave sensitive (S) cones and off-type input from medium/long-wave sensitive (ML) cones (Dacey and Lee, Nature, 1994). Small bistratified cells project to the koniocellular layers of the LGN, where blue-on/yellow-off receptive fields are commonly encountered. It is generally assumed that responses to achromatic stimuli in these cells are linearly proportional to the summed activity of the S-cones within the receptive field, less summed activity of the ML cones within a corresponding field. We tested this hypothesis. Methods: Extracellular recordings of isolated unit responses (N = 45) to coneselective and achromatic drifting gratings were made in Sufentanil-anesthetised marmosets (N = 19). Saturating hyperbolic (Naka-Rushton) functions were fit to S and ML cone isolating gratings. Responses to achromatic (S + ML) and chromatic (S – ML) gratings were compared to predictions obtained by vector combination of cone inputs. Result and conclusion: The best model for cone contrast summation comprised a linear vector sum of cone inputs with different gains, followed by a mild saturating nonlinearity. Gain and timing differences between S and ML inputs both contributed to achromatic response amplitude. The data are consistent with linear summation of cone inputs at (or before) the bipolar-to-ganglion cell synapse, with nonlinear contrast-response at the ganglion cell spike generator. We conclude that a linear vector sum of opponent cone inputs can explain the achromatic responses of blue-on cells. (1716 characters)

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6) In vivo evaluation of Pax6 overexpression in transformation of murine retinal differentiated cells to retinal progenitor cells Ehsan Ranaei Pirmardan, Zahra-Suheila Soheili, Hamid Ahmadieh, Sharham Samiei Department of Molecular Genetics, Faculty of Biological Sciences, TarbiatModares University, Tehran, Iran Ministry of Science, Research and Technology, National Institute of Genetic Engineering and Biotechnology, P.O.Box: 14965/161, Pazhoohesh Boulevard, 17th Kilometers, TehranKaraj Highway, , Tehran-Iran Ophthalmic Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Blood Transfusion Research Center High Institute for Research and Education in Transfusion Medicine, Tehran, Iran.

In vertebrates retina, due to the lack of regeneration systems and self-renewable cells, degenerative diseases often lead to a visual impairment. One of the aims of the regenerative medicine is transformation of a cell type to another. Pax6 transcription factor has a key role in eye development and generation of the entire retinal layer. The main aim of this study was, the investigation of Pax6 overexpression, mediated by adeno-associated virus (AAV-2), in transformation of retinal differentiated cells to retinal progenitor cells in vivo. Recombinant AAV virus harboring PAX6 cDNA and a reporter gene, EGFP, was produced and purified. Virus titration was performed with flow cytometry and qPCR. Induction of acute retinal ganglion cell death and generation of mouse experimental model was performed by N-methyl D-aspartic acid (NMDA) injection. All experiments including virus injection, histology and expression analyses with RT-PCR, IHC and retinal flatmount were performed. We achieved high, acceptable transgene expression in mouse retina. We did not observe any changes in Ki67 expression following PAX6 overexpression in untreated and treated model mice. However, we showed induction of photoreceptors to express SOX2, a persistent marker for multipotential neural stem cells. In our settings, PAX6 overexpression could not reprogram retinal cells to progenitor cells. However, we could show induction of photoreceptors to express SOX2, a universal neural stem cell marker, that confirmed high cellular plasticity in neural retinal cells. It would be a promising result for regenerative medicine approaches in eye.

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7) Reconstruction of natural images from responses of primate retinal ganglion cells Nora Brackbill1, Nikhil Parthasarathy1, Orren Karniol-Tambour1, Colleen Rhoades1, Nishal Shah1, Georges Goetz1, Alexandra Tikidji-Hamburyan1, Alexander Sher3, Alan M. Litke3, and E.J. Chichilnisky1 1

Stanford University, University of California, Santa Cruz

2

Visual signaling by the retina is often probed by studying how retinal ganglion cells (RGCs) respond to visual stimuli. A converse approach is to infer, or reconstruct, the incident stimulus from RGC spikes. Reconstruction provides a view of what information RGCs transmit about the visual scene in the domain of the stimulus, rather than spikes. Here we examine for the first time reconstruction of natural images from the activity of multiple complete RGC populations. Large-scale multi-electrode recordings in an ex vivo preparation of primate retina were used to record light responses from populations of the four major RGC types (ON and OFF parasol and midget) covering a 4x8° region of the visual field. Recorded populations of parasol cells formed nearly complete mosaics over the region of retina recorded; midget cell populations were less complete. Stimuli consisted of flashed natural images (2 preparations), or a sequence of natural images jittered by simulated fixational eye movements (3 preparations). RGC response was given by spike counts over a 100 ms window after image onset. Linear reconstruction was accomplished by least squares regression of RGC responses against image stimuli to obtain reconstruction filters, and subsequent application of these filters to held-out RGC data. Dimension reduction via PCA was performed to minimize cross-validated reconstruction error. As expected, ON and OFF parasol cells encoded distinct ranges of image contrast: reconstruction with only ON (OFF) cells failed to accurately capture dark (light) areas of the image. Overall, however, reconstruction with either cell type yielded similar performance. As expected, reconstruction with ON and OFF parasol cells combined was more accurate than with either alone. Adding ON and OFF midget cells into the reconstruction further increased accuracy. Surprisingly, the visual message transmitted by a given ON or OFF parasol cell, summarized by the shape of its reconstruction filter, was largely unaffected by the inclusion of other cell types in the reconstruction, though the magnitude of the filter was slightly decreased. This suggests that the contribution of a single RGC type to the visual 213

message is largely independent of the parallel information transmitted by other RGC types. Acknowledgements NSF GRFP DGE-114747 and NSF IGERT 0801700 (N.B.), NEI F31EY027166 (C.R.), Stanford Neurosciences Institute Interdisciplinary Fellowship (G.G.), Pew Charitable Trusts Fellowship in Biomedical Sciences (A.S.), NIH R01EY017992 and NSF/NIH CRCNS Grant IIS1430348 (E.J.C.)

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8) Unusual physiological properties of two low-density ganglion cell types in the primate retina Colleen Rhoades1, Nora Brackbill2, Alexandra Tikidji-Hamburyan3, Georges Goetz3, Nishal Shah4, Alexander Sher5, Alan Litke5, E. J. Chichilnisky3 1

Bioengineering, Stanford University, Stanford, CA, United States Physics, Stanford University, Stanford, CA, United States 3 Neurosurgery,Ophthalmology, and Hansen Experimental Physics Laboratory, Stanford University, Stanford, CA, United States 4 Electrical Engineering, Stanford University, Stanford, CA, United States 5 Santa Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz, CA, United States 2

The 5 high-density retinal ganglion cell (RGC) types in primates have been thoroughly studied, but the visual processing performed by the ~15 low-density cell types is less understood. The present work reveals unexpected light response properties and spike generation in two low-density RGC types. Large-scale multi-electrode recording from ex vivo preparations of peripheral macaque retina was used to simultaneously measure light responses in hundreds of RGCs. Reverse correlation of the spike times with a white noise stimulus revealed the spatiotemporal response properties of each cell. Based on these properties, RGCs were classified into distinct types. Classification was confirmed by the mosaic organization of receptive fields within each type. In addition to 5 high-density RGC types, low-density RGCs were also recorded. Two types, one ON and one OFF, are studied further here. The kinetics of light responses in the two cells types were similar to the kinetics observed in simultaneously-recorded ON and OFF parasol cells respectively and were consistent across preparations. The receptive fields of neighboring cells within each type interlocked, providing uniform coverage of visual space. Surprisingly, the receptive fields did not exhibit the classic Gaussian profile, but instead consisted of 3-5 distinct zones of light sensitivity, approximately the size of parasol cells. Simultaneously recorded parasol cells confirmed that the zones did not result from erroneous mixing of spikes from parasol cells and were not produced by photoreceptor damage. Visual stimulation of each zone resulted in the generation of a slightly different spike waveform, suggesting a possible mechanism based on dendritic structure. The zones appear to represent a novel spatial nonlinearity in the retinal circuit. Specifically, with high-contrast gratings, 215

the classic bipolar cell subunit nonlinearity was revealed, as expected. However, with low contrast targeted stimuli, greater nonlinear summation was observed between zones than within them, effectively representing another subunit computation at a larger spatial scale. While the anatomical identity of these RGC types is unknown, their receptive field size and kinetic properties suggest that they correspond to the anatomically identified ON and OFF smooth monostratified cells (Crook et al., 2008). Funding Sources: NEI F31EY027166 (C.R.), NSF GRFP Grant DGE-114747 and NSF IGERT Grant 0801700 (N.B.), Stanford Neurosciences Institute Interdisciplinary Fellowship (G.G.), Pew Charitable Trusts Fellowship in Biomedical Sciences (A.S.), NEI R01EY021271 (E.J.C.)

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9) Stimulation thresholds for different retinal circuitries in primate and mouse retina Florian Jetter1,2, Meng-Jung Lee1,2, Henry Evrard3, Roland Thewes4, Günther Zeck1 1

Neurophysics Research Group, Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Germany 2 Graduate Training Centre of Neuroscience, Tübingen, Germany 3 Functional and Comparative Neuroanatomy, Werner Reichardt Centre for Integrative Neuroscience , Tübingen 4 Chair of Sensor and Actuator Systems, TU Berlin, Berlin, Germany

Motivation: In degenerative diseases like Retinitis Pigmentosa photoreceptors degenerate, while the network of bipolar, horizontal, amacrine and ganglion cells initially remains largely intact. This remaining network can be stimulated electrically to evoke visual percepts, an approach used by different retinal implants. Recently, the use of sinusoidal stimulation has been investigated. Here we investigate how retinal circuitries in the ex vivo primate and mouse retina respond to sinusoidal current stimulation. We correlate the electrically evoked response to light evoked ganglion cell activity. Methods: We interfaced ex vivo primate and mouse retinae to a high-density CMOS-based microelectrode array (CMOS-MEA) in epiretinal configuration. The CMOs-MEA consists of 4225 recording and 1024 stimulation electrodes on an area of 1mm² and allows simultaneous recording and stimulation of RGC activity. Stimulation artefacts are completely removed using lab-written data analysis software. Light stimuli (405 / 470 / 635 nm) are used to analyze the light-induced RGC activity. A detailed analysis of the response of a large population of RGCs (up to 200 RGCs/mm2) and comparison across different stimulus parameters and animal models has been performed. Results: In the non-human primate retina retinal ganglion cells are stimulated by sinusoidal stimulation with a maximal current density of less than 0.5 mA/cm2. The stimulation threshold within one single retinal portion varies by a factor of 2. A similar value for the minimal stimulation threshold has been found for mouse retina (rd10 and C57/Bl6). Interestingly, spontaneously active OFF - RGCs are inhibited by increasing stimulation current, in line with a recent report [Stutzki et al., J.Neurophys. 2016]. Conclusion: Here we analyzed the activation of retinal circuitries by sinusoidal stimulation and relate the activation to the physiological response of the detected ganglion cells.

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10) Encoding natural images by gap junctions in retinal rod photoreceptors through a large-scale network model Yang Yue1, Liuyuan He1, Jian K. Liu3, Kai Du2, Yonghong Tian1, Tiejun Huang1 1

School of Electronics Engineering and Computer Science, Peking University, China Department of Neuroscience, Karolinska Institutet, Sweden 3 Institute for Theoretical Computer Science, Graz University of Technology, Austria 2

The retina encodes information about visual inputs that impinge on the eye through several layers of cells in their response activity. To do so, the photoreceptor cells (PRCs) collect information across their receptive fields (RFs) whereas modulated by dense gap junctions. However, the functional role of these gap junctions is less clear. To date, a systematic detailed measurement of the anatomical and physiological properties of the PRCs in primate animals is still difficult. Here, we explore a modelling approach, extending the pervious detailed physiological model of single rod PRC1,2 into a large scale network with ~1 million of rods. In particular, the spatial distributions of rod PRCs are generated based on previous experiment data in Human3, thus yielding heterogamous density of gap junctions in the rod network. The generated distribution densities of gap junctions in the network are in close match to reported numbers of rod-rod gap junctions in Primates4. The entire network model is implemented in the NEURON simulator. We start by applying spatio-temporal white-noise stimulation to the network to find the spatial receptive fields and temporal filters of rod PRCs. By varying the level of gap junctions, we find both filters are dynamically modulated by intrinsic electrical connections in the rod network. We then apply natural images with different types of noises, including Gaussian noise, Rayleigh noise (similar to Gaussian noise, but skewed to the right), Erlang noise (a special case of Gamma noise) and Salt-Pepper noise (impulse noise). These noises are classical in image processing. Surprisingly, we find there is a direct effect of gap junctions to denoise the given natural images. Moreover, the rod network with gap junctions can automatically filter the classical noises in natural images without prior filterselection. We identify an optimal degree of gap junctions that give the best performance for denoising. Together with these results, we proposed a detailed model of retina in the first stage with rod photoreceptors. Future work will incorporate more cell types towards a full model of retinal circuit.

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11) Topographical variations in multifocal eletrorretinogram amplitude and implicit time with changes in spherical aberration Paulo Fernandes1, Ana Amorim-de-Sousa1, André Amorim António 1, Queirós, Norberto Lopez-Gil2, José M González-Meijome1 1

Clinical and Experimental Optometry Research Laboratory (CEORLab) Center of Physics (Optometry), School of Sciences, University of Minho, Braga, Portugal; 2 CiViUM Research Group, University of Murcia, Spain, Spain.

PURPOSE It is believed that eye growth is a visually guided process and the eye is able to compensate for positive and / or negative blur. The imposition of peripheral blur may have profound effects on the development of refractive error and spherical aberration (SA) causes a blur signal that induces changes in image contrast and it is believed that it may have influence on the onset and progression of myopia. However, the basis of regional variations in the retina leading to discrimination of defocused signals has not yet been fully understood. The objective of this preliminary work is investigate the influence of positive and negative SA in the bioelectric activity of the retina measured with multifocal electroretinogram (mfERG). METHODS: The right eyes of 4 emetropic subjects with visual acuity of logMAR 0.00 or better in both eyes, normal color vision, and good ocular health were analized. The mfERG was recorded using DTL fiber electrodes (Diagnosys LLC) and followed the recommendation of the ISCEV standards. The pupil was dilated with 3 drops of 1% tropicamide at 5-minute intervals. The stimulus pattern, consisting of 103 hexagons scaled with eccentricity was presented on a 19 in. RGB computer monitor using the Espion Imaging System (Diagnosys LLC, Littleton, Mass) using a pseudorandom m-sequence at a rate of 75 Hz at a working distance of 33 cm. The amplitude (nV) and implicit time (ms) of the wave components (N1, P1, N2) of the first Kernel response in 7 concentric rings (maximum eccentricity ~ 33 deg.) were analyzed for different amounts of positive and negative SA: (# 1 = 0.2998μm; # 2 = 0.1470 μm; # 5 = -0.3897 μm; # 6 = -0.1869 μm, SA for a 5 mm pupil) placed 15 mm from the eye. RESULTS: The mean amplitude N1, P1, and N2 components of the first-order mfERG response was significantly different among the 4 different amounts of induced SA principally in central and paracentral regions (rings 1-4, eccentricity ~ 18 deg, p <0.05 in all conditions). Up to ring 4, the N1 amplitude is significantly more negative with increase in positive SA (p<0.05, both conditions) while for P1 an opposite effect was observed; significantly increased with increase of positive SA (p<0.05, both conditions, ring 1 to 3) and becomes more negative with negative SA when compared to baseline. The same effect was observed for N2 amplitude. 219

For implicit time, despite some changes observed, no significant differences were observed in P1, and N2 implicit times in all regions of the retina with the exception for N1 in the central and paracentral rings 1 and 2 (eccentricity ~ 3.3 deg), with higher values positive and negative SA . CONCLUSIONS: From these preliminary evaluations we observed that the central and peripheral retina response changes significantly with increasing levels of spherical aberration and that the retina could use this information as a mechanism that allow the differentiation between positive and negative SA and possibly detect blur.

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12) The role of the microglial Cx3cr1 pathway in the post-natal maturation of retinal photoreceptors Erica L Fletcher, Michelle Waugh, Kirstan A. Vessey, Joanna A. Phipps, Lidia Trogrlic, Una Greferath, Samuel A. Mills, Andrew I. Jobling Department of Anatomy and Neuroscience. The University of Melbourne, Parkville 3010 Victoria, Australia

Microglia are the resident immune cells of the CNS and their response to infection, injury and disease has been well documented. More recently, microglia have been shown to play a role in normal CNS development, with the fractalkine-Cx3cr1 signalling pathway of particular importance. This work describes the interaction between the light sensitive photoreceptors and microglia during eye opening, a time of post-natal photoreceptor maturation. Genetic removal of Cx3cr1 (Cx3cr1GFP/GFP), led to an early retinal dysfunction just after eye opening (P17) and cone photoreceptor loss (P30 onwards). This dysfunction occurred at a developmental time when fractalkine expression was limited to the outer retina, there was an increased microglial presence near the photoreceptor layer and increased microglial-cone photoreceptor contacts. Photoreceptor maturation and outer segment elongation was coincident with increased opsin photopigment expression in wild-type retina, while this was aberrant in the Cx3cr1GFP/GFP retina and outer segment length was reduced. A beadchip array highlighted Cx3cr1 regulation of genes involved in the photoreceptor cilium, a key structure important for outer segment elongation. This was confirmed with quantitative PCR with specific cilium-related genes, Rpgr and Rpgrip1, downregulated at eye opening (P14). While overall cilium structure was unaffected, expression of Rpgr, Rpgrip1 and centrin-3 were restricted to more proximal regions of the transitional zone. This study has highlighted a novel role for microglia in regulating post-natal neuronal development within the retina, with loss of the fractalkine-Cx3cr1 signalling pathway leading to an altered distribution of cilium proteins, failure of outer segment elongation and ultimately cone photoreceptor loss.

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13) Quantitative analysis of microglial morphology and cell type interaction during retinal postnatal development Gloria Colombo, Rouven Schulz, Sandra Siegert Institute of Science and Technology (IST) Austria

Microglia are the resident macrophages of the brain. Besides playing a key role in inflammatory response during injury, microglia have been shown to actively interact with neuronal processes. During brain development, microglia obtain an amoeboid-like morphology, which is classically associated with a pro-phagocytic function. Interestingly, this morphological characteristic is maintained until synaptogenesis finishes in late postnatal development. One big knowledge gap is how microglia interpret the various neurodevelopmental signals, which are necessary to shape synapse wiring, and based on this, how they decide which synapse to remove. Here, we established a quantitative spatial-temporal map of microglial morphology and their interaction to various cell types using the retina. We analyzed microglial shape variations during retinal synaptogenesis using two mouse models, which express GFP under a microglial-specific promoter. We found that microglia show several peaks of activation amongst one is when bipolar cells establishing their synapses. By analyzing the volume of the lysosomal marker CD68 within the microglial surface, we found a correlation between increased lysosomal expression and the amoeboid-like microglial shape. When we investigated whether microglia interact with distinct cell types at certain developmental time points, we observed increased overlapping voxel surface between microglial cells and bipolar cells but not with amacrine cells or photoreceptor terminals. Next, we will perform RNA sequencing to identify genetic networks that specifically switch during defined time-points and to further investigate whether the morphology can predict the functional switch of microglia. Our data will have important implications for understanding the function of microglia in synapse formation and maintenance as well as will provide an import access point to identify sides of injury when microglia are malfunctioning.

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14) Dopamine facilitates transition from rod to cone signaling in the retina Suva Roy, Mariah Hazlett, Greg D Field Duke University, Department of Neurobiology

In the mammalian retina, dopamine (DA) is thought to play a key role in a variety of functions such as light adaptation and circadian response[1-3]. During day or during exposure to light, DA level rises in the retina, while DA level falls at night mediated by melatonin (MT)[4,5]. Changes in the level of DA are detected by DA receptors, a family of GPCRs[6]. This drives multiple changes in the retinal circuitry, such as changes in electrical coupling between cells and modulation of specific membrane conductances[7], suggesting DA’s role in tuning visual processing for day and night. Determining the impact of DA on the output of retinal ganglion cells (RGCs) is therefore crucial for understanding how DA affects circuit functions to shape retinal processing for different light conditions. To test the hypothesis that DA modulates retinal signaling for day and night conditions, we recorded from the retinas of C57BL/6J mice, in an ex-vivo preparation. RGC spiking responses to checkerboard noise and full-field contrast steps were measured using a multi-electrode array (519 electrodes, 30um pitch)[8]. RGCs were functionally classified into distinct types; the classification was validated by the observation of regularly spaced cells within each type, or a ‘mosaic’ organization. To determine the net effect of DA across functionally distinct types of RGCs, a combination of nonselective DA agonist ((-)apomorphine) and MT antagonist (luzindole) was used to simulate a condition similar to circadian day. Responses under this condition were compared to a contrasting state produced by a combination of nonselective DA antagonist (flupenthixol) and melatonin to simulate a condition similar to circadian night. We found that combined exposure to apomorphine and luzindole had several effects on RGC function. First, the mean firing rate of ON and OFF RGCs to repeated checkerboard noise increased. Second, response transience to full-field light steps decreased in ON RGCs and increased in OFF RGCs. Finally, we observed a reduction in pairwise correlated activity with apomorphine and luzindole in several, but not all RGC types. Spatiotemporal receptive field structure was relatively insensitive to activating or antagonizing DA or MT receptors. These results indicate DA and MT receptor activation or antagonism tunes the gain of cone-mediated signals across RGC types. Reduction of pairwise synchrony among RGCs coupled with the observation of stable receptive field structure suggest DA-ergic modulation of gap-junction coupling in the retina primarily tunes 223

correlated spiking and not feature selectivity. Future work aims to uncover how DA and MT signaling shapes rod-mediated RGC responses.

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15) Two ways of A1/A2 visual pigment conversion during light/dark adaptation in fish Kondrashev, Sergey1; Lamash, Nina1,2 1

Zhirmunsky Institute of Marine Biology, National Scientific Center of Marine Biology FEB RAS, Vladivostok, Russia; 2 Papanin Institute of Biology of Inland Waters RAS, Borok, Russia

In many fish species, visual pigments are known to be presented by a mixture of rhodopsins based on the 11-cis retinal (aldehyde of vitamin A1) and porphyropsins based on the 11-cis 3,4-didehydroretinal (aldehyde of vitamin A2). Light is the leading external stimulus for changes of the pigment ratio, and low light level switches this ratio towards A2 in most known cases. It has been recently shown that the expression of a cytochrome P450 family member, Cyp27c1, controls the balance of vitamin A1 and A2 in the eye. We have studied the “inverted” phenomenon of visual pigment changes, observed in a large number of fish species, and that probably depends on the activation of Cyp27c1 enzyme. It was found by microspectrophotometry (MSP) that photoreceptors of the marine fish, masked greenling Hexagrammos octogrammus, contained in summer presumably porphyropsin (A2), thus reaching max = 525 nm for rods and single cones and 562/625 nm for double-cone members due to dynamic mixtures of A1502+A2525, A1522+A2562, and A1560+A2625 pigments. A series of experiments conducted on fish kept in aquaria in different seasons and under natural and controlled light conditions (full darkness, dim light and artificial illumination simulating natural daylight) showed that the ratio of visual pigments shifted to A1-type when the fish were exposed to lower light levels or to a shorter day length. In accordance with MSP findings, the HPLC analysis has shown that the first noticeable shift from A2 to A1 in the pigment ratio occurred after the first two weeks of maintenance at full darkness after bright light exposure. The same results were demonstrated for several fish species from families Hexagrammidae, Stichaeidae, and Tetraodontidae, which have a peculiar lightdepending mechanism of changeable yellow-orange corneal coloration. The mechanisms of the Cyp27c1 expression and interaction of this enzyme with targets in the retina are unknown at present. Our findings indicate at least two possible ways of the activation of this enzyme: both at high and at low light levels. Marine and freshwater species of the puffer fish (Tetraodontidae) with a well studied genome [4] may be regarded as suitable animal models for future studies aimed at controlled A1/A2 conversion. 225

16) Expression of Kv11.1 in retinal ON-bipolar cells Robert M. Duvoisin, Gaoying Ren, Tammie L. Haley, Weihong Xiong, Maria Borisovska, Cyrus McHugh, and Catherine W. Morgans Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, Oregon, U.S.A.

Our study aims to determine if ON-bipolar cell responses to light are influenced by dendritic potassium channels. We focused on Kv11.1 (ERG1, hERG) as it has been shown previously to be expressed in bipolar cells. Two antibodies against different epitopes of Kv11.1 were validated by immunofluorescence and western blotting using Kv11.1-transfected HEK293 cells. Kv11.1 expression in mouse retina was confirmed by western blots of retinal proteins, which revealed a band of approximately 150 kD with both antibodies, consistent with expression of Kv11.1 isoform a (ERG1a). The distribution of Kv11.1 in the retina was determined by immunofluorescence confocal microscopy. Both the inner and outer plexiform layers (IPL and OPL) of the mouse retina were labeled. In the OPL, Kv11.1 co-localized with TRPM1 and GPR179 in the dendritic tips of ON-bipolar cells, and occasionally with TRPM1 in ON-bipolar cell bodies. Kv11.1 immunofluorescence was unchanged in TRPM1 knockout and PKCα knockout retina. The physiological role of Kv11.1 in bipolar cells was assessed by measuring the effect of the Kv11.1 blocker, E-4031, on patch-clamp recordings of bipolar cells and electroretinogram (ERG) recordings. Patch-clamp recordings from bipolar cells revealed an E-4031 sensitive potassium current. The putative Kv11.1 current was present in both intact cells and cells where the axon terminal was severed, indicating that the current is likely to arise from the dendrites. Injection of E-4031 (Kv11.1 blocker) into one eye caused a delay and decrease in amplitude of the ERG b-wave compared to the contralateral eye that was injected with PBS only. The effect of E-4031 on the ERG was absent in ERGs from PKCα knockout mice. Our results indicates a role for Kv11.1 in shaping the light response of ON-bipolar cells, and that in rod bipolar cells, Kv11.1 activity may be regulated by PKCα phosphorylation.

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17) Light/dark adaptive and circadian-mediated horizontal cell signaling switches back and forth between cones at night and ON-cone bipolar cells in the day Stuart C. Mangel Dept Neurosci, Ohio State Univ, Columbus, OH USA.

Purpose. Horizontal cells (HCs) can signal cone bipolar cells (cBCs) directly and indirectly by providing feedback inhibition to cones. Although HCs evoke cBC surround responses, the identity of the HC transmitter and whether the two HC pathways have similar functions remain unclear. My lab has shown that dopamine D1Rs (on ON-cBC dendrites) and D4Rs (on cones), by modulating intracellular PKA, increase the expression and activity of GABAARs on 1) ON-cBC dendrites in the day following maintained (>30 min) illumination (Chaffiol et al, 2017, Current Biology, in press) and 2) cone terminals at night following maintained darkness (Mangel et al, 2015, ARVO). We thus studied whether HC signaling to ON-cBCs and to cones depend on GABAARs, D1Rs, D4Rs, the background light level, and the time of day or night. Methods. The effects of artificially polarizing HCs on nearby ON-cBCs in rabbit retinal slices and on nearby cones in intact goldfish retinas were studied in the day and night in the presence and absence of gabazine (GBZ, GABAAR antagonist), SCH23390 (SCH, D1R antagonist), spiperone (SP, D4R antagonist) and MFA (gap junction blocker) (HCs: sharp pipettes; ON-cBCs, cones: patch pipettes). Results. Preliminary rabbit HC/ON-cBC paired recordings revealed that in the day 1) artificially hyperpolarizing HCs reduced ON-cBC surround responses (as did GBZ or SCH), and 2) in the absence of light stimuli and with APB in the Ringer, depolarizing and hyperpolarizing HCs depolarized and hyperpolarized ON-cBCs, respectively (i.e. sign-conserved signaling), effects that were blocked by GBZ or SCH. Preliminary fish HC/cone recordings revealed that in the absence of light stimuli and with MFA in the Ringer HC polarizations altered cone voltage in a signinverted manner at night in the dark and in the day following SP (effects that were blocked by GBZ), but not in the day without SP. Conclusions. The results suggest that 1) in the day when the D1Rs of ON-cBCs are activated by maintained illumination HCs provide a GABAAR-mediated signconserving (excitatory) signal to ON-cBCs that contributes to surround responses, and 2) at night when the D4Rs of cones are NOT activated by the retinal circadian clock HCs provide a GABAAR-mediated sign-inverting (inhibitory) signal to cones that decreases cone light responses. Because cones and ON-cBCs do not exhibit surrounds at night, the findings also suggest that in addition to operating at different times, HC signaling to cones and to ON-cBCs have different functions. 227

18) Pannexin 1 channels are essential for negative feedback from horizontal cells to cones Maarten Kamermans, Valentina Cenedese, Wim de Graaff, Tamas Csikós, Georg Zoidl Netherlands Institute for Neuroscience, Amsterdam

The mechanism of negative feedback from horizontal cells to cones has puzzled the retinal community for decades. General agreement exists that horizontal cell hyperpolarization leads to a shift of the activation potential of the Cone calcium channels to more negative potentials. Two hypotheses were put forward to account for this shift: an ephaptic mechanism and a pH based mechanism. Vroman et al (2014) showed that both mechanism were working simultaneously. The ephaptic mechanism mediates ultrafast negative feedback (synaptic delay < 1ms) while the pH dependent mechanism is very slow ( = 200 ms). The key protein in the ephaptic mechanism is connexin 55.5. Indeed knocking out Cx55.5 results in a severe reduction of negative feedback. This reduction was almost entirely due to the reduction of the fast feedback response. The slow feedback response remained more or less intact. Vroman et al proposed that Panx1 is the key protein for the pH mediated mechanism. Here we report about two mutant zebrafish we have generated where Panx1 channels have been knocked out. Zebrafish has two isoforms of Panx1: Panx1a and Panx1b. Both mutant zebrafish develop normally. Negative feedback responses in cones were strongly reduced in all Panx1-KO animals. In depth analysis of the remaining feedback response revealed that the slow component of feedback is substantially reduced while the fast component was only reduced to some extent. These results confirm that negative feedback from horizontal cells to cones is mediated by two mechanisms working together: an ultrafast ephaptic mechanism and a very slow pH dependent Panx1/ATP mediated mechanism.

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19) Irradiance tuning of narrowband oscillations in the mouse visual thalamus Storchi R, Allen AE, Martial FP, Wynne J, Humphries MD, Brown TM, & Lucas University of Manchester, Faculty of Biology, Medicine and Health In spite of a large body of research throughout the last 30 years the relevance, if any, of narrowband gamma oscillations to visual coding remains enigmatic. An important step to address this challenging question is to gain a better understanding of whether and how these oscillations are driven by key attributes of the visual scene. Recent work from our and other labs has shown that ambient light (irradiance) is a robust drive for these oscillations [1,2]. Namely the intensity of uniform ambient illumination, even when no spatial contrast is present, provides a graded modulation of power and frequency of gamma narrowband in retina, dorsal geniculate nucleus (dLGN) and primary visual cortex. This modulation is positively correlated with light intensity and is at least partially explained by melanopsin excitation. We now address the question of how and to what extent identical patterns of spatiotemporal contrast, also known to drive narrowband oscillations [3,4], are represented by gamma narrowband at different levels of ambient illumination. To this aim we set out to estimate gamma narrowband from spike activity in mouse dLGN, the main sensory input to primary visual cortex, and Lateral Posterior nucleus (LP), a higher order thalamic relay also involved in visual processing. We displayed a sweeping visual object and an approaching-distancing one at different levels of positive and negative spatiotemporal contrasts and we recorded simultaneous responses by large pools of single units by using a high density 256 channels probe in anaesthetized animals. We first asked whether pools of single units whose firing rate responses are similarly tuned to spatiotemporal contrast can produce narrowband gamma and, if so, how frequency and power of these oscillations are defined by irradiance and spatiotemporal contrast. In order to identify those homogeneous pools we developed an automated functional clustering procedure building upon previous works by Newman [5] and Humphries [6] and based on the concept of network modularity [7]. We found that these homogeneous pools could produce gamma narrowband at all irradiance tested but most reliably at mid-high irradiance levels (~2*1014 photons/cm2/s) compared with lower irradiance condition (~2*1012 photons/cm2/s). The power of gamma narrowband was strongly correlated with firing rate responses as homogeneous pools responding both to positive and negative contrast objects, frequently found in our recordings, also exhibited a matching modulation in narrowband power. In these same pools however the 229

frequency of gamma narrowband exhibited a monotonic dependence from contrast, being lowest for the darkest objects and highest for the brightest ones. These results were consistent for sweeping and approaching-distancing objects suggesting a systematic divergence in tuning of power and frequency of gamma narrowband.

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20) The absolute limits of increment and decrement detection at low light levels Sanna Koskela1, Markku Kilpeläinen2, Anton Laihi1, Daisuke Takeshita1 & Petri Ala-Laurila1,3 1

Department of Biosciences, University of Helsinki, Helsinki, Finland. 2Department of Psychology, University of Helsinki, Helsinki, Finland. 3 Department of Neuroscience and Biomedical Engineering, Aalto University School of Science, Espoo, Finland.

Retinal outputs can be divided into On and Off pathways that respond to increases in light intensity (increments) and decreases in light intensity (decrements) with opposite polarities. Several functional asymmetries in coding increments and decrements have been previously identified between these two pathways. These asymmetries have been linked to functional differences between increment and decrement perception as measured in human psychophysics. However, most of the studies have been done at photopic light levels and direct comparisons between retinal circuit function and psychophysical measurements in matching conditions hardly exist. Our aim was to determine the fundamental limits of detecting light increments and decrements at the level of retinal On and Off circuits as well as human psychophysics. We used dark-adapted, flat-mounted mouse retinas and targeted On and Off sustained alpha ganglion cells (RGCs). We determined the threshold for light increments and decrements using brief (20-ms) spatially homogenous positive and negative flashes across dim background light levels in cell-attached patch clamp configuration. We measured the increment and decrement thresholds for human observers to similar light increments and decrements using a ganzfeld (fullfield) stimulus. We found that the sensitivity thresholds for increments and decrements were symmetric for On and Off RGCs at low background lights. Similarly, we found a symmetry between increment and decrement thresholds in human psychophysics. We will discuss the constraints posed by retinal On and Off pathways to downstream processing and psychophysical performance in increment and decrement detection.

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21) The impact of light adaptation on information encoded by RGC populations Kiersten Ruda, Greg Field Duke University, Department of Neurobiology

Correlated activity in retinal ganglion cells (RGCs) has been proposed to serve many visual functions, from conveying unique visual features to redundantly encoding signals to mitigate the impact of noise. In support of the idea that correlated activity may tune visual encoding, its strength and timescale depend strongly on RGC type and light level. Here, we determine the impact of correlations on retinal encoding by finding the information that correlated activity conveys about a stimulus and its dependence on light adaptation. In particular, we test the idea that RGC responses are more redundant at scotopic levels. This theory is based on the observation that visual signals are noisier at scotopic conditions, and posits that redundancy may provide a signal to downstream areas that is less susceptible to noise in single RGCs. We test this theory by finding the redundancy of retinal responses across RGC types and light levels. We recorded from isolated mouse and rat retinas using a 512 multi-electrode array, which allowed simultaneous recording from hundreds of RGCs. This permitted a robust classification of RGCs into distinct types, which was verified by their mosaic organization. Visual stimuli consisted of checkerboard noise and natural movies; each stimulus was presented across a range of light levels spanning nighttime to daytime conditions. The information conveyed about the visual stimuli by individual and pairs of RGCs was estimated and compared across responses to identical stimuli presented at different light levels. Consistent with previous work, we show that single RGCs convey less information about stimuli at scotopic versus photopic light levels. Contrary to expectations, we found that redundancy in RGC population responses did not increase at scotopic light levels. Surprisingly, some RGC types exhibited synergistic responses under scotopic conditions, meaning that the joint activity from RGC pairs conveyed more information than expected from their individual responses. We further searched for features of the stimulus and responses associated with the highest information in RGC activity, and compared them to predictions from receptive fields. These findings suggest that rather than increasing redundancy to combat noise at 232

scotopic conditions, increased synergy partly offsets the lower fidelity of individual RGCs. More broadly, this work begins to elucidate how the retina robustly encodes stimuli across a large range of light levels.

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22) Differential adaptation across direction selective retinal ganglion cells in mouse Xiaoyang Yao1, Amanda McLaughlin3, Gautam Awatramani3, Greg Field2 1

Graduate Program in Neurobiology, Duke University Department of Neurobiology, Duke University School of Medicine 3 Department of Biology, University of Victoria 2

Detecting motion and accurately estimating its direction are likely critical for survival across the range of light levels presented during day and night. In the retina, motion direction is encoded by the population activity of multiple types of direction selective retinal ganglion cells (dsRGC). These populations consist of four types of ON-OFF dsRGCs and three types of ON dsRGCs. Determine how the responses and direction tuning of individual cells depends on light level, and how these dependencies influence motion encoding in the population, is critical for understanding how motion signals are processed during day and night. We used large-scale multi-electrode arrays to record the spiking of hundreds of RGCs simultaneously from the ex vivo mouse retina. ON and ON-OFF dsRGCs were identified based on their direction tuning and speed tuning. The response properties of all dsRGCs were measured using a battery of visual stimuli including drifting gratings, moving bars, and dim flashes across a range of light levels spanning starlight to daylight. We observed that light adaptation exerted differential effects on direction tuning properties of ON-OFF dsRGCs preferring different directions. Superior preferring ON-OFF dsRGCs became more broadly tuned under scotopic conditions, while the other three types exhibited stable tuning. By recording responses from transgenic retina in which connexin36 was conditionally KO in superior preferring DSGCs, we show that the broader tuning of superior ON-OFF dsRGCs at scotopic light levels is largely caused by a combination of gap junction coupling and diminished GABAergic inhibition. Specifically, weaker GABAergic inhibition permits more spikes at the level of individual cells of non-preferred directions, which is amplified by gap-junction coupling. To determine the impact this tuning width change on motion decoding by dsRGCs, we examined motion discrimination and detection performance of ON-OFF dsRGC populations across light levels. This analysis indicated that broader direction tuning curves are beneficial for motion encoding at low light levels. 234

These results provide new insights into how light adaptation impacts the encoding and decoding of motion in the mammalian retina.

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23) Circadian control of cone kinetics Maria M. Arietti, Sahar Farajnia, Maarten Kamermans Retinal signal processing lab, Netherlands institute for Neuroscience, Amsterdam, The Netherladnds; NeuroTime Erasmus Mundus

It is well known that cone responses are faster in the light-adapted than in the dark-adapted retina due to light/dark adaptational processes in the phototransduction cascade. In this study we investigated whether there is also a circadian control of cone kinetics. Electrophysiological recordings were performed in goldfish retinas during the day and night (12h-12h; LD, DD). Naturalistic stimuli were used to determine cone frequency response relations. Voltage light responses of cones were faster during the day than during the night. This difference remained in DD (n=17, p<0.001). To determine the underlying mechanism, we measured the cone frequency response relations under currentand voltage-clamp conditions. Responses under current-clamp are shaped both by phototransduction and voltage-gated currents whereas responses under voltage-clamp are fully determined by the phototransduction cascade. Both current- and voltage- clamp recordings showed faster kinetics in the day than in the night (n=40; p<0.01). However, in DD the change in kinetics only occurred under current-clamp conditions. This indicates that voltage-gated channels control cone kinetics in a circadian fashion. We found that Ih, the current flowing through HCN channels, is circadianmodulated in cones being larger in the day than in the night both in LD and DD (n=93; p<0.01). Previously we have shown that Ih induces contrast adaptation in cones (Howlett et al., 2017). Since Ih is circadian regulated, we found reduced contrast adaptation at night compared to the day both in LD and DD conditions (p<0.01). We next showed the rhythm in Ih and cone kinetics is dopaminedependent. Dopamine is involved in the circadian modulation of Ih which is larger during the day making cones fast and allowing contrast adaptation. Circadian modulation of cone membrane properties seems to prepare the retina to transmit the appropriate visual signal depending on the time of the day.

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24) A quantitative model for adaptation of in cone photoreceptors to contrast Wouter Kamermans, Milagros Arietti, Sahar Farajnia, Marcus Howlett, Maarten Kamermans Netherlands institute for Neuroscience, Amsterdam

Recently we have shown that a hyperpolarization activated current is responsible for a novel type of adaptation in cone photoreceptors. This adaptational mechanism gets most effectively activated by high contrast stimuli. Hyperpolarization induced by this stimulus activates Ih, a current mediated by HCN-channels, which results in a speeding up of the responses of the cones. With Ih blocked this change in kinetics does not occur (Howlett et al. 2017). Here we present a quantitative model that fully describes this behavior. The model consists of ion-channels simulated as Markov processes. The Ih model is directly based on our own experimental data in which we characterized Ih using both pharmacological and biophysical approaches. These data reveal two less known properties of Ih: inactivation and mode switching. Mode switching is the phenomena in which the activation potential of a channel switches between two values depending on the activation state of the channel. A good fit of the model and our experimental data was only obtained when we included these two features. This resulted in an 8 state Markov model. Both mode switching and inactivation turn out to be essential for the adaptational behavior of cones. In general it is believed that Ih suppresses low frequencies. However, here we show that activation of Ih with both mode switching and inactivation only leads to minor reduction of low frequencies but a large increase in gain at high frequencies. Since these features are likely to be present in many other HCN-channels, the results of this study might have a much broader implication than only photoreceptor adaptation.

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25) The spatial distribution of regular and displaced ganglion cells in the rodent retina Christian Puller, Sabrina Duda, Martin Greschner Department of Neuroscience, Carl von Ossietzky University, Oldenburg, Germany

Recent advances in the field of retinal research have led to a greatly increased number of known ganglion cell types and insight has been gained into their distribution patterns across the retina, where several cell types exhibit distinct density gradients or peaks. Here, we analyzed the spatial organization of ganglion cells across the retina and determined the position of cell bodies in the ganglion cell layer (GCL) and in the inner nuclear layer (INL). We investigated the localization of ganglion cells by immunohistochemistry and light microscopy in adult mouse and guinea pig retinas. Antibodies to label all ganglion cells (RBPMS) or certain subtypes, such as OPN, SMI-32, or Foxp2, were applied to whole-mounted specimens. We also recorded the spike responses of several hundred ganglion cells in the isolated retina using a large scale multielectrode array. The spatio-temporal receptive fields and autocorrelation functions were used to physiologically classify distinct ganglion cell types. We obtained overall density distributions of ganglion cells in the GCL of mouse and guinea pig retinas similar to previous studies (e.g. do Nascimento et al., 1991; Salinas-Navarro et al., 2009). The ON and OFF sustained alpha ganglion cells showed a peak density in the GCL of the dorso-temporal mouse retina as reported earlier (Bleckert et al. 2014). Moreover, we found a remarkably high number of the cells’ somata displaced to the INL in the dorso-temporal region of the retina. Displaced ganglion cells occurred primarily at the far peripheral edge of the retina (e.g. Dräger and Olsen, 1981). Here, in this more central region of the retina, more than half of the population of displaced ganglion cells were alpha-type cells. This differs from other retinal locations, where alpha-type cells constituted only a minor fraction of the population of displaced cells. The displaced alpha-like ganglion cells comprised both ON and OFF types. Preliminary experiments to investigate the functional alignment of displaced ganglion cells with regularly placed cells suggest that the receptive field mosaics of alpha-type ganglion cell showed no major disruptions.

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26) Retinal functional changes in a mouse model of α-Synuclein overexpression Hartwig Seitter1, Nadia Stefanova2 and Alexandra Koschak1 1

University of Innsbruck, Institute of Pharmacy, Department of Pharmacology and Toxicology, Innsbruck, Austria; 2 Innsbruck Medical University, Division of Neurobiology, Department of Neurology, Innsbruck, Austria

Multiple System Atrophy (MSA) is a neurodegenerative disease with proposed causal involvement of α-Synuclein (α-SYN) aggregate formation. In Parkinson’s disease (PD), another α-Synucleinopathy, visual symptoms such as diminished contrast sensitivity are well described while for MSA visual manifestations have thus far not been prominent. In the clinical examination of patients, visual tests are an important tool to distinguish PD and MSA. Our experiments are aiming to determine the visual function of a MSA mouse model of human α-SYN overexpression. We detected strong aggregation of α-SYN in the retina and set out to determine the effect of the α-SYN aggregation on retinal physiology. We used transgenic mice, expressing human α-SYN under the proteolipid protein promotor (PLP-α-SYN) and corresponding wild type (WT) controls of two months of age. Mice were dark adapted and retinas were extracted under dim red light. Retinal ganglion cell spiking responses and retinal local field potentials (“in vitro ERG”) were recorded using micro-electrode arrays (MultiChannel Systems MEA2100), while the retina was stimulated with a variety of visual stimuli designed to test contrast sensitivity. The stimuli included flashes, sinusoidal full field stimuli and drifting gratings, with different contrasts each. Visual stimulation was done at two distinct light levels corresponding to scotopic and photopic luminance ranges. In vitro ERG responses to full field flashes of different contrasts showed no difference between WT and PLP-α-SYN retinas. However, we found significant changes in a frequency modulated sinusoidal stimulus which were light leveldependent. While the scotopic responses were comparable, the photopic responses of PLP-α-SYN retinas were significantly more delayed (0.023 < p < 0.048, Wilcoxon ranksum test; WT 81.5 ± 7.8 ms, PLP-α-SYN 114.1 ± 7.9 ms; means of 29 peaks ± std). Retinal ganglion cell responses in PLP-α-SYN mice on the other hand were comparable to WT both in frequency domain modulation as well as in ganglion cell contrast sensitivities. The unchanged flash in vitro ERG highlights that outer retinal function of PLP-αSYN animals is not severely impaired. The lack of a strong impact on ganglion cell responses conforms to the normal visual function in MSA patients. The changes in 239

the in vitro ERG to frequency modulation however revealed a so far unknown functional change that could be a potential diagnostic marker to be tested in human patients. Acknowledgements: FWF (P26881, AK; F4414, NS), University of Innsbruck, Center of Molecular Biosciences (CMBI).

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27) Establishing a comprehensive ganglion cell typology through single-cell functional and transcriptomic techniques Jillian Goetz and Greg Schwartz Northwestern University Department of Ophthalmology

As the output neurons of the retina, retinal ganglion cells (RGCs) are not only critical to vision but also intriguingly diverse. Morphological and physiological interrogations of RGCs have led to estimates of over 30 subtypes, each with distinctive response profiles to light stimuli. For example, RGCs have been identified that show preferential responses to light onset and offset, specific directions and speeds of motion, orientations of edges, and other features of a visual scene. Additionally, transcriptomic surveys of individual RGCs indicate vast diversity in the expression profiles of these neurons, and genetic markers have been identified to target a nontrivial proportion of RGC subsets. However, the majority of functional and morphological RGC subtypes cannot be reliably or specifically targeted genetically. Given the advanced state of murine transgenic tools, it would be a straightforward progression from finding a specific marker of a cell type to understanding that cell type’s physiological nuances, its projection pattern in the central brain, its consequences in visual processing, and the developmental programs that lead to its generation – if only the genetic markers were readily available. The goal of this study is to further our understanding of the transcriptomic diversity underlying RGC functionality by analyzing the transcriptional profiles of known RGC subtypes. We first identify the functionality of individual RGCs by their spike responses to a battery of light stimuli, then isolate each RGC, reverse transcribe and amplify its mRNA, and perform next-generation sequencing on the resulting cDNA library. Our preliminary results indicate that this method reliably generates a full transcriptomic library complete with pan-ganglion cell markers as well as markers of known RGC subtypes, including exclusive expression of genes known to be expressed in particular subsets of ON-OFF direction-selective RGCs. As the study progresses, we will identify novel genetic markers for functionally distinct RGC subtypes. This increased understanding of the correlations between individual neural transcriptomes and physiological functionality will lead to unparalleled insights about neuronal diversity among RGCs. Lessons learned from linking physiological responses to transcriptomics will be broadly applicable to other large-scale efforts to classify neurons throughout the brain. 241

28) Different sets of visual features are routed in two behaviorally relevant circuits Katja Reinhard, Chen Li, Emily Burke, Steven Heynderickx, Karl Farrow Neuro-Electronics Research Flanders, Leuven, Belgium

Visually guided behavior is based on the extraction of relevant features from the visual scene and the routing of this information to the correct motor centers. At the first stage of visual processing, the retina splits the visual scene into over 30 distinct features. Each feature is embodied by a different ganglion cell type that sends visual information to one or several brain targets. To determine the rules of routing we have studied two disynaptic neuronal circuits that link the mouse retina via the superior colliculus to two midbrain nuclei – the lateral posterior nucleus (pulvinar) and the parabigeminal nucleus. It has been demonstrated that each of these midbrain nuclei receives input from different sets of collicular neurons, while optogenetic activation of each projections leads to similar avoidance behavior in mice. Here we have applied a trans-synaptic viral tracing strategy to specifically label the retinal ganglion cells at the beginning of each of the two pathways. By analyzing morphological properties of the labelled cells, we can identify the ganglion cell types that are part of each of the circuits. Our data shows that each brain area receives information from a limited, partially overlapping set of retinal ganglion cells, where each set contains approximately 6 ganglion cell types. These results indicate that the circuitry of the superior colliculus act to route specific sets of visual feature to different downstream targets.

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29) Retinal inputs to wide-field cells in the superior colliculus Chen Li, Katja Reinhard, Emily Burke, Steven Heynderickx, Karl Farrow Neuro-Electronics Research Flanders, Leuven, Belgium

The retina splits the visual scene into over 30 distinct features, where each feature is carried by a different ganglion cell type. As distinct aspects of the visual scene are involved in guiding different behaviors it is expected that a limited set of retinal ganglion cells provides inputs to different visual-motor circuits in central brain regions. Here, we identified the retinal input to one genetically defined set of neurons in the superficial layer of the mouse superior colliculus, the wide-field cells. Selective activation of this neuronal population has been demonstrated to trigger avoidance behaviors via projections through the lateral posterior nucleus of the thalamus. We applied viral vector based neural circuit tracing tools to label the ganglion cells projecting to these wide-field cells. The labelled cells in the retina were then characterized using a combination of their morphology, visual response properties, and antibody-staining of molecular markers. This approach allowed us to identify a small set of retinal ganglion cell types that provide specific visual information to wide-field cells in the superior colliculus.

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30) Following stage II retinal waves during development with a biophysical model 1 D. Karvouniari, 2L. Gil, 3O. Marre, 3S. Picaud, 1B. Cessac, 1

INRIA, Sophia Antipolis INLN, Sophia Antipolis 3 Institut de la Vision, Paris 2

Retinal waves are spontaneous bursts of activity propagating in the developing retina and playing a central role in shaping the visual system. They appear early in development and gradually disappear upon maturation. They are classified in 3 consecutive stages (I,II,III), mainly characterized by different synaptic transmissions and transient networks of specific cells. However, in order to elucidate the dominant mechanisms shaping retinal waves within a specific time window of maturation, as well as the transition between them, it is important to investigate the continuous variations of biophysical characteristics due to development within each stage. Focusing on stage II, we propose a biophysical model, grounded on experiments, and accessible to dynamical systems analysis, featuring a network of cholinergic-coupled Starburst Amacrine Cells (SAC) with a calcium-controlled slow After HyperPolarization current (sAHP). In agreement with biophysics SACs burst spontaneously and their interaction via acetylcholine gives rise to waves. A bifurcation analysis exhibits 3 key biophysical parameters having a big impact on SACs spatiotemporal activity: the fast K+ conductance, the acetylcholine synaptic strength (varies upon maturation), and the rest potential (varies upon pharmacology). This analysis leads us to reproduce a bench of experimental results . It allows us to explain the wide variability in interwave intervals observed across species with a unique generic mechanism (figure not shown). More generally, the nonlinear dynamics generates heterogeneous local spatial structures inside which retinal waves propagate (Fig 1B). This induces a wide variability in waves characteristics (size, duration) even though our network is perfectly homogeneous. Therefore, we show that although variability is de facto present in biological systems, it is not necessary to explain the appearance of spatial structures and waves, as well as their variability (Fig 1). We analyze how the evolution of cholinergic conductance due to the maturation of nicotinic receptors dramatically changes the retinal wave characteristics. Especially, there is a very narrow interval of acetylcholine conductance where retinal waves size obey a power law distribution, suggesting a specific (homeostatic) mechanism stabilizing temporarily the SACs network in this specific range. Finally, we discuss several experimental predictions of our model. 244

31) Tracking migrating cells in AMD patients using adaptive optics imaging (image processing approaches) Michel Paques, Florence Rossant, Kate Grieve, Hasan Sawan. Hôpital des Quinze Vingts National. Institut Supérieur d’Electronique de Paris (ISEP). UPMC.

Adaptive optics (AO) imaging has improved our understanding of tissue changes during age-related macular degeneration (AMD), through detection of novel features such as slowly migrating melanin-loaded cells (MLCs) which are only observed in patients with AMD. Several patients with dry AMD were imaged regularly with the rtx1 camera (Imagine Eyes, France) for a few months, at a rate of around one image a week. Time-lapse movies were created out of those images. Image processing approaches were developed to track individual cells in order to extract statistical data about the moving cells, and check if there is any possible role played by those cells in AMD advancement. This study revealed that the complex, dynamic process of redistribution of MLCs throughout the posterior pole precedes and accompanies the emergence and progression of atrophy. Therefore, these clumps are probably also a biomarker of RPE damage.

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32) The diagnostic value of optical coherence tomography angiography in diabetic retinopathy – A systematic review David Gildea Institute of Ophthalmology, Moorfield’s Eye Hospital, London, United Kingdom

Purpose; Accurate investigative tools are essential for the early diagnosis and monitoring of diabetic retinopathy (DR). Optical coherence tomography angiography (OCTA) is a recently developed technology that enables visualization of the retinal microvasculature. The purpose of this systematic review was to assess the diagnostic value of OCTA in DR. Methods; A comprehensive review of the literature was performed according to the PRISMA guidelines. Results; OCTA has demonstrated the ability to identify microvascular features of DR such as microaneurysms, neovascularization, and capillary non-perfusion. OCTA is enabling quantitative evaluation of the microvasculature of diabetic eyes. It has the ability to detect early microvascular changes, in eyes with or without clinically evident DR. It has also been shown to detect progressive changes in the foveal avascular zone, and vascular perfusion density, with worsening severity of disease. It provides 3-dimensional visualisation of the individual retinal vascular networks, and is thereby enhancing our understanding of the role of the deeper vasculature in the pathogenesis of diabetic retinopathy and maculopathy. Conclusions; Limitations exist with current OCTA technology, in respect to the small field of view, image quality, projection artifact, and inaccuracies in deep vasculature analysis. While questions remain regarding its practical applicability in its present form, with continuing development of the technology, the diagnostic value of OCTA in DR is likely to become evident.

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33) Large-scale offline and online spike sorting for extracellular recordings validated on ground truth data Baptiste Lefebvre1, Giulia L. B. Spampinato2, Elric Esposito2, Jens Duebel2, Olivier Marre2 and Pierre Yger2 1

Laboratoire de physique statistique, Département de physique de l'ENS, Ecole normale supérieure, PSL Research University, Université Paris Diderot, Sorbonne Paris Cité, Sorbonne Universités, UPMC Univ. Paris 06, CNRS, 75005 Paris, France 2 Institut de la Vision, INSERM UMRS 968, CNRS UMR 7210, 75012 Paris, France

Understanding how assemblies of retinal ganglion cells encode information requires recording of large populations of cells in the retina. In recent years, multielectrode arrays and large silicon probes have been developed to record simultaneously from thousands of electrodes packed with a high density. Those devices challenge the classical way to do spike sorting. We developed a fast and accurate spike sorting algorithm (available as an open source software named SpyKING CIRCUS) to solve this issue [1]. It is a templatematching based algorithm, highly scalable and automated such that the time spent by the user on manual curation is reduced to the minimal and do not scale with the number of electrodes. The algorithm has been validated both in vitro and in vivo based on simultaneous extracellular and patch clamp recordings. In this context, the solution to the sorting problem is partially known. We show that we reach near optimal performance. SpyKING CIRCUS thus appears as a general solution to sort, offline, spikes from large-scale extracellular recordings. However, understanding how retina encodes visual information requires also to explore a high dimensional stimulus space. Since closed-loop experiments enable this exploration, we implemented an “online” mode to sort spikes in real time while the data are acquired. The software is parallelized to use optimally the computing resources: all the different processing steps of the algorithm are distributed across different nodes/machines. The modified algorithm is composed of two concurrent workflows running continuously: 1) “a template-finding” process to extract the cell templates (i.e. the pattern of activity evoked over many electrodes when one neuron fires an action potential) over the recent time course; 2) a “template-matching” process where the templates are matched onto the raw data to identify the spikes. It is therefore a promising solution for future closed-loop experiments involving recordings with hundreds of electrodes. We are currently testing the performance of the algorithm on various ground truth datasets. 247

34) “convis: a python toolbox to simulate and optimize convolution filter based vision models” Jacob Huth, Timothée Masquelier, Angelo Arleo 1

CERCO UMR5549, CNRS, University Toulouse 3, France Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, Paris, France 2

We developed “convis” [1], a Python toolbox for large scale simulation and efficient fitting of LNcascade models with large, non-separable spatio-temporal filters and non-linearities of different complexity. The models are implemented as abstract computational graphs in theano and allow for flexible inspection and manipulation while computation intensive tasks are executed on GPUs. With automatic differentiation, even very large linear filters can be fitted efficiently to experimental data. “convis” offers a range of pre-build models, such as LN-cascade models with feedback and delays as well as a reimplementation of the “VirtualRetina” model including contrast gain control and a spiking mechanism. The models in the toolbox are extensible with additional computational layers, parameterized 3d filter kernels, error functions and optimization routines. We show that exploration of the derivatives of the error with respect to the different parameters can optimize the fitting process by determining which optimization processes are concave and which are reasonably close to 2nd degree polynomials. This allows us to use very large linear filters for 3d convolutions, as these are easy to fit, yet can capture complex spatio-temporal receptive fields. The package is available via Pypi and github [1].

[1] convis github repository http://github.com/jahuth/convis/

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35) Cooperative coding of moving edges in the superior colliculus Daniel de Malmazet, Karl Farrow NERF, KU Leuven, VIB, IMEC

The visual system of vertebrates is adept at extracting salient features of a visual scene such as spatial orientations and motion direction. The retina is the first stage of visual processing and targets brain areas including the superior colliculus. Here the visual scene is represented by a set of overlapping, retinotopically organized feature maps. Orientation selective neurons in the mouse superior colliculus form an inhomogeneous map where, unlike in other visual areas, neurons with the same orientation selectivity cluster together such that a single orientation is represented at each retinotopic location. However, the topographical organization of other feature maps, and their relationship to each other remains unknown. Using two-photon calcium imaging, we recorded the activity of neurons spanning more than half of the superior colliculus, while simultaneously measuring their receptive field and determining their orientation and direction selectivity. We found that the preferred axis of motion, of direction selective neurons is dependent on their retinotopic position. When comparing preferred directions with orientation in the same retinotopic location, direction selective neurons showed a strong preference for directions of movement orthogonal to the preferred orientation of nearby orientation selective neurons. These findings uncover a second inhomogeneous map accounting for motion detection that can be superimposed with the pre-established spatial orientation map.

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36) The neuroretina in multiple system atrophy: morphological implications of Plp-α-Syn mice Kathrin Kaehler1, Hartwig Seitter1, Adolf Sandbichler2, Nadia Stefanova3 and Alexandra Koschak1 1

University of Innsbruck, Institute of Pharmacy, Department of Pharmacology and Toxicology, Center for Chemistry and Biomedicine, Innsbruck, Austria 2 University of Innsbruck, Institute of Zoology, Innsbruck, Austria 3 Innsbruck Medical University, Division of Neurobiology, Department of Neurology, Innsbruck, Austria

Neurodegenerative diseases like Parkinson's disease (PD) and Multiple System Atrophy (MSA) have been shown to exhibit physiological and morphological neuronal abnormalities. These abnormalities can result from exceeding aggregation of α-synuclein (α-SYN), a 140aa presynaptic protein that exerts toxic function in case of dysregulation. Both PD and MSA are associated with a variety of visual symptoms and a potential role of the retina as a biomarker for progression of Parkinson's disease is recently discussed. We therefore aimed at investigating the underlying mechanisms in homozygous transgenic mice overexpressing human α-SYN under the proteolipid protein (PLP)-promoter (PLPα-SYN) compared to wild type (WT) animals of two different age groups (two months, one year). By performing immunohistochemical analyses on vertical retinal sections we discovered that distinct α-SYN signal occurred in different retinal cell layers of PLP-α-SYN mice, but not in WT mice. This is remarkable as the PLP promotor driving the α-SYN expression in oligodendrocytes was reported to be inactive in the retina. PLP expression stopped at the optic nerve/retina junction, where we observed a colocalization with α-SYN. Notably the cell type distinctly stained in the inner retina was rod bipolar cells, which was confirmed by PKCα co-staining. Quantitative real-time PCR using specific TaqMan® probes will help to verify the α-SYN expression in rod bipolar cells. Interestingly, immunoreactivity for α-SYN was more distinct in peripheral rod bipolar cells than in central ones. Additional immunohistochemical experiments included the investigation of the glial fibrillary acidic protein (GFAP), a marker for activation of Müller glia that can indicate neuroinflammatory processes, and Iba1, a marker for microglial activation. GFAP-positive fibers spanning the peripheral retina were pronounced in aged animals in WT and even more in PLP-α-SYN. This might also reflect the direction of accumulation of human α-Syn in rod bipolar cells, which proceeds in the same way. In preliminary experiments Iba1 expression seemed comparable. Based on the upregulated expression of GFAP in PLP-α-SYN animals, TUNEL staining was performed to elucidate whether the extent of apoptosis 250

changes as a result of the α-SYN overexpression. Due to the known impairments of the dopaminergic system in α-synucleinopathies we investigated also dopaminergic neurons. Although the cell bodies seemed deformed in PLP-α-SYN animals their number was comparable. Tyrosine-hydroxylase-positive processes however appeared to reach into deeper strata of the inner plexiform layer. Our findings clearly implicate an impairment of retinal neurons in the PLP-α-SYN MSA model, which may also underlie visual deficits reported in MSA patients.

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37) Effect of neuromodulators on light responses and second messengers in the mouse retina visualized by optogenetic sensors

Safaa Belaidi, Anna Sieben, Benjamin Usai, Frank Müller Institute of Complex Systems, Cellular Biophysics (ICS-4), Forschungszentrum Jülich, Germany

Second messengers play a major role in cellular signaling pathways and in retinal adaptation. In the retina, a large number of neurotransmitters are utilized by different cell types. Dopamine and adenosine are promising candidates that may control light and dark adaptation, respectively. These neuromodulators are released at different sites in the retina and under different conditions. They act on G-proteincoupled receptors, leading to either an increase or a decrease in the concentration of cyclic adenosine monophosphate (cAMP), directly coupled to changes in the activity of protein kinase A (PKA). In addition to the cAMP/PKA pathway, retinal neurons use calcium (Ca2+) as intracellular messenger. Although much is known about the effects of dopamine at cellular and synaptic levels, little is known about the effects of adenosine in the retina. We studied the effect of dopaminergic agonists on the light response of retinal ganglion cells in vitro using the patch-clamp method. We used the fluorescent genetically encoded Akinase activity reporter AKAR4 in combination with imaging techniques to examine the effect of dopamine and adenosine on the cAMP metabolism in cultured retinal neurons in real time. For the visualization of changes in the internal concentration of Ca2+ in cultured retinal neurons, cells were loaded with the chemical Ca2+-indicator Fluo-4-AM. In the intact retina changes in intracellular Ca2+ were detected using a transgenic mouse line that expresses the FRET-based Ca2+-sensor TN-L15 in ganglion cells. Agonists at dopamine D1 receptors reduced the light response of darkadapted retinal ganglion cells and shifted the stimulus-response curve towards brighter stimuli, features that are usually observed in light-adapted retinae. As D1 receptors are expressed in both synaptic layers, we assumed that the reduction of the light responses might be mediated by dopaminergic modulation of glutamate receptor activity. Indeed, in calcium imaging experiments in the intact retina dopamine differentially modulated the activity of glutamate receptors of the AMPA, kainate, and NMDA type, indicating that dopamine modulates the relay of light responses through the retinal network. Interestingly, the effect of adenosine on the same receptor types was quite 252

different. Many cultured retinal cells responded to dopamine with changes in internal cAMP and Ca2+. Different responses could be observed that were based on different signaling pathways. Only few cells responded to adenosine with changes in the internal cAMP concentration.

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SYMPOSIUM INNOVATIONS ET SOLUTIONS THERAPEUTIQUES 1) Les prothèses rétiniennes de nouvelle génération : Prevot Paul-Henri – Institut de la Vision, Paris Yannick Lemer - Fondation Ophtalmologique A. de Rothschild, Paris Les implants photovoltaïques sous-rétiniens créent des réponses physiologiques au niveau de la rétine et cortex visual de rongeurs aveugles. Pour préparer la phase clinique de ces implants, nous avons testé l’efficacité de ces implants à travers des expériences ex vivo utilisant des rétines de primates non humains et par des tests comportementaux chez trois primates non humains. Nous montrons que les implants fabriqués par Pixium Vision peuvent stimuler indirectement les cellules ganglionnaires en testant des modèles de rétines dégénérées de primates non humains. Un primate non humain a montré un clair comportement la vision restaurée grâce à un implant en réalisant des saccades dans la direction de l’implant. Ces résultats préparent les futurs tests cliniques.

2) Restauration visuelle par thérapie Optogénétique : Antoine Chaffiol – Institut de la Vision, Paris Chez les patients atteints de dystrophies rétiniennes de type rétinopathie pigmentaire ou de pathologies plus complexes comme la dégénérescence maculaire liée à l’âge (DMLA), les photorécepteurs de la rétine dégénèrent. Depuis des années, une approche thérapeutique, celle des prothèses rétiniennes, a permis à des patients devenus aveugles de retrouver une certaine perception visuelle, et de nouvelles générations de prothèses sont en cours de développement afin de parvenir à une fonctionnalité améliorée en particulier concernant la définition des images perçues. A l’institut de la Vision, nous travaillons également sur une stratégie alternative récente et très prometteuse, la thérapie optogénétique, reposant sur l’expression d’une protéine photosensible d’algue ou de bactérie dans les cellules résiduelles du circuit rétinien. Grâce à cette méthode, différents types cellulaires peuvent être ciblés, selon le degré de dégénérescence de la rétine, et il devient alors possible de prendre le contrôle optiquement sur l’activité électrique de cellules rétiniennes jusqu’ici dormantes puisque ne recevant plus leur afférence des photorécepteurs, et de transmettre les signaux jusqu’au cerveau. Après avoir développé cette 254

approche à l’institut de la vision sur des modèles de souris aveugles, nous l’évaluons actuellement sur des primates non-humains dont les caractéristiques de l’œil et de la rétine sont très proches de ceux de l’homme en termes de taille, fonction et réaction inflammatoire.

3) Dystrophie rétinienne : La survie des cônes et le RdCVF : Emmanuelle Clérin, Delphine Pagan, Ying Yang, Seiki Achiedo, Julie Degardin, Quénol Cesar, Manuel Simonutti, Géraldine Millet-Puel, Frédéric Blond, Najate Aït-Ali, José-Alain Sahel & Thierry Léveillard – Institut de la Vision, Paris La rétine des vertébrés comprend deux types distincts de photorécepteurs dont la morphologie les caractérise: les bâtonnets et les cônes. Les bâtonnets sont très sensibles et donc saturés à la lumière du jour. Les cônes sont à l’origine de la vision des couleurs, mais aussi de la vision des détails de jour, donc de l’acuité visuelle. Pour relayer le signal lumineux, les opsines qui captent les photons incidents, sont enchâssées dans une bicouche lipidique riche en acides gras polyinsaturés. Ceci exerce une contrainte importante sur le système car le taux d’insaturation des acides gras est proportionnel à leur tendance à s’oxyder et donc à se dégrader. Les segments oxydés sont donc éliminés par phagocytose réalisée par l’épithélium pigmenté rétinien et le segment externe est renouvelé quotidiennement. Ainsi, les photorécepteurs sont des capteurs très performants de la lumière au prix d’une dépense énergétique intense. Ceci a des répercutions sur les pathologies rétiniennes. Pour la dégénérescence rétinienne héréditaire la plus fréquente, la rétinopathie pigmentaire, les 60 mutations identifiées entrainent chacune directement la mort des bâtonnets et indirectement celle des cônes. Nos travaux ont mis en évidence un phénomène unique d’altruisme intercellulaire: les bâtonnets nourrissent les cônes. La mort des bâtonnets entraine donc la famine pour les cônes. Le gène nucleoredoxin-like1 (NXNL1) code pour deux protéines par épissage alternatif, la protéine rodderived cone viability factor (RdCVF) est un facteur sécrété par les bâtonnets qui augmente la capacité des cônes à emmagasiner et métaboliser le glucose, ceci pour stimuler la resynthèse quotidienne de leurs segments externes. Sans RdCVF, le renouvellement ne se fait plus: l’acuité visuelle est perdue. De manière particulièrement étonnante, le second produit du gène est une enzyme de type thiorédoxine qui répare les atteintes cellulaires dues à l’oxydation. Le lien entre l’activité trophique de RdCVF, l’un des produits du gène NXNL1 et de l’activité 255

enzymatique de la thiorédoxine RdCVFL est à l’origine du concept de signalisation métabolique et redox car le pouvoir redox de RdCVFL provient aussi du glucose. Nous montrons par délivrance de vecteurs viraux que la combinaison des deux produits du gène NXNL1 permet de ralentir la perte de l’acuité visuelle des cônes chez un modèle animal de la rétinopathie pigmentaire. Nous avons franchi un pas décisif vers le transfert de cette thérapie vers la clinique.

4) Production de rétines in vitro à partir de cellules pluripotentes humaines : Un nouvel outil thérapeutique. Sacha Reichman– Institut de la Vision, Paris Les maladies dégénératives de la rétine comme les rétinopathies pigmentaires, la dégénérescence maculaire liée à l'âge ou le glaucome aboutissent à la perte irréversible de cellules rétiniennes indispensables à la vision. Au 21e siècle, la thérapie cellulaire s’installe comme une approche prometteuse qui consiste à ramener les cellules perdues par chirurgie. L’une des sources de production de ces cellules thérapeutiques est l’utilisation des cellules souches humaines induites à la pluripotence (iPS). Dérivées d’une simple biopsie de peau, les cellules iPS peuvent être différenciées en rétine in vitro notamment en utilisant un protocole simple et innovant développé au sein de notre équipe à l’Institut de la Vision à Paris. Cette méthode permet de générer en un mois des organoides rétiniens (structures en 3D) composés de cellules progénitrices pouvant se différencier dans tous les types cellulaires suivant la rétinogénèse humaine. Par la suite, ces progéniteurs différenciés en cellules d'intérêt thérapeutique (photorécepteurs et cellules ganglionnaires) peuvent être purifiées et transplantées dans l’œil. Leur intégration pourrait permettre de rétablir une "activité neurorétinienne" pour récupérer la vision. De manière complémentaire, la production de cellules rétiniennes « malades » à partir de cellules iPS de patients atteints de rétinopathies héréditaires offre l'opportunité de développer de nouveaux modèles in vitro humains en plus des modèles animaux préexistants. Cette approche de modélisation est nécessaire, d'une part pour comprendre les mécanismes moléculaires et cellulaires qui soustendent la maladie. Et d'autre part, pour permettre le développement de tests automatisés indispensables aux campagnes de criblages à haut-débit de composés actifs pour l’émergence de nouveaux traitements pharmacologiques. En conséquence, grâce à ces nouveaux outils cellulaires, nous espérons que le 256

traitement des maladies dégénératives rétiniennes ne sera plus seulement un espoir mais une réalité.

5) Stratégies d’ingénierie tissulaire de l’œil à base de cellules souches embryonnaires humaines et à visée thérapeutique : Conception et essais cliniques en cours et futurs: Karim Ben M Barek, Walter Habeler, Alexandra Plancheron, Mohamed Jarraya, Florian Regent, Angélique Terray, Ying Yang, Laure Chatrousse, Sophie Domingues, Yolande Masson, José-Alain Sahel, Marc Peschanski, Olivier Goureau and Christelle Monville – Institut de la Vision, Paris Les maladies rétiniennes telles que la dégénérescence maculaire liée à l’âge (DMLA) et les rétinites pigmentaires (RP) touchent des millions de personnes à travers le monde. La DMLA, première cause de cécité acquise dans les pays développés, a pour origine l’interaction complexe de facteurs environnementaux et génétiques. À l’opposé, les RP sont causées par des mutations monogéniques et affectent une population jeune. Dans la DMLA et certaines formes de RP, la mort ou la dysfonction primaire de l’épithélium pigmentaire rétinien (EPR) induit la mort des photorécepteurs. Il n’y a pas de traitement curatif disponible actuellement pour les RP et la majorité des patients DMLA. Parmi les différents axes de recherches proposés, la thérapie cellulaire visant à remplacer les cellules dysfonctionnelles ou mortes par des cellules saines est prometteuse. Différentes approches pour produire des cellules fonctionnelles de grade clinique à partir de cellules souches et à l’échelle industrielle seront abordées. Nous verrons que la formulation finale de ces cellules (feuillet complexe ou suspension cellulaire) joue également un rôle crucial dans la réponse thérapeutique. Enfin, dans le cadre du développement d’une thérapie cellulaire, la sécurité du produit (absence de prolifération ou de dispersion des cellules anormale) doit également être évaluée avant les premiers essais chez l’homme.

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6) Imagerie et nouveaux critères d’évaluations médicaux : Michel Pâques, Kate Grieve– Institut de la Vision et CHNO des 15‐20, Paris L'imagerie à haute résolution de la rétine par optique adaptative permet d'observer de manière totalement non invasive des phénomènes dynamiques à l'échelle microscopie comme les migrations de cellules, dans des maladies aussi communes que la dégénérescence maculaire liée à l'âge. Nous avons ainsi analysé des déplacements cellulaires sur des périodes de plusieurs mois, et nous émettons l'hypothèse que l'analyse des déplacements et de la taille des cellules pourrait nous permettre de mieux comprendre leur nature. Un aspect particulièrement intéressant est la possible corrélation entre l'aggravation de la maladie et les migrations observées, car dans certains cas nous avons pu observer que cette migration précédait l'apparition de plaques d'atrophie caractéristiques de la maladie. Différentes stratégies d'analyse d'image sont mises en oeuvre afin d'extraire des biomarqueurs pertinents reflétant l'évolution de la maladie et les mécanismes cellulaires et tissulaires sous-jacents.

7) Impact des troubles visuels sur la vie journalière des patients atteints de dystrophies rétiniennes : Saddek Mohand-Saïd – CHNO des 15‐20, Paris

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COMPANIES & SPONSORS presenting and participating at the ERM 2017

FOUNDATION FIGHTING BLINDNESS The Foundation Fighting Blindness was established in 1971. It has since raised more than $700 million for research aimed at preventing, treating, and curing blindness caused by retinal degenerative diseases. In excess of 10 million Americans, and millions more worldwide, experience vision loss due to retinal degeneration. Through its support of focused and innovative science, the Foundation drives the research that has and will continue to provide treatments and cures for people affected by retinitis pigmentosa, macular degeneration, Usher syndrome, and other inherited retinal diseases worldwide. Stephen Rose, Ph.D. Chief Research Officer [email protected] www.fightblindness.org

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ESSILOR Essilor, a world leader in ophthalmic optics, designs and manufactures a wide range of ophthalmic lenses and provides equipment and services for eye care professionals, helping to correct, protect and prevent risks to the visual health of more than one billion people worldwide every day. As a true industry pioneer with a 170-year history, Essilor is committed to meeting a key challenge of our times: poor vision. It offers unique products and solutions, ranging from high-technology corrective lenses and the most widely accessible products to photochromic lenses that protect against UV rays and harmful blue-violet light. The Group’s presence in nearly 70 countries enables Essilor to operate close to the eye care professionals and consumers it serves. Committed to raising awareness of the importance of good vision, it also works around the globe to improve access to solutions most adapted to consumer needs. Its unique mission of Improving Lives by Improving Sight is central to the long-term development of the Group and provides a powerful driver to improve visual health for everyone. With a strong commitment to people, the Group’s performance is powered by its dynamic approach to innovation, partnerships and the engagement and entrepreneurial spirit of its 64 000 employees. 260

In the pics are the following persons, from the top to the bottom: Coralie Barrau, Research Engineer, Optics and Photobiology, Essilor R&D International, [email protected] Eva Lazuka, Global KOL and Professional Relations, Essilor International, [email protected] (Editor www.pointsdevue.com) Veronika Marek, Research Engineer, Science & Collaborations Strategy, Essilor R&D international, [email protected] Thierry Villette PhD, Director Science & Collaborations Strategy, Essilor R&D International, [email protected]

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PIXIUM VISION

Khalid ISHAQUE The Chief Executive Officer

Blindness affects roughly 39 million people worldwide and despite many technology and scientific advancements in the world of science, the standard solution for these patients had represented for a long time a simple cane and/or a guide dog. Neither a biblical miracle, nor science fiction, Pixium Vision is the only company in the world today to have developed in parallel two innovative retinal Bionic Vision Systems: IRIS® II, a 150 electrode Epi-Retinal Bionic Vision System designed to be exchangeable, which obtained the CE mark in 2016, and PRIMA, a sub-retinal miniaturized and totally wireless photovoltaic implant, is in pre-clinical stage with first in human clinical studies expected to start in 2017.

These systems incorporate active implantable prostheses and bio-inspired neuromorphic camera sensor mimicking the functioning of human retina, intended to compensate for blindness resulting from the outer retinal degeneration like retinitis pigmentosa (RP) today, and in the near future dry agerelated macular degeneration (AMD). These bionic systems are aimed at significantly improving the independent, mobility, and quality of life of these blind patients.

Priscilla PAGNACCO Product Manager, Marketing

Located in Paris, near the Vision Institute, and collaborating with several renowned scientists from prestigious academic and technology institutions around the world, Pixium Vision’s mission is to create a world of bionic vision for those who have lost their sight, with a functional optic nerve, and enabling them to regain partial visual perception and to lead more independent lives.

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Within an eco-system of global scientific and technological excellence, supported also by intellectual property and know-how composed of more than 250 patents, the Company harnesses the rapid advances in visual processing, microelectronics / nanoelectronics, optoelectronics, neurobiology and intelligent software algorithms to develop systems that could ultimately provide blind people with a new form of prosthetic/bionic vision to lead more independent lives. Contact information: 74 rue du Faubourg Saint-Antoine 75012 Paris, France Tel : +33 1 76 21 47 68 Fax : +33 1 43 42 05 26 Email : [email protected] http://www.pixium-vision.com/en

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MAXWELL BIOSYSTEMS MaxWell Biosystems develops and distributes the most advanced electrophysiology instrumentation for readouts with high signal quality, high resolution, and high throughput. Our CMOS-based microelectrode array (MEA) systems are used to advance basic research and accelerate drug discovery. The MaxOne system, which includes MEA chips for biological samples, recording unit and PC, and MaxLab Live data analysis software, is ideal for retinal applications. The large sensor area of the MEA chip, packed with 3300 electrodes/sq-mm, can be used to record large retinal areas with densely packed retinal ganglion cells (RGCs). Specific retinal applications include: label-free and long-term identification of retinal ganglion cell mosaics, screening of transgenic animal retina, efficacy assessment of visual restoration techniques, and functional assays using human iPSC-derived retinal tissue. Michele Fiscella, Ph.D. VP Scientific Affairs michele.fiscella @mxwbio.com

Marie Obien, Ph.D. VP Marketing and Sales marie.obien @mxwbio.com

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Learn more about MaxOne and retinal applications at our booth.

MULTI CHANNEL SYSTEMS Multi Channel Systems, a division of Harvard Bioscience, Inc., focuses on the development of precision scientific measuring instrumentation and equipment in the field of electrophysiology for research groups at universities and for the pharmaceutical industry. We provide solutions for extra-cellular recordings with microelectrode arrays in vitro and in vivo as well as for electrical stimulation. Moreover, we offer devices for the automatic injection in oocytes and intracellular recording of oocyte ion channels. Because of their modular principle, our products can be extended and adjusted to your specific experimental needs. Together with our distribution partners, we serve laboratories, research institutes and the pharmaceutical industry all over the world. Over 20 years of experience and our international distribution network make us the global market leader in the field of non-clinical electrophysiology with microelectrode arrays. Emma David Multi Channel Systems MCS GmbH a division of Harvard Bioscience, Inc. Aspenhaustrasse 21 72770 Reutlingen Germany Phone: +49 7121 909 25-25 Fax: +49 7121 909 25-11 Email: [email protected] Website: www.multichannelsystems.com

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3BRAIN 3Brain is the world's first company having designed and realized high-resolution microelectrode arrays (MEAs). 3Brain's technology revolutionized brain research by allowing researchers to visualize signal propagations occurring at a submillisecond scale between thousands of cells for the first time. 4096 recording electrodes, sampled simultaneously at 18kHz, provide orders of magnitude more data points compared to conventional passive Multielectrode Arrays (MEAs), thus leading to the recording, over a large bandwidth, of electrophysiological signals ranging from slow largepopulation field potentials to fast single-cell spiking activity.The BioCAM X platform delivers unique insights into several invitro electrophysiological models, such as neuronal cell cultures, brain slices and retina preparations. Alessandro Maccione CSO and co-founder

Marco Aquila Sales Manager

Email: [email protected] Website: www.3brain.com

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IRIS PHARMA Iris Pharma Specializes in Offering Preclinical and Clinical Ophthalmology Research Services Worldwide Iris Pharma is a world-leading Contract Research Organization (CRO) offering preclinical and clinical ophthalmology research services. Iris Pharma works hand-in-hand with customers and has helped bring more than 70 ocular drugs and medical devices to international markets to date. The company is able to do this as efficiently as possible by maintaining a global view of the development process and by bringing personalized advice. Iris Pharma indeed addresses all ophthalmology challenges by offering programs and study designs that perfectly suit the needs of customers: therapeutic value assessment, full preclinical regulatory package, product value creation or à la carte studies. The CRO has expertise in all ophthalmic indications - from the ocular surface to the posterior segment of the eye and supports customers, newcomers or experts in ophthalmology, ranging from small start-up companies to large pharmaceutical companies in moving forward products that directly or indirectly affect eye health. Yann Quentric, M.Sc. President, Iris Pharma Holding SAS

In addition to a wide portfolio of services such as proof Erwann Wydauw of concept studies in animal models, GLP preclinical Pharm.D., MBA, studies, clinical trials and post-market studies, M.Sc. Head of Clinical bioanalytical testing, cellular and molecular biology Operations assays, Iris Pharma also offers consulting services to help customers determine the best indication and the best route for molecules and products in development. To support more than 300 customers worldwide, Iris Pharma maintains a GLP-accredited laboratory for 267

preclinical research and bioanalytical testing and conducts high-quality clinical trials in accordance with ICH GCP guidelines. For more information, visit www.iris-pharma.com. Iris Pharma Allée Hector Pintus 06610 La Gaude France Tel.: +33493594959 Fax: +33493594950 email: [email protected] www.iris-pharma.com

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european retina meeting 2017 - ERM 2017

EUROPEAN RETINA MEETING 2017 October 5 -7, 2017 Paris, France Organisers: Serge Picaud, Alvaro Rendon (Institut de la Vision, France) Petri Ala-Lauri...

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