Circuit specific approaches to retinal diseases

Final Report Summary - RETICIRC (Circuit specific approaches to retinal diseases)

Executive Summary:
The RETICIR project focuses on neuronal mechanisms of vision from photoreceptor level to visual cortex. Physiological knowledge of the visual system was used to address pathophysiological neuronal mechanisms of diseases affecting vision. In the project we identified molecular and cellular disease mechanism of retinitis pigmentosa, and we demonstrated restoration of vision in retinas with degenerated photoreceptors from single neuronal responses in the retina all the way to cortical visual evoked responses and behavioural assays.
Blindness in man often results from dysfunction of the retina. For a number of retinal diseases, the retinal circuitry is a major part of the problem. Effective strategies for treatment of those diseases and for all attempts to restore vision, understanding of the retinal circuitries is essential. Our strategy in this project was that understanding the structure and function of specific retinal circuits should be used to find therapies to retinal diseases, like for instance retinitis pigmentosa. Specific circuits can be only studied with cell type specific promoters and therefore the methodology that we used is the combination of in vivo marked cell types with genetic interference with the function of these cells and state of the art physiological and behavioural approaches.
The project consisted of four work packages. WP01 was dedicated to management while WP02, WP3 and WP04 were directed to science. WP02 - “Retinal circuit analysis” has generated basic morphological, physiological and system biological knowledge needed to perform the translational, disease related work of WP3 and WP4. WP03 - “Photoreceptor degeneration, the bystander effect” focused on disease mechanisms. It studied the spread of degeneration through the photoreceptor network during retinitis pigmentosa. Finally, WP04 - “Rescuing retinal function using cell specific expression of light sensitive proteins“ focused on developing a novel strategy of restoration of vision in people with degenerated photoreceptors. In all of these WPs great progress has been made.
The major progress of WP02 can be summarized in two global achievements. In the retina photoreceptors are the light sensitive neurons. These neurons transform light into electrical signals. These signals are fed in to a neuronal network of horizontal cells and bipolar cells. This is the first processing stage of the retina. We have identified and characterized the critical processing steps in this first processing stage: Contrast adaptation, Gain control, Subtractive inhibition, Synaptic amplification, and Lateral integration. This allowed us, for the first, time to develop a full quantitative model of the outer retina. Such model is essential for the development of optogenetic treatments in WP04. The second major achievement deals with the inner retina. There we described an approach sensitive ganglion cell and resolved its neuronal mechanism. This work illustrates that in the inner retina highly specialized neuronal sub-networks exist. Knowledge of such specialized sub-networks is essential for any strategy to restore function of a diseased retinal network.
Retinitis pigmentosa leads to degeneration of rods followed by a secondary degeneration of cones. Especially the loss of cones leads to severe visual problems. Prevention of the loss of cones would increase the quality of life of retinitis pigmentosa patients enormously. WP03 aimed at testing the so called bystander hypothesis. This hypothesis suggests that cones receive signals from the dying rods, a “dead signal” such that the cones also degenerate. If this were the case then one could stop the cone degeneration by blocking the coupling between rods and cones. This was tested in an animal without gap-junctions between photoreceptors. The absence of photoreceptor coupling did not rescue the cones, showing that the metabolic coupling by gap-junctions of photoreceptors is not the cause of the secondary degeneration of cones.
Showing proof of principle for the reestablishment of retinal function in retinas with degenerated photoreceptors was one of the goals of WP04. A commonly used mice model for retinitis pigmentosa is the RD-mouse. In these mice, photoreceptors degenerate. However, it was found that the photoreceptors do not disappear completely but that cell bodies and synaptic terminals remain functionally connected to the rest of the retina. By transfecting these degenerated photoreceptors with constructs encoding for halorhoropsin, we were able to restore lights responses of retinal neurons. The retinal ganglion cells in these mice showed normal center surround properties and these mice were able to perform visual tasks again. In pilot experiments it was shown that a similar restoration of vision could be obtained in cultured human retinas.
Overall RETICIRC has delivered what it promised: better understanding of the retinal circuitry, direct test of the bystander hypothesis and proof of principle of the optogenetic approach to restore vision in blind patients.

Project Context and Objectives:
The RETICIR project focuses on retinal neuronal mechanisms of vision (Figure 1). Physiological knowledge of the visual system will be used to address pathophysiological neuronal mechanisms of diseases affecting vision. We broad specialists on the mechanisms of vision and retinal circuitry together to collaborate with researchers investigating the (patho)physiological mechanisms of retinal diseases in order to design new therapeutic approaches and rehabilitating strategies. Such a team ensured that the knowledge obtained in fundamental research benefits disease related research and will flow naturally into therapeutic solutions and eventually improves health of the European citizen. This team is fully equipped to target a wide variety of diseases of the visual system. For the present project the team focuses on degenerative retinal diseases like Retinitis Pigmentosa (RP), as will be shown later, therapeutical strategies for such diseases depend strongly on knowledge of specific retinal circuitry. In the project we will identify molecular and cellular disease mechanism, cellular targets, and will demonstrate restoration of vision from single neuronal responses in the retina all the way to cortical visual evoked responses in mice and behavioral assays in mice and zebrafish.
Concept
The retina is our most complex sensory organ. Blindness in man often results from dysfunction of this complex neural network. Some retinal diseases depend only on malfunctioning of photoreceptors. However, for a number of retinal diseases, the retinal circuitry is a major part of the problem. For effective strategies for treatment of those diseases and for all attempts to restore vision, understanding of the retinal circuitries is essential.
Therefore, the core of our strategy is that understanding the structure and function of specific retinal circuits should be used to find rational therapies to retinal diseases. We focused our efforts on specific circuits in the retina where system level description from basic science can be directly used to translational studies. Specific circuits can be only studied with the aid of cell type specific promoters and therefore the methodology that enables us to follow the above described approach is the combination of in vivo marked cell types with genetic interference with the function of these cells and state-of-the-art physiological and behavioural approaches.
The way we built our team reflects our strategy: First, we have basic science teams that developed animals where specific retinal cell types of specific circuits are marked or modified. Second, we have teams that are experts in optical and physiological recordings as well as behavioural readouts which will analyze these animals. Third, we have teams that are experts in studying the retinal disease mechanisms in these animals.
Translation
To develop effective treatments based on animal research, one needs to translate the results from animal studies to the human condition. Although the general organization of the retina is highly conserved though all vertebrates (Figure 2), some differences occur between species. Studying only one experimental animal imposes the risk that one studies such a specific feature of that particular retina instead of a general feature that is likely to exist also in man. To overcome this risk we have chosen to use both zebrafish and mouse as animal models. Both are well established genetic models and both are used in retinal research. Clinically used diagnostic systems are available in both animal models (ERG, VEP and behavioural readouts). By using the results of both animal systems, we will be able to study general mechanism in retinal circuitry, generate fundamental knowledge about general principles of retinal neural processing and increase the chance for successful translation to the human condition. The second reason to use two animal systems is that some experiments can be performed better in zebrafish and some better in mouse. For instance: outer retina is better approached in fish whereas for the mouse a wide variety of genetic tools are available. Furthermore, the advantage of the zebrafish is the possibility to generate numerous animal generations with mutated genes in a relatively short period whereas a large number of different disease models are available for mouse
Objectives
The retina is a complex multilayered neural network that transforms light into electrical signals usable for the brain. In the outer retina, photoreceptors project to bipolar cells (BC) and horizontal cells (HC) whereas in the inner retina BCs project to amacrine cells (AC) and ganglion cells (GC) (Figure 3) The pathway formed by photoreceptors, BC and GCs is the so-called “through pathway”. HCs and ACs form two layers of lateral interactions. The general understanding is that lateral interactions form the basis for the centre-surround organization of the receptive field of many neurons in the visual system (For a review see: Chalupa and Werner, 2003). However, recent evidence suggests much more sophisticated functions of these lateral pathways as well (Kamermans et al., 1998; VanLeeuwen et al., 2007). The lateral interactions in the outer retina seems to function as an adaptation and general gain control mechanisms whereas the lateral interactions in the inner retina are involved in different kinds of feature extraction (Roska et al., 2000; Roska and Werblin, 2001; Fried et al., 2005; Roska et al., 2006). This notion is further strengthened by the fact that, although only 5 general classes of neurons are present in the retina, the number of specialized subtypes is enormous. Instead of having a through pathway and two lateral inhibitory pathways, the retina consists of many parallel pathways and a variety of lateral circuits with highly specialized tasks (Figure 4).
The aim of this project is to develop circuit specific approaches to retinal diseases. We will focus on the retinal disease, RP, for which effective treatment depends on detailed knowledge of some of these specialized networks, and which is a major causes of blindness in man. In this integrated project we will be able to make major progress in preventing degeneration of cones and develop strategies to restore vision.
The program has 3 key objectives addressed in 3 work packages (WP2 - 4)
• WP2: Retinal circuit analysis
• WP3: The bystander effect and photoreceptor degeneration
• WP4: Rescuing retinal function using cell specific expression of light sensitive proteins
The core of our strategy is that understanding the structure and function of specific retinal circuits enables us to find rational therapies to retinal diseases. The team has access to cell specific promoters for all retinal neurons to be studied in this project. We will, for the first time, be able to analyze and interfere specifically with one cell class while leaving the other intact and study under these conditions the role of those neurons in disease mechanisms.
WP02 “Retinal circuit analysis”
The objective of WP02 was to generate basic morphological, physiological and system biological knowledge needed for the translational, disease related work in WP03 and WP04. The pathways we focused on in this WP are outer retinal integration and gain control. In many retinal degenerative diseases, photoreceptors degenerate while the rest of the retinal network remains relatively intact. We argued that when we would be able to make either photoreceptors or bipolar cells light sensitive using optogenetical techniques, we might restore vision in patients suffering from those diseases. In such a strategy, although light sensitivity might be restored, part of the retinal processing is lost. To restore vision, one needs to restore that processing capability. Stimulating optogenetically reactivated cones will not generate physiological responses since the adaptation processes in cones are lost and the kinetic response of the artificial photopigment differs significantly from the response of the phototransduction cascade. For reactivated bipolar cells the problem is even larger, since all outer retinal processing has been lost. To compensate for these losses we envision that after optogenetic treatment patient will have to use a optical stimulator. This stimulator converts images into stimulus patters adapted such that cones and bipolar cells respond with their normal response kinetics. Such a stimulus device will incorporate a model of the photoreceptors and the outer retina. WP02 aims at obtaining a quantitative description of the process in the outer retina and formulating an outer retinal model. To test such a model one needs fundamental knowledge of the many retinal sub-circuits in the inner retina. Therefore we aimed at describing the properties of specific ganglion cells.
WP03 “Photoreceptor degeneration, the bystander hypothesis”
In order to develop strategies for the prevention of degeneration in retinitis pigmentosa (RP), WP03 focused on elucidation of functions of the first cellular component in the outer retinal circuits, the photoreceptors. The degeneration of photoreceptors in RP comprises two stages: 1. The degeneration of the rod photoreceptors resulting in night blindness and 2. The degeneration of cone photoreceptors, which is the more disabling stage as the human vision is mainly based on cone vision. The analyses performed concentrated on understanding the mechanisms mediating the spread of the disease from rod to cone photoreceptors, to gain information for therapeutic strategies. A state of the art hypothesis, the bystander hypothesis, proposes that cones die in retinitis pigmentosa as a result of signals which are transported from dying rods to cones via gap junctions (Ripps, 2002; Hamel, 2007). These gap junctions are most likely composed of hexameric complexes of connexins and the physiological function under healthy conditions is that rod signals are transported piggyback in the cone pathway under light conditions where the cones are inactive (Schneeweis and Schnapf, 1995; Hornstein et al., 2005). In mice, the coupling complex is composed of Cx36 on the cone side and rod-cone coupling is disturbed in Cx36 deficient mice (Feigenspan et al., 2004; Trumpler et al., 2008). WP03 targeted the question, to which extend rod-cone coupling is involved in cone degeneration in retinitis pigmentosa. As a prerequisite for pharmacological interventions, the rod component of the coupling complex was investigated and the physiological properties of the putative candidates pannexin2 and pannexin1 were analyzed.
To access the impact of rod-cone coupling, two different mice strains, which are established models for retinitis pigmentosa with different progression of the disease, were analyzed. The cross breeding with a mice strain with a genetic modification that leads to a disruption of rod-cone coupling, allowed the detailed investigation of the influence of coupling on the onset as well as on the progression of cone degeneration. The analyses revealed no differences in time-dependent process of cone degeneration when rod-cone coupling is disrupted, neither in the fast degenerating rd1 nor the slowly degenerating rho-/- strain. We conclude, that the bystander hypothesis describes a minor effect in retinitis pigmentosa and thus, pharmacological modification of rod-cone coupling would be no reasonable and effective strategy to interfere with the progress of cone degeneration (K. Kranz et al., 2012, J.Neurosci. in prep). Despite the use of different state-of-the-art techniques and approaches the component of the coupling complex on rod side has not yet been identified. However, we were successful in the exclusion of a set of the 20 murine connexins as possible rod connexin candidates, whereas Cx45 and Cx30.2 are still under investigation as candidates (Bolte et al., 2012, Mol Vision, in prep). Pannexin2 and Pannexin1, two proteins which share the mayor structural domains of the connexins were excluded as a part of the rod-cone coupling complex. For the first time, the distribution of these proteins in the outer retina was studied in detail. Physiological experiments in heterologous expression systems enlightened, that Pannexin2 does not form hemichannels with the necessary voltage gated functions (Bolte et al., 2012, J Comp Neurol, in prep). The physiology of pannexin1 was studied in wild-type and Panx1 knock-out mice and showed, that this channel functions as a negative regulator in retinal circuits. We postulate a physiological role in the rod-pathway or in the feedback from horizontal cells to photoreceptors. (Kranz et al., 2012, J Comp Neurol, in prep).
WP04 “Rescuing retinal function using cell specific expression of light sensitive proteins”
The goal of WP4 is to develop the scientific background for restoring vision in retinitis pigmentosa, a blinding disease in which photoreceptor function is lost but much of the retinal circuits remain, using cell-type-targeted light-activated channels or pumps. Our initial goal was to target channelrhodopsin-2, a light-activated channel to ON-bipolar cells, in mouse and zebrafish in models of retinitis pigmentosa as well as to wild type mice. This approach would recover visual function to the ON retinal channels in models of retinitis pigmentosa, leaving the OFF channels non-functional. A surprising discovery that we could reactivate both the ON, OFF as well as ON-OFF retinal channels with targeting halorhodopsins, a light activated pump, to surviving cone cell bodies in mouse models of retinitis pigmentosa changed our plans. Therefore we switched to a more ambitious goal namely to reactive both ON, OFF and ON-OFF channels in mice, to develop viral vectors that can be used in humans and finally to test these vectors in ex vivo human retinal preparation as the first step towards translation.
Due to the discovery mentioned above we modified two of our original tasks to be more appropriate for human translation. Firstly, we decided to concentrate on human retina models as opposed to zebrafish models due to its potential for translation to the clinic. Secondly, we concentrated on adeno-associated viral (AAV) delivery instead of transgenic mice since AAVs can be potentially used in the clinic. However, we also developed transgenic mouse models in order to test for potential toxicity. All the other tasks remained the same as proposed except with the new approach: halorhodopsins targeting to remaining cones.

Project Results:
WP02 “Retinal circuit analysis”
The objective of WP02 was to generate basic morphological, physiological and system biological knowledge needed for the translational, disease related work in WP03 and WP04. The pathways we focused on in this WP are outer retinal integration and gain control. In many retinal degenerative diseases, photoreceptors degenerate while the rest of the retinal network remains relatively intact. We argued that when we would be able to make either photoreceptors or bipolar cells light sensitive using optogenetical techniques, we might restore vision in patients suffering from those diseases. In such a strategy, although light sensitivity might be restored, part of the retinal processing is lost. To restore vision, one needs to restore that processing capability. Stimulating optogenetically reactivated cones will not generate physiological responses since the adaptation processes in cones are lost and the kinetic response of the artificial photopigment differs significantly from the response of the phototransduction cascade. For reactivated bipolar cells the problem is even larger, since all outer retinal processing has been lost. To compensate for these losses we envision that after optogenetic treatment patient will have to use a optical stimulator. This stimulator converts images into stimulus patters adapted such that cones and bipolar cells respond with their normal response kinetics. Such a stimulus device will incorporate a model of the photoreceptors and the outer retina. WP02 aims at obtaining a quantitative description of the process in the outer retina and formulating an outer retinal model. To test such a model one needs fundamental knowledge of the many retinal sub-circuits in the inner retina. Therefore we aimed at describing the properties of specific ganglion cells.
WP03 “Photoreceptor degeneration, the bystander hypothesis”
In order to develop strategies for the prevention of degeneration in retinitis pigmentosa (RP), WP03 focused on elucidation of functions of the first cellular component in the outer retinal circuits, the photoreceptors. The degeneration of photoreceptors in RP comprises two stages: 1. The degeneration of the rod photoreceptors resulting in night blindness and 2. The degeneration of cone photoreceptors, which is the more disabling stage as the human vision is mainly based on cone vision. The analyses performed concentrated on understanding the mechanisms mediating the spread of the disease from rod to cone photoreceptors, to gain information for therapeutic strategies. A state of the art hypothesis, the bystander hypothesis, proposes that cones die in retinitis pigmentosa as a result of signals which are transported from dying rods to cones via gap junctions (Ripps, 2002; Hamel, 2007). These gap junctions are most likely composed of hexameric complexes of connexins and the physiological function under healthy conditions is that rod signals are transported piggyback in the cone pathway under light conditions where the cones are inactive (Schneeweis and Schnapf, 1995; Hornstein et al., 2005). In mice, the coupling complex is composed of Cx36 on the cone side and rod-cone coupling is disturbed in Cx36 deficient mice (Feigenspan et al., 2004; Trumpler et al., 2008). WP03 targeted the question, to which extend rod-cone coupling is involved in cone degeneration in retinitis pigmentosa. As a prerequisite for pharmacological interventions, the rod component of the coupling complex was investigated and the physiological properties of the putative candidates pannexin2 and pannexin1 were analyzed.
To access the impact of rod-cone coupling, two different mice strains, which are established models for retinitis pigmentosa with different progression of the disease, were analyzed. The cross breeding with a mice strain with a genetic modification that leads to a disruption of rod-cone coupling, allowed the detailed investigation of the influence of coupling on the onset as well as on the progression of cone degeneration. The analyses revealed no differences in time-dependent process of cone degeneration when rod-cone coupling is disrupted, neither in the fast degenerating rd1 nor the slowly degenerating rho-/- strain. We conclude, that the bystander hypothesis describes a minor effect in retinitis pigmentosa and thus, pharmacological modification of rod-cone coupling would be no reasonable and effective strategy to interfere with the progress of cone degeneration (K. Kranz et al., 2012, J.Neurosci. in prep). Despite the use of different state-of-the-art techniques and approaches the component of the coupling complex on rod side has not yet been identified. However, we were successful in the exclusion of a set of the 20 murine connexins as possible rod connexin candidates, whereas Cx45 and Cx30.2 are still under investigation as candidates (Bolte et al., 2012, Mol Vision, in prep). Pannexin2 and Pannexin1, two proteins which share the mayor structural domains of the connexins were excluded as a part of the rod-cone coupling complex. For the first time, the distribution of these proteins in the outer retina was studied in detail. Physiological experiments in heterologous expression systems enlightened, that Pannexin2 does not form hemichannels with the necessary voltage gated functions (Bolte et al., 2012, J Comp Neurol, in prep). The physiology of pannexin1 was studied in wild-type and Panx1 knock-out mice and showed, that this channel functions as a negative regulator in retinal circuits. We postulate a physiological role in the rod-pathway or in the feedback from horizontal cells to photoreceptors. (Kranz et al., 2012, J Comp Neurol, in prep).
WP04 “Rescuing retinal function using cell specific expression of light sensitive proteins”
The goal of WP4 is to develop the scientific background for restoring vision in retinitis pigmentosa, a blinding disease in which photoreceptor function is lost but much of the retinal circuits remain, using cell-type-targeted light-activated channels or pumps. Our initial goal was to target channelrhodopsin-2, a light-activated channel to ON-bipolar cells, in mouse and zebrafish in models of retinitis pigmentosa as well as to wild type mice. This approach would recover visual function to the ON retinal channels in models of retinitis pigmentosa, leaving the OFF channels non-functional. A surprising discovery that we could reactivate both the ON, OFF as well as ON-OFF retinal channels with targeting halorhodopsins, a light activated pump, to surviving cone cell bodies in mouse models of retinitis pigmentosa changed our plans. Therefore we switched to a more ambitious goal namely to reactive both ON, OFF and ON-OFF channels in mice, to develop viral vectors that can be used in humans and finally to test these vectors in ex vivo human retinal preparation as the first step towards translation.
Due to the discovery mentioned above we modified two of our original tasks to be more appropriate for human translation. Firstly, we decided to concentrate on human retina models as opposed to zebrafish models due to its potential for translation to the clinic. Secondly, we concentrated on adeno-associated viral (AAV) delivery instead of transgenic mice since AAVs can be potentially used in the clinic. However, we also developed transgenic mouse models in order to test for potential toxicity. All the other tasks remained the same as proposed except with the new approach: halorhodopsins targeting to remaining cones.

Scientific results/foreground
WP02 “Retinal Circuit Analysis”
WP02 made great progress in the development of a quantitative description of the outer retinal processing. The various achievements are summarized below.
1. Cone responses under naturalistic stimulus conditions
Visual information in natural scenes is distributed over a broad range of intensities and contrasts. This distribution has to be compressed in the retina to match the dynamic range of retinal neurons. In this study we examined how cones perform this compression and investigated which physiological processes contribute to this operation. M- and L-cones were stimulated with a natural time series of intensities (NTSI) and their responses were recorded. The NTSI displays an intensity distribution which is skewed towards the lower intensities and has a long tail into the high intensity region. Cones transform this skewed distribution into a more symmetrical one by compressing the tail in the high intensity range. The voltage responses of the cones were compared to those of a linear filter and a non-linear biophysical model of the photoreceptor. The results show that the linear filter under-represents contrasts at low intensities compared to the actual cone whereas the non-linear biophysical model performs well over the whole intensity range used. Quantitative analysis of the two approaches indicates that the non-linear biophysical model can capture 91±5% of the coherence rate (a biased measure of information rate) of the actual cone, where the linear filter only reaches 48±8%. These results demonstrate that cone photoreceptors transform an NTSI in a non-linear fashion. The comparison between current clamp and voltage clamp recordings and analysis of the behavior of the biophysical model indicates that both the calcium feedback loop in the outer segment and the hydrolysis of cGMP are the major components that introduce the specific non-linear response properties found in the goldfish cones. Endeman & Kamermans (2010) J. Physiol. 588(3):435-446.
2. Contrast adaptation
In natural scenes light intensity and contrast are statistically independent. The visual system seems to utilize this phenomenon as adaptive mechanisms for luminance and contrast operate independently in many classes of visual neurons. Retinal luminance adaption begins at the photoreceptor level whereas retinal contrast adaption is believed to start at later stages of processing as cones have been reported not to adapt to contrast. However, we find that this might not be entirely the case. Light responses of cone photoreceptors from isolated retina were recorded by whole cell voltage and current clamp techniques. Both naturalistic chromatic time series of intensities and monochromatic artificial time series of intensities were used. Different contrast levels were generated by using different modulation depths with equal mean luminance. Transfer functions were derived. When analysing the overall response amplitudes of cones in voltage clamp conditions the shape of the transfer function does not change. However, in current clamp cones attenuated higher frequency components of the lower contrast stimuli to a greater extent than they did for the higher contrast stimuli indicating that cones shift their bandwidth depending on the contrast of the stimulus. In conclusion: Cones adapt to different contrast conditions not by scaling their response amplitudes but by altering their frequency bandwidth. The difference between current and voltage clamp recording indicates that this process is mediated by cone membrane properties and not the phototransduction cascade. Such an adaptive filtering system is consistent with van Hateren’s (1992) proposed adaptive filter employed to maximise sensory information in different signal to noise ratio conditions. Howlett & Kamermans (in prep).
3. Cone/HC network under naturalistic stimulus conditions
Natural images are statistically redundant; they display strong correlations in space, time and wavelength. Much of the processing in the early stages of visual processing, in particular those in the retina, is concerned with reducing these correlations. Its resources (number of neurons, dynamic range of neurons, synaptic contacts per neuron, etc.) here fore are limited, thus requires the retina to efficiently use those resources. A way to use neural resources efficiently is to remove redundant information across a population of neurons by making the responses of retinal neurons statistically independent, i.e. any particular piece of information is not duplicated in several neurons. A classical example in which this seems to happen concerns the coding of chromatic information with opponent color mechanisms.
The spectral sensitivity of the L, M and S cones overlap substantially, creating highly correlated, statistically dependent, responses. Efficient coding can occur by transforming the cone responses so that they are statistically independent. Buchsbaum and Gottschalk (1983) demonstrated mathematically that such a transformation can be established by color opponent mechanisms. This transformation might be carried out by retinal horizontal cells, which at least in some species display color opponency. Goldfish possess three types of cone dominated HCs, of which two are color opponent. We experimentally tested if responses to chromatic stimuli become statistically more independent from cones to horizontal cells and thereby improve coding efficiency. Here fore we analyzed responses of cones and horizontal cells of the goldfish, which were stimulated with a naturalistic chromatic timeseries. It was found that the separation in chromatic and luminosity channels was far from complete. Both monophasic and biphasic horizontal cells responded to luminosity and chromatic stimuli and were dominated by luminocity information. Endeman et al., (in prep)
4. Gain control at the photoreceptor synapse
The retina can function under a variety of adaptation conditions and stimulus paradigms. To adapt to these various conditions, modifications in the phototransduction cascade and at the synaptic and network levels occur. We focused on the properties and function of a gain control mechanism in the cone synapse. We show that horizontal cells, in addition to inhibiting cones via a “lateral inhibitory pathway,” also modulate the synaptic gain of the photoreceptor via a “lateral gain control mechanism.” The combination of lateral inhibition and lateral gain control generates a highly efficient transformation. Horizontal cells estimate the mean activity of cones. This mean activity is subtracted from the actual activity of the center cone and amplified by the lateral gain modulation system, ensuring that the deviation of the activity of a cone from the mean activity of the surrounding cones is transmitted to the inner retina with high fidelity. Sustained surround illumination leads to an enhancement of the responses of transient ON/OFF ganglion cells to a flickering center spot. Blocking feedback from horizontal cells not only blocks the lateral gain control mechanism in the outer retina, but it also blocks the surround enhancement in transient ON/OFF ganglion cells. This suggests that the effects of the outer retinal lateral gain control mechanism are visible in the responses of ganglion cells. Functionally speaking, this result illustrates that horizontal cells are not purely inhibitory neurons but have a role in response enhancement as well. Van Leeuwen et al., 2009 J Neurosci 29(19):6358-6366
5. Ephaptic feedback
In the vertebrate retina, horizontal cells generate the inhibitory surround of bipolar cells, an essential step in contrast enhancement. For the last decades, the mechanism involved in this inhibitory synaptic pathway has been a major controversy in retinal research. One hypothesis suggests that connexin hemichannels mediate this negative feedback signal; another suggests that feedback is mediated by protons. Mutant zebrafish were generated that lack connexin 55.5 hemichannels in horizontal cells. Whole cell voltage clamp recordings were made from isolated horizontal cells and cones in flat mount retinas. Light-induced feedback from horizontal cells to cones was reduced in mutants. A reduction of feedback was also found when horizontal cells were pharmacologically hyperpolarized but was absent when they were pharmacologically depolarized. Hemichannel currents in isolated horizontal cells showed a similar behavior. The hyperpolarization-induced hemichannel current was strongly reduced in the mutants while the depolarization-induced hemichannel current was not. Intracellular recordings were made from horizontal cells. Consistent with impaired feedback in the mutant, spectral opponent responses in horizontal cells were diminished in these animals. A behavioral assay revealed a lower contrast-sensitivity, illustrating the role of the horizontal cell to cone feedback pathway in contrast enhancement. Model simulations showed that the observed modifications of feedback can be accounted for by an ephaptic mechanism. A model for feedback, in which the number of connexin hemichannels is reduced to about 40%, fully predicts the specific asymmetric modification of feedback. To our knowledge, this is the first successful genetic interference in the feedback pathway from horizontal cells to cones. It provides direct evidence for an unconventional role of connexin hemichannels in the inhibitory synapse between horizontal cells and cones. This is an important step in resolving a longstanding debate about the unusual form of (ephaptic) synaptic transmission between horizontal cells and cones in the vertebrate retina. Klaassen et al., 2011 Plos Biology 9(7): e1001107; Klaassen, Fahrenfort & Kamermans, Brain Res. (Submitted).
6. Modulation of the cone synapse
6.1. The role of the Ca-dependent Cl-current
In neuronal systems, excitation and inhibition must be well balanced to ensure reliable information transfer. The cone/horizontal cell interaction in the retina is an example of this. Because natural scenes encompass an enormous intensity range both in the temporal and the spatial domains, the balance between excitation and inhibition in the outer retina needs to be adaptable. How this is regulated is unknown. In this paper, it is shown that GABA-gated and Ca2+-dependent Cl--currents in cones modulate the strength of negative feedback from horizontal cells to cones. Using electrophysiological techniques in the isolated retina of the goldfish it was found that application of GABA reduced the size of light-induced feedback responses both in cones and horizontal cells. Conversely application of picrotoxin, a blocker of GABAA and GABAC receptors, had the opposite effect. In addition, blocking GABA release from horizontal cells by blocking GABA-transporters also led to an increase in the size of feedback. To test whether the GABA-ergic-modulation of feedback was solely mediated by activation of a Cl--conductance in cones, the effect of another Cl--current in cones on feedback was studied. Opening Ca2+-dependent Cl--channels in recorded cones reduced the size of feedback in cones similar as GABA application. Since the independent manipulation of Ca2+-dependent Cl--currents in individual cones yielded results comparable to bath-applied GABA, it was concluded that activation of either Cl--current by itself is sufficient to reduce the size of feedback. The results can fully be accounted for by an ephaptic feedback model in which a Cl--current shunts the current flow in the synaptic cleft. Modulation of the feedback strength by GABA might play a role during light/dark adaptation, whereas the Ca2+-dependent Cl--current could function to alter the feedback strength during high contrast stimuli and might contribute to afterimage formation. Endeman, Fahrenfort & Kamermans (submitted to J Physiol)
6.2. Properties of Panx1 channels
In the retina, chemical and electrical synapses couple neurons into functional networks. New candidates encoding for electrical synapse proteins have recently emerged. In the present study, we determined the localization of the candidate protein pannexin1 (zfPanx1) in the zebrafish retina and studied the functional properties of zfPanx1 exogenously expressed in N2a cells. ZfPanx1 was identified on the surface of horizontal cell dendrites invaginating deeply into the cone pedicle near the glutamate release sites of the cones, providing in vivo evidence for hemichannel formation at that location. This strategic position of zfPanx1 in the photoreceptor synapse could potentially allow modulation of cone output. Using whole cell voltage clamp and excised patch recordings of transfected N2a cells, we demonstrated that zfPanx1 forms voltage activated hemichannels with a large unitary conductance in vitro. These channels can open at physiological membrane potentials. Functional channels were not formed following mutation of a single amino acid within a conserved protein motif recently shown to be N-glycosylated in rodent Panx1. Together, these findings indicate that zfPanx1 displays properties similar to its mammalian homologues and can potentially play an important role in functions of the outer retina. Nora Prochnow et al., (2009) Neurosci 162(4):1039-1054
6.3. Spillover feedback
We have provided the final proof that negative feedback from horizontal cells to cones leads to the modulation of the Ca-current of cones via an ephaptic feedback mechanism (Verweij et al., 1996; Kamermans et al., 2001; Fahrenfort et al., 2009; Klaassen et al., 2011; Klaassen et al., 2012 submitted)). These experiments were performed in retinas where part of the rods was removed in order to obtain better access to the cones. Recently we changed our procedure such that we could record from cones without removing rods. By doing so, we discovered a novel component of negative feedback. The first component is a sustained shift of the Ca-current to negative potentials. This component does not induce any change in current at potentials below -60 mV. The second component is a transient increase in conductance at negative potentials. It becomes larger the more negative the holding potential is.
We found that surrounding cones receiving a feedback signal from horizontal cells that leads to the increase of their glutamate release. This increased glutamate release activates a glutamate transporter (EAAT) in the central cone which is associated with a transient increase in Cl-conductance. In addition to this, the increased glutamate release leads to an increase in glutamate conductance in the horizontal cell dendrites, inducing an influx of Ca2+ and an opening of Panx1 hemichannels. ATP released via these hemichannels activates either a purinergic or adenosine receptor on cones which leads to an influx of Ca2+ in the cones. This Ca2+-influx leads to an increase in glutamate release.
Such a feedback system has positive and negative components. The spillover feedback from surrounding cones leads to inhibition of feedback. The opening of Panx1 hemichannels leads to an increase in negative feedback whereas the activation of the purinergic or adenosine pathway also leads to an increase in negative feedback. All these mechanisms together ensure a stable but highly potent feedback mechanism. Vroman & Kamermans (in prep). In addition to this functional work, the identity of the glutamate transporters in zebrafish has been identified by cloning the EAATs of zebrafish. A paper describing this work is in preparation. Neuhauss and co-workers (in prep).
7. Coupling of horizontal cells and modulation
Horizontal cells of the same subtype form autonomous networks, and we suppose that the gap junctions involved in connecting these independent circuits are composed of different connexins. Four horizontal cell-specific connexins, termed zfCx52.6 zfCx55.5 zfCx52.7 and zfCx52.9 have been identified in the zebrafish retina (Zoidl et al., 2004; Shields et al., 2007; Klaassen et al., 2011). In addition we identified and cloned a new connexin isoform, designated zebrafish Cx53.4 (zfCx53.4). This connexin represents a splice isoform of zfCx52.6. zfCx53.4 is expressed in all subtypes of horizontal cells, in which it might act as a component of dendro-dendritic and axo-axonal gap junctions. No staining for zfCx53.4 was observed within the cone pedicles, suggesting that zfCx53.4 does not form hemichannels within the ribbon synaptic complex. Janssen-Bienhold and co-workers (in prep).
Horizontal cell coupling changes during/light dark adaptation. We gained major insight into the identification of the signal transduction pathways, which underlie plasticity. The Cx coupling horizontal cells has been identified as a phosphoprotein, the phosphorylation of which is differentially regulated by ambient light conditions: While light adaptation results in a highly phosphorylated status of the Cx coupling horizontal cells, the protein reveals sparse phosphorylation in the dark. But, both phosphorylation levels are clearly different from the fully dephosphorylated Cx. The effect of light can be mimicked by incubation of dark-adapted retinae with Dopamine (DA) and cAMP, respectively. The DA- induced phosphorylation is mediated by the D1-receptor/adenylate cyclase/cAMP/PKA signal transduction cascade, as it can be inhibited by the D1R-antagonist SCH23390 and specific PKA-inhibitors. The phosphorylation correlates with an increased integration of the Cx into the plasma membrane of N2a cells and with an increase of the Cx immunoreactivity into the membranes of horizontal cells in the OPL of the fish retina. Our results indicate that already the integration of Cxs into the plasma membrane and the assembly of hemichannels require phosphorylation of Cxs. Janssen-Bienhold and co-workers (in prep).
8. Horizontal cell ablation
To assess the importance of horizontal cells for network formation in the outer retina and proper retinal function, we generated a mouse model in which horizontal cells could be eliminated from the fully developed retina. Following horizontal cell ablation, an extensive remodeling of the outer retinal contacts occurs: The outer plexiform layer (OPL) starts thinning two weeks after horizontal cells have been ablated, and progressive degeneration of synaptic contacts leads to significant thinning of the OPL within six month. Concomitant, photoreceptor degeneration becomes obvious, as revealed by a 30% reduction of nuclei in the outer nuclear layer (ONL) and the loss of the columnar structure. Rods retract their axons from the OPL and partially degenerate. Remaining rod spherules are localized in close contact to cone pedicles. The number of cones is unaffected by ablation of horizontal cells, but cone pedicles appear atrophic and their arrangement is changed to complex clusters in the remaining OPL. The synaptic contacts of cones are markedly changed: all invaginating synapses are missing completely six month after horizontal cell ablation and bipolar cells make multiple flat contacts near the synaptic ribbons. Some OFF bipolar cells show cell-type specific restructuring in response to ablation of horizontal cells: while Type IIIa cells remain their contacts to cones, Type IIIb cells show more pronounced sprouting of dendrites into the ONL. ON bipolar cells lose their invaginating contacts with cone pedicles and rod spherules; instead, they extend their dendrites into the ONL and form ectopic synapses within the ONL to compensate for lost contacts in the OPL. Rod bipolar cells sprout extensively into the ONL, with dendrites extending up to the outer limting membrane. In contrast to these strong effects of horizontal cell ablation on the photoreceptor to bipolar cell synapse, multi-electrode recordings from ganglion cells of isolated retinae showed only moderate effects on ganglion cell responses: whereas the firing pattern of OFF ganglion cells did not differ at all, ON ganglion cells were slightly slower, and higher light intensities were needed to evoke the half-maximal response in ON ganglion cells. In line with the mild effect of horizontal cell ablation on the output of the retinal network, visual performance (visual acuity and contrast sensitivity) is also only slightly affected. Thus, we conclude that the adult retina has a distinct capacity for neuronal plasticity (remodeling and ectopic synapse formation), which serves to maintain visual function even when a complete neuronal network has been eliminated from the retina. Janssen-Bienhold and co-workers (in prep)
9. ON-bipolar cell signaling
The signal of the rod and cones is transmitted to two types of bipolar cells: ON-bipolar cells and OFF-bipolar cells The ON-bipolar cells depolarize due to light stimulation whereas the OFF-bipolar cells hyperpolarize to light stimulation. The response sign in the ON-bipolar cells is determined by a metabotropic glutamate receptor signaling cascade. Since the ON-bipolar cells are, together with the cones, the prime target for optogenetic restoration of vision, one needs to get insight in the properties of this signaling cascade. Therefor we focused on the type of 1) glutamate receptor, 2) the ion channel driven by the signaling cascade and an newly discovered G-protein coupled receptor active in the signaling cascade in the ON-bipolar cell dendrite.
9.1. The mGluR6 family in zebrafish
In order to assess the contribution of metabotropic glutamate signalling to the visual ON-response in the outer retina we cloned all mGluR receptors, performed a phylogenetic and expression analysis (Haug et al., 2012). Of particular interest are the mGluR6 family members, since they have been implicated since a number of years in the mammalian ON-response. We identified two paralogs in the zebrafish, with only mGluR6b being expressed in appreciable amounts in larval bipolar cells. Downregualtion by morpholino antisense technology led to a partial decrease in the ERG b-wave, confirming a function in the ON pathway. Interestingly, both paralogs are expressed at all stages at higher levels in ganglion cells, including a number of know downstream activated genes (e.g. GalphoO). Preliminary results with paralog specific antibodies confirm the results from RNA localization (Huang et al., 2012).
9.2. TrpM1 channels
Congenital stationary night blindness (CSNB) is a clinically and genetically heterogeneous group of retinal disorders characterized by nonprogressive impaired night vision and variable decreased visual acuity. We report here that six out of eight female probands with autosomal-recessive complete CSNB (cCSNB) had mutations in TRPM1, a retinal transient receptor potential (TRP) cation channel gene. These data suggest that TRMP1 mutations are a major cause of autosomal-recessive CSNB in individuals of European ancestry. We localized TRPM1 in human retina to the ON bipolar cell dendrites in the outer plexifom layer. Our results suggest that in humans, TRPM1 is the channel gated by the mGluR6 (GRM6) signaling cascade, which results in the light-evoked response of ON bipolar cells. Finally, we showed that detailed electroretinography is an effective way to discriminate among patients with mutations in either TRPM1 or GRM6, another autosomal-recessive cCSNB disease gene. These results add to the growing importance of the diverse group of TRP channels in human disease and also provide new insights into retinal circuitry. Van Genderen (2009) Am J Human Gen 85(5):730-736
9.3. Gpr179 receptor
We found here that mutations in GPR179, encoding an orphan G protein receptor, underlie a form of autosomal-recessive cCSNB. The Gpr179nob5/nob5 mouse model was initially discovered by the absence of the ERG b-wave, a component that reflects depolarizing bipolar cell (DBC) function. We performed genetic mapping, followed by next-generation sequencing of the critical region and detected a large transposon-like DNA insertion in Gpr179. The involvement of GPR179 in DBC function was confirmed in zebrafish and humans. Functional knockdown of gpr179 in zebrafish led to a marked reduction in the amplitude of the ERG b-wave. Candidate gene analysis of GPR179 in DNA extracted from patients with cCSNB identified GPR179-inactivating mutations in two patients. We developed an antibody against mouse GPR179, which robustly labeled DBC dendritic terminals in wild-type mice. This labeling colocalized with the expression of GRM6 and was absent in Gpr179nob5/nob5 mutant mice. Our results demonstrate that GPR179 plays a critical role in DBC signal transduction and expands our understanding of the mechanisms that mediate normal rod vision. Peachy et al., (2012) Am J Human Gen 90, 331–339; Peachy et al., (2012) Am J Human Gen (submitted)
10. Outer retinal model
The release of glutamate by the cone ribbon synapse is modulated by horizontal cells through negative feedback, which may comprise several mechanisms. In the ephaptic hypothesis, activation of calcium channels in the cone terminal is shifted by the extracellular voltage gradient within the invagination at the cone ribbon synapse. The voltage gradient is controlled by the current flowing into the horizontal cell dendritic tips through AMPA and hemi-Cx channels. It also depends on the resistance of the extracellular space and the input resistance of horizontal cells. Although several important parameters have been measured that support this ephaptic mechanism, exactly how each parameter contributes to the ephaptic negative feedback in a dynamic system is unknown. We developed a dynamic computational model of ephaptic feedback for an array of cones converging onto horizontal cells. The model included the membrane potential of cones and the horizontal cells, currents in extracellular space (~1000 MOhm), realistic L-type calcium and calcium-activated chloride channels in the cone terminal, AMPA and hemi-Cx channels in the horizontal cell dendritic tips, and a Kir channel in the horizontal cell soma. We calibrated the model to produce realistic synaptic release rates (cones: ~100ves/s, dark), and horizontal cell voltages (Hz cells: -35 mV/dark, -80 mv/full-field bright light). We then tested different combinations of the synaptic and membrane conductances for their effect on feedback. When a central cone in the model was clamped at a typical dark resting potential (-40 mV), and the surrounding cones were given a short pulse to -55mV to simulate a bright flash, the horizontal cell hyperpolarized, increasing the central cone's release rate (~50%), indicating negative feedback. The light response activated the horizontal cell's Kir channels, further hyperpolarizing it, and increasing the current through the AMPA and hemi-Cx channels. This in turn generated a more negative external voltage at the cone membrane, which after a short delay increased the cone's calcium. The Kir channel turns out to be very important since it forms the exit route for the current entering the horizontal cell via the hemi-Cx channels. We have constructed a realistic dynamic model of ephaptic feedback which may help to provide intuition about the function of this circuit. The model predicts that the role of inwardly rectifying potassium channels in the horizontal cell is to facilitate negative feedback to the cone. Smith & Kamermans, (in prep)
11. Approach sensitive ganglion cells
The detection of approaching objects, such as looming predators, is necessary for survival. Which neurons and circuits mediate this function? We combined genetic labeling of cell types, two-photon microscopy, electrophysiology and theoretical modeling to address this question. We identify an approach-sensitive ganglion cell type in the mouse retina, resolve elements of its afferent neural circuit, and describe how these confer approach sensitivity on the ganglion cell. The circuit’s essential building block is a rapid inhibitory pathway: it selectively suppresses responses to non-approaching objects. This rapid inhibitory pathway, which includes AII amacrine cells connected to bipolar cells through electrical synapses, was previously described in the context of night-time vision. In the daytime conditions of our experiments, the same pathway conveys signals in the reverse direction. The dual use of a neural pathway in different physiological conditions illustrates the efficiency with which several functions can be accommodated in a single circuit. Münch et al., 2002, Nat Neurosci 12(10) 1308-1318.
Milestones and Deliverables
Due to technical problems in WP03 some changes have been made in the milestones and deliverables of WP02. Since the coupling protein between rods and cones has not been identified we could not specifically interfere with rod cone coupling and therefore not determine its contribution of outer retinal processing. However, it was found that Panx1 and Panx2 channels were abundantly present in the photoreceptor/horizontal cell/bipolar cells synapse. Given the fact that these proteins are located at a very strategic position in this synaptic complex and that their function is unknown, we shifted the attention from the rod/cone coupling towards the role of Panx channels in the cone synaptic terminal. Given these changes, all milestones and deliverables of WP02 have been finished and delivered.
WP03 “Photoreceptor degeneration, the bystander hypothesis”
“Retinitis pigmentosa” describes a heterogeneous group of inherited degenerative diseases of the retina with the same clinical manifestations. The origins of all forms of retinitis pigmentosa are mutations in rod photoreceptor specific genes, 44 of which have been identified so far (see http://www.sph.uth.tmc.edu/retnet/sum-dis.htm for updated genes and loci; Sahni et al. 2011). The disease starts with the degeneration of rods leading to night blindness. At later stages the cone photoreceptors also degenerate. Prevention of the cone degeneration is an important aspect in the therapy of retinitis pigmentosa as the quality of life of patients is more affected by the later loss of cones than by the early stages of the disease. WP03 “photoreceptor degeneration- the bystander hypothesis” aimed to investigate strategies to interfere with the progress of retinitis pigmentosa at the side of the photoreceptors. The studies were based on the bystander hypothesis, which is of current debate in vision research (Hamel, 2007). This hypothesis states, that the electrical coupling of rods and cones mediates the spread of the degeneration from dying rods to the cones. In rat models of RP it was shown, that a rod derived cone viability factor can prevent cone degeneration (Leveillard et al., 2004). This factor is putatively transfered from rods to cones via the protein complex, which mediates rod-cone coupling. Under healthy conditions the coupling of rods to cones enables the use of the cone vertical pathways under light conditions where the opsins of the cones remain in their resting states. The existence of this pathway in mice was proven indirectly by intracellular recordings of rod signals from the somato-dendritic compartment of horizontal cells, which only contacts cone photoreceptors (Trumpler et al., 2008). The gap junction channels mediating coupling consist of two hexameric complexes, with connexin 36 representing the hemichannel forming protein in cones (Feigenspan et al., 2004), and mice deficient of this protein do not exhibit rod-cone coupling (Trumpler et al., 2008).
The aim of WP03 was to test the impact of rod-cone coupling in mouse models of retinitis pigmentosa in order to identify therapeutic strategies applicable to interrupt the spread of the disease from rods to cones. The identification of the protein which makes up the coupling complex on the rods´ side and the characterization of the physiological properties of this complex were prerequisites for the search for pharmaceuticals that disrupt the rod-cone coupling.
1. Impact of rod-cone coupling on cone degeneration in rd mouse models – Testing the bystander hypothesis
To assess the impact of rod-cone coupling on the spread of the disease in mouse models of retinitis pigmentosa, two different mouse models of the disease were used: Rhodopsin knock-out mice (rho-/-), which show a moderate progression of the degeneration of the outer nuclear layer, were used to shed light on the onset of cone degeneration, whereas mice of the fast degenerating rd1 strain were used to evaluate the impact of rod-cone coupling on later stages of cone degeneration. These strains were cross-breed with a rod-cone coupling deficient strain and biochemical and histological analyses were performed with the two resulting strains (rho-/-/Cx36-/-) and (rd1/Cx36-/-) in comparison with the respective strains with functional rod-cone coupling (rho-/- and rd1). These studies revealed the final conclusion that the knock-out of Cx36 has no significant impact on cone degeneration in general and the time course of degeneration is unaffected. Therefore, the disruption of rod-cone coupling is no suitable approach to prevent the loss of cones in retinitis pigmentosa, and there is no need for the search for respective pharmaceuticals.
The number of outer segments (OS) of the remaining cone photoreceptors was evaluated as a first indication for cone degeneration in rho-/-/Cx36+/+and rho-/-/Cx36-/-versus wild type retinae. To assess the effect of coupling on the time course of degeneration, the densities of OS were quantified by counting m- and s-opsin labeled OS in selected regions of interest in dorsal and ventral parts of retinal whole-mounts derived from 5, 9 and 12 weeks old mice.In 9 and 12 week old rho-/-/Cx36+/+ and rho-/-/Cx36-/- animals, a significant progress of cone degeneration in comparison to retinae of wild-type mice was evident. The densities of OS between both transgenic mouse lines showed no significant difference. Thus, the findings obtained from the study of the early phase of cone degeneration show that deletion of Cx36 has no influence on the initiation of cone degeneration. This indicates that rod-cone coupling is not involved in the onset of cone degeneration.
Next, the impact of rod-cone coupling on retinal morphology was tested in rho-/- mice versus rho-/-/Cx36-/- double knock mice by means of different immunohistological techniques. Double-labeling experiments with antibodies against glycogen phosphorylase (glypho), (which specifically labels cone photoreceptors in the outer nuclear layer (ONL)), and antibodies against velis-3 (to label photoreceptor terminals and the outer limiting membrane (OLM)), revealed a decrease in the thickness of the ONL of the rhodopsin mutants (rho-/-/Cx36+/+) with increasing age. But, a comparison of the ONL morphology of rho-/-/Cx36+/+ and rho-/-/Cx36-/- retinae revealed no distinct differences between the transgenic mouse lines. The effects of photoreceptor degeneration on the morphology of second-order neurons were assessed by labeling rod bipolar cells and horizontal cells with antibodies against PKCalpha and calbindin. Here, remodeling of some rod bipolar cell and horizontal cell dendrites, which extended into the ONL, was observed in 5 week old animals of both genotypes. But, these abnormally grown dendrites disappeared at increasing age of the animals and with progress of degeneration in both transgenic mouse lines. Again, the data revealed no apparent differences between the morphologies of Cx36-expressing and Cx36-deficient rho-/- mice, and photoreceptor degeneration affected the dendritic organization of horizontal cells and rod bipolar cells in the same way.
The impact of rod-cone coupling on the progress of cone degeneration was investigated in the rd1 mouse model. This mouse strain serves as a fast degenerating model of retinitis pigmentosa. The time course of degeneration in rd1 versus Cx36 deficient rd1 (rd1/Cx36-/-) mice was determined by quantification of immunolabeled cones at different ages. In both strains the progressive loss of cones peaked at the age of 21 to 30 days, making this interval the ideal time point to study the effect of rod cone coupling on later stages of degeneration. Quantification of cone loss in and comparison of cell death rate by means of TUNEL staining between wild-type mice and the two rd1/Cx36+/+ and rd1/Cx36-/- mutants at the age of 21 and 30 days clearly showed: (1) a significant decrease in the number of cones in rd1/Cx36+/+ and rd1/Cx36-/- mutants when compared to wild-type mice of the same age, but no significant differences between the two transgenic mouse lines, (2) a significant increase in the number of TUNEL-positive cells in the ONL in both rd1/Cx36+/+ and rd1/Cx36-/- mutants when compared to wild-type mice of the same age, but no significant differences between the two transgenic mouse lines.
In summary, our findings indicate that the disruption of rod-cone coupling neither prevents the initial stage of cone degeneration in rho-/-/Cx36-/- nor cone cell death during the progressive phase of cone degeneration in rd1/Cx36-/- mice. Hence, we conclude that rod-cone coupling is not involved in the secondary cone degeneration, and that the bystander hypothesis is not valid for cone degeneration in mouse models of retinitis pigmentosa.
2. Identification of the protein components of the rod-cone coupling complex
The gain of knowledge about the molecular components mediating rod-cone coupling and the mechanisms regulating this connection are important objectives in respect to the following two aspects: (1) Understanding this synaptic connection and its regulation is essential for a detailed analysis of the outer retinal networks and their functional importance for vision; (2) an influence of this connection on the progress of photoreceptor degeneration observed in patients with retinitis pigmentosa is discussed among neuroscientists since several years, but evidence for this hypothesis is still ambiguous.
In the context of this project, we could show that the assumption of an impact of a functional rod-cone coupling complex on the progression of the disease is false. But, for the understanding of retinal function this objective remains interesting.
Rod-cone coupling provides an important contribution to retinal circuits and adaptation, as it enables the retina to use the cone bipolar cell pathways under dark conditions. Light conditions and the circadian clock regulate this connection so that under higher light intensities the cone-pathway is exclusively used by cones (Wang and Mangel, 1996; Ribelayga et al., 2008). On the cones side the coupling complex consists of Cx36 (Feigenspan et al., 2004), but the hemichannel composition on the rod side remains unknown.
Following the experimental strategies that lead to the detection of Cx36 in cone photoreceptors, the first strategy was a molecular biological strategy to search for connexin-mRNA in rod samples of the routinely used mouse strain C57Bl/6. Two different experimental approaches were applied: the identification of connexin-transcripts with wobble primers which match groups of the connexins, and the identification of transcripts with primers which were generated specifically for all of the 20 connexins encoded in the murine genome. Cx32, Cx36, Cx43 and Cx45 were identified by these approaches. But laser scanning microscopy performed with immune-labeled retinal sections excluded the expression of these connexins in rods. Instead, the expression of Pannexin2 in rods was shown at the level of RNA and protein, but physiological studies ruled out that Pannexin2 is capable to form ion channels which function as hemichannels within the rod-cone coupling complex.
The dilemma, that no coupling protein was identified, was faced with the use of the 129/SV strain for our molecular studies. In contrast to C57BL/6 mice, which are frequently used for gene manipulation, but show weak circadian rhythm, 129/SV mice exhibit a prominent influence of circadian rhythm on rod-cone coupling (increase of rod-cone coupling during the night; Ribelayaga and Mangel 2010). mRNA analyses were performed on rod samples derived from dark-adapted retinae of 129/SV which were in the first three hours of the night cycle. RT-PCR with gene specific primers designed to detect all 20 reported mouse connexins revealed the expression of Cx36-, Cx45-, Cx50- and Cx57-mRNA in these samples. However, immunohistological analyses on retinal sections derived from the same mice showed, that Cx36 is exclusively found in cone photoreceptors and that Cx45, Cx50 and Cx57, although localized in the OPL, are not positioned close enough (co-localized) in relation to Cx36 to fulfil the requirements of the sought-after rod connexin. Thus, we concluded, that the detected connexin transcripts more likely respresent contaminations from photoreceptor adjacent cells, namely bipolar cells (Cx45) and horizontal cells (Cx50 and Cx57).
Due to the failure of the second molecular biological strategy the following strategies were developed to detect the rod connexin: 1) Following the presumption that the connexin might be a splice variant, as all anti-connexin-antibodies we used bind to the less conserved C-terminal domain of the respective connexin, 3´Race-PCR was performed with connexin subfamily specific primers (e.g. ?-, ?-, ? -and ?-group specific connexin primers) and with sequence specific primers for the reported Cx36-interacting connexins, which are Cx30.2 Cx36 and Cx45. 2) Cx36 was immunoprecipitated from retinal samples and the resulting protein complexes were analysed in western blots and with mass spectroscopy, with the goal to identify all the proteins, including the rod connexin. Different systems of immunoprecipitation were tested. Until now, we neither detected splice variants in cDNA-samples of whole retinae and rod photoreceptors, nor were we able to identify further connexins in complexes with Cx36. But, the use of RACE-RT-PCR and immunoprecipitation are ongoing strategies in our lab, and we are still optimizing the experimental procedures.
Based on our molecular and histological data, we can exclude many connexins as components of rod connexons (compare Bolte et al., 2012; in prep. for submission to Mol Vis). Otherwise, we cannot exclude the possibility that the immunofluorescence microscopic techniques, we used to localize Cx45, Cx50 and Cx57 in the mouse retina, may not be sensitive enough to detect the small gap junctions between rods and cones. To overcome this difficulty and to verify our immunofluorescence data, we started immunoelectron microscopic analyses with anti-Cx45 and anti-Cx50 antibodies and are currently evaluating previously obtained ultra-structural data on the distribution of Cx57 in the outer plexiform layer of the retinae of C57BL/6 mice.
To summarize, the expression of none of the known 20 murine connexins could be definitely verified in rod photoreceptors, which retards the physiological description of the coupling complex between rods and cones. But in WP03 we were successful in the exclusion of several of the 20 murine connexins and the pannexins as possible candidates for the coupling protein on rod side. Based on the work that was performed during RETICIRC there are ongoing studies to identify the rod connexin.
3. Functional analysis of candidate proteins
In the beginning of RETICIRC the Pannexin1 and Pannexin2 were candidates for rod-cone coupling in mice. They are members of the recently discovered pannexin family of proteins, which is the mammalian homolog of the innexins, the insect gap junction proteins. The mRNA of both proteins was reported in rod and cone photoreceptors (Dvoriantchikova et al., 2006). Meanwhile we can rule out a function of both proteins in the coupling complexes between rods and cones, because physiological studies ruled out a hemichannel function of Pannexin2 and Pannexin1 protein was not found in photoreceptors. However, we nearly finished the analyses of the distribution of both proteins in the mouse retina, and the data are currently prepared for publication.
3.1. The former coupling candidate Pannexin2 is expressed in all cells that make synaptic contacts in the OPL
Pannexin2 (Panx2) is a neuronal protein in mammals, which forms oligomeric complexes in membranes. Full-length Panx2 mRNA was amplified from samples of mouse retina and photoreceptors and cloned into mammalian expression vectors. Patch-clamp recordings of transfected N2A cells revealed, that panx2 neither forms gap junction channels nor voltage gated hemichannels. These findings are in line with recently published data (e.g. Ambrosi et al., 2009) and make it unlikely to regard Panx2 as a candidate for rod-cone coupling anymore. However, with this study we are the first group that showed Panx2 protein expression in the mouse retina.
By means of different immune-histological techniques we could identify Panx2 protein in all cell types which make contacts in the outer plexiform layer namely the photoreceptors, horizontal cells and bipolar cells. The subretinal distribution of Panx2 was studied in vertical sections and whole mount preparations, where Pannexin2 was labeled with a custom made antibody and the subcellular distribution was analyzed in preparations of dissociated retinal cells double-labelled for Panx2 and cell-type specific markers. In dissociated retinal cells, Panx2 was present in glycogen phosphorylase labeled cones as well as in PSD95 labelled rod cells. The analyses of the retinal ultrastructure revealed Panx2 immunoreactive signals in PSD95 labeled photoreceptor terminals and the bassoon stained ribbon synapses in the OPL. Panx2 expression was also observed close to cone pedicles, which were either labeled with peanut agglutin or with glycogen phosphorylase. Immunoelectron microscopy using anti-Panx2 antibodies showed small patches of Panx2 labeled cell membranes of rod spherules and cone pedicles. Thus, Panx2 is expressed in both types of photoreceptors, particularly at the synaptic endings. Labelling of horizontal cells different tissue preparations turned out, horizontal cells express Panx2. Subcellularly, Panx 2 is localized at the dendritic tips and axon terminal endings of invaginating horizontal cells. This was shown by means of laser scanning microscopy with double labeled preparations and by means of immunoelectron microscopy.
Due to the distribution of Panx2 on the presynaptic terminals of photoreceptors as well as the postsynaptic localization on horizontal cell tips, we propose that Panx2 may have a function in the synaptic transmission in the outer retina. The publication of this study (Bolte et al., 2012, J. Comp. Neurol, in prep) will be postponed until the final analysis of immunosignals in a genetically modified Panx2 deficient mouse is finished.
3.2. The putative candidate protein Pannexin1 affects signal transmission in the outer retina
Pannexin1 is a hexameric membrane protein, which is capable of the formation of hemichannels. In the RETICIRC project the subretinal distribution of this protein was analysed by means of different methods, including single cell RT-PCR, immunofluorescence and immunoelectron microscopy. Panx1 is localized in horizontal and distinct bipolar cells, where it is localized in the tips of the dendritic and axonal endings of horizontal cells and throughout the membrane of type 3a OFF bipolar cells, which were identified by their expression of the proton gated channel HCN4. In order to classify the specific subtype of bipolar cells which expresses Panx1, a bundle of antibodies had to be used as markers for the respective cell types and dye-injections into bipolar cells were performed to exclude further cell types, for which no markers exist. For the histological part, Panx1 knock-out mice were used as an indicator for the specificity of the anti-Panx1 antibodies.In vivo and in vitroelectroretinographic recordings (ERG) of Panx1 deficient were compared to those of Panx1 wild-type mice, to identify the impact of Panx1 on retinal transmission. In vivo ERG recordings of Panx1 knock-out mice under scotopic conditions showed increased amplitudes in a- and b-waves when compared to Panx1 wild-type mice. The changes of the b-waves observed in Panx1 knock-out mice were confirmed by the recordings obtained in in vitro ERGs from the inner retina. Taken together these results suggest a physiological role of Panx1 in the rod pathway. Furthermore, the distinct localization of Panx1 channels in mouse horizontal cells, which is in agreement with recent findings in the zebrafish retina (Prochnow et al., 2009) suggests an involvement of Panx1 channels in the negative feedback mechanism from horizontal cells to photoreceptors.
Milestones and Deliverables
A milestone in WP03 is the findings which lead to the conclusion that rod-cone coupling has no impact on cone degeneration in different mouse models of retinitis pigmentosa. Thus the bystander hypothesis is to be disclaimed. The second milestone, the full description of the coupling complex between rods and cones is postponed. Despite many different efforts and the use of state-of-the-art techniques, the rod connexin is not yet identified. But possible candidates like pannexin2, Cx36, Cx50 and Cx57 were successfully excluded and we state, that Cx45 and Cx30.2 are left as possible candidates. We hope to identify the rod connexin with the improved strategies within the next half year. The third milestone dealt with the identification of therapeutical substances to interfere with rod-cone coupling. As this coupling does not influence retinal degeneration in two different mouse models, its disruption is no longer a therapeutic strategy. This milestone has been cancelled. Given these changes all milestones and deliverables have been finished and delivered.
WP04 - Rescuing retinal function in retinal degeneration by expressing Channelrhodopsin-2 in ON-BCs
We achieved the following results which will be summarized in the next paragraphs:
1. We have restored visual responses to dormant cones in rd1and Rho-/-Cnga3-/- mice.
2. We have demonstrated that the light re-sensitized cones can efficiently drive the ON, OFF and ON-OFF retinal channels at the level of ganglion cells.
3. We have demonstrated that the resensitized retina is capable of performing sophisticated image processing functions such as computing motion direction.
4. We have shown that the regained retinal signal is transmitted to the visual cortex in rd1 mice.
5. Using two different behavioural essays we have shown that the regained retinal activity is able to drive visual behavior in rd1 mice.
6. Using both viral delivery and a mouse line we developed we demonstrated that the expression of halorhodopsin does not induce toxicity in normal cones.
7. We have shown regained retina function up two years of age in mice.
8. We have developed a human ex vivo retinal preparation.
9. We have shown that our viral vectors could resensitize human cones to light.
10. We have developed four different variants of viral vectors for testing in in vivo in primates. All these vectors were tested in rd1 mice and all were able to restore ganglion cell responses to light.
11. We have screened hundreds of patients with retinitis pigmentosa that could be eligible for therapy.
12. We initiated in vivo primate studies to test specificity, expression efficacy and safety.

The disease
Retinitis pigmentosa is the result of diverse mutations in more than 44 genes expressed in rod photoreceptors; these then degenerate, causing loss of night vision. Subsequently, cone photoreceptors, which are responsible for color and high acuity daytime vision, progressively lose their photoreceptive outer segments, leading to overall blindness. Despite this loss of sensitivity, cone cell bodies remain present longer than rods in both humans and animals, but it was not known whether these light-insensitive cells can be reactivated and if information from them can still flow to downstream visual circuits for a significant time window after the loss of photosensitivity.
Our approach
To restore light-evoked activity in light-insensitive cone photoreceptors we genetically targeted a light-activated chloride pump, enhanced Natronomonas pharaonis halorhodopsin (eNpHR), to photoreceptors by means of adeno-associated viruses (AAVs). Light-activated chloride pumps are rational candidates for reactivating vertebrate photoreceptors as both eNpHR-expressing cells and healthy photoreceptors hyperpolarize in response to increases in light intensity. We selected two animal models of Retinitis pigmentosa for gene therapy, both of which lead to retinal degeneration (RD). Cnga3-/-; Rho-/- double-knockout mice served as a model of slow forms of RD (s-RD mice), and Pde6brd1 (also known as rd1) mice as a model of fast forms of RD (f-RD mice). Targeted expression of eNpHR was accomplished using three cell-specific promoters: Human rhodopsin (hRHO); human red opsin (hRO); and mouse cone arrestin-3 (mCAR).
We resensitized cones to light
We tested whether eNpHR drives cone light responses in RD retinas at an age (P53-P264) when many (s-RD) or all (f-RD) rods have already died. eNpHR-EYFP-expressing photoreceptors in s-RD and f-RD retinas displayed large, sustained, and significantly faster photocurrents than wild-type cones. The photocurrents peaked at 580 nm. Photoreceptors expressing only EGFP did not react to light. The magnitude of photocurrents in eNpHR-transduced s-RD and f-RD mice were similar. The current size was independent of the holding voltage, a finding that is consistent with the view that the photocurrents are mediated by ion pumps. Photocurrents and voltages were modulated across three logarithmic units of intensities. In the absence of functional outer segments, which normally generate currents that depolarize photoreceptors in the dark, RD photoreceptors were expected to stay hyperpolarized. A hyperpolarized state would limit the ability of eNpHR currents to modulate synaptic transmission. However, the recorded eNpHR-expressing RD photoreceptors were depolarized in the dark.
We tested light response in ganglion cells
To test for potential signal flow from photoreceptors to ganglion cells, we recorded excitatory currents from ganglion cells, the output neurons of the retina. Ganglion cells in eNpHR-transduced s-RD and f-RD retinas displayed robust, light-evoked excitatory currents, indicating functional outer and inner retinal synaptic connections. The magnitudes of these excitatory currents were comparable to those in wild-type retinas. There were no measurable light-evoked currents in the ganglion cells of retinas transfected with EGFP. The retina incorporates two major information channels that diverge at the level of bipolar cells, and are physically separated by depth within the inner plexiform layer (IPL). ON cells are activated by light increments, while OFF cells are activated by light decrements. The activity of ganglion cells is coded by action potentials (“spikes”) that form the output signal of the retina and the input signal to higher brain centers. Strikingly, light stimulation evoked excitatory currents and spiking activity in ganglion cells of eNpHR-transduced RD retinas either at light increments, light decrements, or both (P53-P264). The dendritic processes of morphologically reconstructed ganglion cells were properly aligned in the corresponding ON or OFF strata of the IPL. Ganglion cell spike frequencies spanned similar ranges in s-RD, f-RD, and wild-type retinas. Some ganglion cell types respond preferentially to changes in illumination and therefore spike transiently after a light step while others represent the level of illumination and therefore spike in a more sustained manner. The dynamics of spiking activity in eNpHR-transduced retinas varied from transient to sustained, as in wild-type retinas. Light-stimulation of eNpHR-reactivated photoreceptors in RD retinas even evoked ganglion cell spiking activity at later stages of degeneration (s-RD: We tested if the resensitized retina can process information
Lateral inhibition is a conserved feature of vertebrate retinas that is important for spatial contrast (“edge”) enhancement. When spots of increasing sizes were presented to eNpHR-expressing RD retinas, the response magnitude of ganglion cells reached a maximum and then gradually decreased, a sign of lateral inhibition. Lateral inhibition also results in ON-center OFF-surround responses that we were also able to observe in eNpHR-expressing RD retinas. Another example of spatial processing is the directional-selective responses of types of ganglion cells to motion stimuli. The activity of directional selective ganglion cells is important for the optokinetic reflex. When eNpHR-transduced RD retinas were stimulated with bars moving in different directions some ganglion cells responded preferentially to motion in a particular direction but produced little activity when the bar moved in the opposite direction. This response asymmetry suggests that the retinal circuit for directional selectivity is, at least partially, maintained in RD retinas.
We tested if the resensitized retina can send visual information to the cortex
The slow and fast RD mouse models not only differ in the time course of photoreceptor degeneration, but also in the amount of light-driven activity present during development. s-RD mice have no light-sensitive rod-cone system during development while f-RD mice lose the rod-cone function gradually, and are blind by four weeks of age. In the retina, the responses in eNpHR-activated s-RD and f-RD mice were similar, suggesting that the development of the tested retinal functions, like direction selectivity, may not require light-driven input from rods and cones. Light stimulation of the eyes resulted in visually evoked potentials in eNpHR-expressing f-RD mice but not in EGFP-transduced controls (P42-P118). In contrast to the retina, we could not measure light-driven cortical activity in eNpHR-transduced s-RD mice.
We tested if the resensitized retina can drive visual behavior
In dark-light box tests, eNpHR-expressing f-RD and s-RD mice performed significantly better than the corresponding EGFP-expressing control groups (P44-P143), and the increased performance depended on the illumination level. In the optomotor reflex test, only eNpHR-expressing f-RD mice performed better than EGFP-expressing control mice at a variety of drum speeds (P69-P153). Wild-type and f-RD responses both peaked at the same speed. The optimum spatial frequency was higher for wild-type animals (0.26 cpd) compared to f-RD (0.13 cpd). These experiments demonstrated that resensitized photoreceptors are able to drive visually-guided behavior in f-RD and, to some extent, in s-RD mice.
We tested toxicity
We first compared the retinas of eNpHR-transduced wild-type mice (six weeks after AAV administration) with those of normal wild-type mice. The number of photoreceptors was similar in both conditions and, in addition to the light-induced spiking activity, ganglion cells in eNpHR-transduced wild-type retinas had a wider action spectrum, a gain of function at longer wavelengths, than ganglion cells in uninjected wild-type retinas. This suggests that both intrinsic opsins and eNpHR are at work. Next, we compared the retinas of transgenic mice expressing NpHR in photoreceptors under the control of a bovine rhodopsin promoter with wild-type retinas and found similar numbers of photoreceptors (at P140). These results suggest that in the studied time window neither the unmodified NpHR nor eNpHR induce additional photoreceptor degeneration.
We tested halorhodopsin function in ex vivo human retinas
We tested our AAVs on human ex vivo retinal explants, which we could keep in culture for 2-3 weeks. Due to this short time window and the relatively long period of time required to efficiently express eNpHR from AAVs, we had to use immunohistochemistry to visualize eNpHR-EYFP protein expression in the cultured human retinas. Of the three promoters, mCAR directed expression of eNpHR specifically in human photoreceptors. To reduce the time required to obtain robust eNpHR-EYFP expression, necessary for two-photon laser targeted electrophysiology, we inserted the mCAR-eNpHR construct into a lentiviral vector. Using this new vector, high levels of eNpHR expression were found specifically in human photoreceptors after only 1-2 days of incubation. Brightly labeled photoreceptors in the parafoveal region displayed photocurrents and photovoltages with spectral tuning reflecting eNpHR activation. We could not measure any photocurrents from control human retinas, even at the time when the retina was isolated.
We screened for patients with retinitis pigmentosa that have remaining cones
We screened a database created in Paris, which contained records of retinal images acquired by optical coherence tomography (OCT), Goldman visual field tests, multifocal electroretinograms (ERG), full field ERG, and visual acuity tests. We identified legally blind patients with no visible outer segments on OCT pictures, but cone cell bodies in the central region. These criteria may be used in the future for selecting patients who could benefit from this therapy.
Milestones and Deliverables
Note that as explained above we switched to halorhodopsins, instead of using zebrafish we used human retina culture model and instead of analyzing transgenic mice we analyzed AAV injected mice. Given this change all milestones and deliverables have been finished and delivered.

Potential Impact:
RETICIRC has been very successful. New neuronal mechanisms have been discovered, long standing hypothesis has been tested, and proof of principle for a new approach for restoration of vision has been delivered. These achievements have been published in 24 papers of which a large part has appeared in the very best journals available: Nature (1), Science (1), Cell (1), Nature Neuroscience (1), Nature Methods (1), American Journal of Human Genetics (2) and PLoS Biology (1). A large number of additional papers are in preparation or submitted. This is an excellent score for a three year project.
Apart from these publications, the results have been presented on many scientific meetings in Europe and the rest of the world.
RETICIRC has paved the way for future translational work that will be focused on the optogenetically reactivation of retinal neurons in order to restore vision in patients with Retinitis Pigmentosa. Demonstrating the clinical value of optogenetic tools to restore vision could also pave the way for their application in the treatment of other neurological and sensory disorders. RETICIRC has provided basic insights in the functioning of the retinal network and provided proof of principle in animal models for an optogenetical therapeutic solution for retinitis pigmentosa. These successes together with the recent progress in corrective gene-therapy in ophthalmology in human subjects (Bainbridge et al., 2008; Maguire et al., 2008; Cideciyan et al., 2008) should facilitate the fast progress to a method for treatment of retinitis pigmentosa patients.
As mentioned in the introduction, retinitis pigmentosa is a degenerative disease that leads to severe visual impairment or even blindness. The quality of life of retinitis pigmentosa patients is severely affected, and this seriously invalided patient group requires considerable care. The symptoms of retinitis pigmentosa can occur at any age but mostly appear in young adults. Unfortunately, there is no known cure for retinitis pigmentosa. The only available treatment is high daily dose of vitamin A palmitate (15,000 IU). This treatment is controversial and is only partially effective in a very small fraction of patients. Therefore, there is a high medical need to identify, develop and bring to market an innovative product that could treat retinitis pigmentosa patients and restore their vision.
The ophthalmic pharmaceutical market is expected to grow strongly during the years to come. New technologies and products will be developed, especially in the area of retinal degenerative diseases. The proposed program will generate the basic and preclinical data for such a product or group of products. While RETICIRC was directed towards a treatment for retinitis pigmentosa it is highly likely that technological spin-offs will occur that can be used to treat other retinal diseases, such as Age-related Macular Degeneration (AMD). AMD is considered the leading cause of blindness in elderly people in developed countries and, like retinitis pigmentosa, is also characterized by progressive degeneration of photoreceptors. For some forms of AMD (15%) partially effective treatments are available by inhibiting the VEGF pathway (Macugen® Lucentis®) or by photodynamic therapy (Visudyne®), but for the remaining 85% of patients there are currently no effective treatments.
To ensure that the knowledge and technological developments will find their way to the patients we need to start a translational project that will show proof of principle of the optogenetic approach in human retinas and deal with all the safety and regulatory issues such that clinical trials could be started in 3-5 year time. The aim of these future clinical trials is to establish Proof of Concept (POC) in man. We estimate that POC could be established within a 5 to 6 year period and that the first product could reach the market in a 8 to 10 year period, assuming that a translational project can be started as follow up of RETICIRC.

List of Websites:
http://www.reticirc.eu/

Final Report Summary - RETICIRC (Circuit specific approaches to retinal diseases)

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