Targeted Expression of Channelrhodopsin-2 to the Axon Initial Segment Alters the Temporal Firing Properties of Retinal Ganglion Cells

The axon initial segment (AIS) is essential for initiating action potentials and maintaining neuronal excitability in axon-bearing neurons in the CNS. There is increasing interest in the targeting of optogenetic tools to subcellular compartments, including the AIS, to gain precise control of neuronal activity for basic research and clinical applications. In particular, targeted expression of optogenetic tools in retinal ganglion cells (RGCs) has been explored as an approach for restoring vision after photoreceptor degeneration. Thus, understanding the effects of such targeting on spiking abilities and/or patterns is important. Here, we examined the effects of recombinant adeno-associated virus (rAAV)-mediated targeted expression of channelrhodopsin-2 (ChR2)-GFP with a Na_V channel motif in mouse RGCs. We found that this targeted expression disrupted Na_V channel clustering at the AIS and converted the spike firing patterns of RGCs from sustained to transient. Our results suggest that the clustering of membrane channels, including Na_V channels, at the AIS is important for the ability of RGCs to generate sustained spike firing. Additionally, the targeting of optogenetic tools to the AIS with the Na_V channel motif may offer a way to create transient light responses in RGCs for vision restoration.     

Visual Properties of Transgenic Rats Harboring the Channelrhodopsin-2 Gene Regulated by the Thy-1.2 Promoter

Channelrhodopsin-2 (ChR2), one of the archea-type rhodopsins from green algae, is a potentially useful optogenetic tool for restoring vision in patients with photoreceptor degeneration, such as retinitis pigmentosa. If the ChR2 gene is transferred to retinal ganglion cells (RGCs), which send visual information to the brain, the RGCs may be repurposed to act as photoreceptors. In this study, by using a transgenic rat expressing ChR2 specifically in the RGCs under the regulation of a Thy-1.2 promoter, we tested the possibility that direct photoactivation of RGCs could restore effective vision. Although the contrast sensitivities of the optomotor responses of transgenic rats were similar to those observed in the wild-type rats, they were enhanced for visual stimuli of low-spatial frequency after the degeneration of native photoreceptors. This result suggests that the visual signals derived from the ChR2-expressing RGCs were reinterpreted by the brain to form behavior-related vision.     

Differential targeting of optical neuromodulators to ganglion cell soma and dendrites allows dynamic control of center-surround antagonism

Retinal degenerative diseases cause photoreceptor loss and often result in remodeling and deafferentation of the inner retina. Fortunately, ganglion cell morphology appears to remain intact long after photoreceptors and distal retinal circuitry have degenerated. We have introduced the optical neuromodulators channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR) differentially into the soma and dendrites of ganglion cells to recreate antagonistic center-surround receptive field interactions. We then reestablished the physiological receptive field dimensions of primate parafoveal ganglion cells by convolving Gaussian-blurred versions of the visual scene at the appropriate wavelength for each neuromodulator with the Gaussians inherent in the soma and dendrites. These Gaussian-modified ganglion cells responded with physiologically relevant antagonistic receptive field components and encoded edges with parafoveal resolution. This approach bypasses the degenerated areas of the distal retina and could provide a first step in restoring sight to individuals suffering from retinal disease.     

Retinal Ganglion Cells are Resistant to Photoreceptor Loss in Retinal Degeneration

The rapid and massive degeneration of photoreceptors in retinal degeneration might have a dramatic negative effect on retinal circuits downstream of photoreceptors. However, the impact of photoreceptor loss on the morphology and function of retinal ganglion cells (RGCs) is not fully understood, precluding the rational design of therapeutic interventions that can reverse the progressive loss of retinal function. The present study investigated the morphological changes in several identified RGCs in the retinal degeneration rd1 mouse model of retinitis pigmentosa (RP), using a combination of viral transfection, microinjection of neurobiotin and confocal microscopy. Individual RGCs were visualized with a high degree of detail using an adeno-associated virus (AAV) vector carrying the gene for enhanced green fluorescent protein (EGFP), allowed for large-scale surveys of the morphology of RGCs over a wide age range. Interestingly, we found that the RGCs of nine different types we encountered were especially resistant to photoreceptor degeneration, and retained their fine dendritic geometry well beyond the complete death of photoreceptors. In addition, the RGC-specific markers revealed a remarkable degree of stability in both morphology and numbers of two identified types of RGCs for up to 18 months of age. Collectively, our data suggest that ganglion cells, the only output cells of the retina, are well preserved morphologically, indicating the ganglion cell population might be an attractive target for treating vision loss.     

Long-term gene therapy causes transgene-specific changes in the morphology of regenerating retinal ganglion cells

Recombinant adeno-associated viral (rAAV) vectors can be used to introduce neurotrophic genes into injured CNS neurons, promoting survival and axonal regeneration. Gene therapy holds much promise for the treatment of neurotrauma and neurodegenerative diseases; however, neurotrophic factors are known to alter dendritic architecture, and thus we set out to determine whether such transgenes also change the morphology of transduced neurons. We compared changes in dendritic morphology of regenerating adult rat retinal ganglion cells (RGCs) after long-term transduction with rAAV2 encoding: (i) green fluorescent protein (GFP), or (ii) bi-cistronic vectors encoding GFP and ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF) or growth-associated protein-43 (GAP43). To enhance regeneration, rats received an autologous peripheral nerve graft onto the cut optic nerve of each rAAV2 injected eye. After 5-8 months, RGCs with regenerated axons were retrogradely labeled with fluorogold (FG). Live retinal wholemounts were prepared and GFP positive (transduced) or GFP negative (non-transduced) RGCs injected iontophoretically with 2% lucifer yellow. Dendritic morphology was analyzed using Neurolucida software. Significant changes in dendritic architecture were found, in both transduced and non-transduced populations. Multivariate analysis revealed that transgenic BDNF increased dendritic field area whereas GAP43 increased dendritic complexity. CNTF decreased complexity but only in a subset of RGCs. Sholl analysis showed changes in dendritic branching in rAAV2-BDNF-GFP and rAAV2-CNTF-GFP groups and the proportion of FG positive RGCs with aberrant morphology tripled in these groups compared to controls. RGCs in all transgene groups displayed abnormal stratification. Thus in addition to promoting cell survival and axonal regeneration, vector-mediated expression of neurotrophic factors has measurable, gene-specific effects on the morphology of injured adult neurons. Such changes will likely alter the functional properties of neurons and may need to be considered when designing vector-based protocols for the treatment of neurotrauma and neurodegeneration.     

Restoring the ON Switch in Blind Retinas: Opto-mGluR6, a Next-Generation,Cell-Tailored Optogenetic Tool

Photoreceptor degeneration is one of the most prevalent causes of blindness. Despite photoreceptor loss, the inner retina and central visual pathways remain intact over an extended time period, which has led to creative optogenetic approaches to restore light sensitivity in the surviving inner retina. The major drawbacks of all optogenetic tools recently developed and tested in mouse models are their low light sensitivity and lack of physiological compatibility. Here we introduce a next-generation optogenetic tool, Opto-mGluR6, designed for retinal ON-bipolar cells, which overcomes these limitations. We show that Opto-mGluR6, a chimeric protein consisting of the intracellular domains of the ON-bipolar cell–specific metabotropic glutamate receptor mGluR6 and the light-sensing domains of melanopsin, reliably recovers vision at the retinal, cortical, and behavioral levels under moderate daylight illumination.     

A C-terminal motif targets Hedgehog to axons, coordinating assembly of the Drosophila eye and brain

The developmental signal Hedgehog is distributed to two receptive fields by the photoreceptor neurons of the developing Drosophila retina. Delivery to the retina propagates ommatidial development across a precursor field. Transport along photoreceptor axons induces the development of postsynaptic neurons in the brain. Hedgehog is composed of N-terminal and C-terminal domains that dissociate in an autoproteolytic reaction that attaches cholesterol to the N-terminal cleavage product. Here, we show that the N-terminal domain is targeted to the retina when synthesized in the absence of the C-terminal domain. In contrast to studies that have focused on cholesterol as a determinant of subcellular localization, we find that the C-terminal domain harbors a conserved motif that overrides retinal localization, sending most of the autocleavage products into vesicles bound for growth cones or synapses. Competition between targeting signals at the opposite ends of Hedgehog apparently controls the match between eye and brain development.     

Photochemical restoration of visual responses in blind mice

Retinitis pigmentosa (RP) and age-related macular degeneration (AMD) are degenerative blinding diseases caused by the death of rods and cones, leaving the remainder of the visual system intact but largely unable to respond to light. Here, we show that AAQ, a synthetic small molecule photoswitch, can restore light sensitivity to the retina and behavioral responses in?vivo in mouse models of RP, without exogenous gene delivery. Brief application of AAQ bestows prolonged light sensitivity on multiple types of retinal neurons, resulting in synaptically amplified responses and center-surround antagonism in arrays of retinal ganglion cells (RGCs). Intraocular injection of AAQ restores the pupillary light reflex and locomotory light avoidance behavior in mice lacking retinal photoreceptors, indicating reconstitution of light signaling to brain circuits. AAQ and related photoswitch molecules present a potential drug strategy for restoring retinal function in degenerative blinding diseases.     

Classification of nAChRβ2-immunoreactive retinal ganglion cells and their tectal projections in chicks

The relationship between the type of retinal ganglion cell (RGC) and the retinoreceptive layer of the tectum is investigated by the immunostaining of RGCs with nicotinic acetylcholine receptor_β2 (nAChR_β2) antibody and intracellular staining by DiI and also by anterograde degeneration and biotinylated dextran amine labeling of retinotectal fibers in chicks. The results strongly suggest that many of the RGCs that express immunoreactivity to nAChR_β2 send axons to tectal layer 7 and are mainly classified into the simple-type of Groups II and III, which contain the cells providing middle-sized to large dendritic fields with simple dendritic arborization. These nAChR_β2-immunoreactive RGCs receive visual information via the multiple sublayers of the inner plexiform layer.     

Taurine Provides Neuroprotection against Retinal Ganglion Cell Degeneration

Retinal ganglion cell (RGC) degeneration occurs in numerous retinal diseases leading to blindness, either as a primary process like in glaucoma, or secondary to photoreceptor loss. However, no commercial drug is yet directly targeting RGCs for their neuroprotection. In the 70s, taurine, a small sulfonic acid provided by nutrition, was found to be essential for the survival of photoreceptors, but this dependence was not related to any retinal disease. More recently, taurine deprivation was incriminated in the retinal toxicity of an antiepileptic drug. We demonstrate here that taurine can improve RGC survival in culture or in different animal models of RGC degeneration. Taurine effect on RGC survival was assessed in vitro on primary pure RCG cultures under serum-deprivation conditions, and on NMDA-treated retinal explants from adult rats. In vivo, taurine was administered through the drinking water in two glaucomatous animal models (DBA/2J mice and rats with vein occlusion) and in a model of Retinitis pigmentosa with secondary RGC degeneration (P23H rats). After a 6-day incubation, 1 mM taurine significantly enhanced RGCs survival (+68%), whereas control RGCs were cultured in a taurine-free medium, containing all natural amino-acids. This effect was found to rely on taurine-uptake by RGCs. Furthermore taurine (1 mM) partly prevented NMDA-induced RGC excitotoxicity. Finally, taurine supplementation increased RGC densities both in DBA/2J mice, in rats with vein occlusion and in P23H rats by contrast to controls drinking taurine-free water. This study indicates that enriched taurine nutrition can directly promote RGC survival through RGC intracellular pathways. It provides evidence that taurine can positively interfere with retinal degenerative diseases.     

Cell-type specific dendritic contacts between retinal ganglion cells during development.

The extent of a neuron's dendritic field defines the region within which information is processed. The dendritic fields of functionally distinct ON and OFF center retinal ganglion cells (RGCs) form separate mosaics across the retina. Within each mosaic, neighboring dendritic fields overlap by a constant amount, sampling the visual field with the appropriate coverage. Contact-mediated lateral inhibition between neighboring RGCs has long been thought to regulate both the extent and overlap of dendritic fields during development. Here we show that dendro-dendritic contact exists between developing RGCs and occurs in a manner that would regulate the formation of ON and OFF mosaics separately. Dye-filled neighboring ON and OFF ferret alpha RGCs were reconstructed using multiphoton microscopy. At all neonatal ages examined, we observed dendro-dendritic contacts between RGCs of the same sign (ON/ON; OFF/OFF), but never between cells of opposite signs (ON/OFF). Terminal dendrites of one cell often touched a dendrite of its neighbor as they intersected. In some instances, the distal dendrite of one cell formed a fascicle with the proximal process of its neighbor. Alpha cells did not form contacts with neighboring beta cells of the same sign. Together, these observations suggest that dendro-dendritic contact between RGCs is cell-type specific. Dendritic contacts were observed even before the alpha cell arbors were completely stratified, suggesting that cell-cell recognition may take place early in their development. For each cell type, the relative overlap of dendritic fields was constant with age, despite a two-fold increase in field area. We suggest that dendro-dendritic contacts may be sites of intercellular signaling that could regulate local extension of dendrites to maintain the relative overlap of RGCs within a mosaic during development.     

Receptive field microstructure and dendritic geometry of retinal ganglion cells

We studied the fine spatial structure of the receptive fields of retinal ganglion cells and its relationship to the dendritic geometry of these cells. Cells from which recordings had been made were microinjected with Lucifer yellow, so that responses generated at precise locations within the receptive field center could be directly compared with that cell's dendritic structure. While many cells with small receptive fields had domeshaped sensitivity profiles, the majority of large receptive fields were composed of multiple regions of high sensitivity. The density of dendritic branches at any one location did not predict the regions of high sensitivity. Instead, the interactions between a ganglion cell's dendritic tree and the local mosaic of bipolar cell axons seem to define the fine structure of the receptive field center.     

Efficient gene targeting mediated by adeno-associated virus and DNA double-strand breaks

Gene targeting is the in situ manipulation of the sequence of an endogenous gene by the introduction of homologous exogenous DNA. Presently, the rate of gene targeting is too low for it to be broadly used in mammalian somatic cell genetics or to cure genetic diseases. Recently, it has been demonstrated that infection with recombinant adeno-associated virus (rAAV) vectors can mediate gene targeting in somatic cells, but the mechanism is unclear. This paper explores the balance between random integration and gene targeting with rAAV. Both random integration and spontaneous gene targeting are dependent on the multiplicity of infection (MOI) of rAAV. It has previously been shown that the introduction of a DNA double-stranded break (DSB) in a target gene can stimulate gene targeting by several-thousand-fold in somatic cells. Creation of a DSB stimulates the frequency of rAAV-mediated gene targeting by over 100-fold, suggesting that the mechanism of rAAV-mediated gene targeting involves, at least in part, the repair of DSBs by homologous recombination. Absolute gene targeting frequencies reach 0.8% with a dual vector system in which one rAAV vector provides a gene targeting substrate and a second vector expresses the nuclease that creates a DSB in the target gene. The frequencies of gene targeting that we achieved with relatively low MOIs suggest that combining rAAV vectors with DSBs is a promising strategy to broaden the application of gene targeting.     

Can the data of Campbell and Robson be explained without assuming fourier analysis?

Certain experiments on the detection of low-contrast gratings, occasionally cited as evidence of Fourier analysis within the visual system, are interpreted without the assumption of Fourier analysis. Theoretical curves are obtained and compared with the published experimental points, showing mostly satisfactory agreement. The computations utilize Gaussian receptive fields (on-center and off-center) for the retinal ganglion cells, spatial summation, center-surround antagonism, quasilinear response at low contrasts (X-cells), and the assumption that the first significant convergence is primarily between cells of like response type and like receptive field geometry.     

Morphological properties of medial amygdala-projecting retinal ganglion cells in the Mongolian gerbil

The amygdala is a limbic structure that is involved in many brain functions, including emotion, learning and memory. It has been reported that melanopsin-expressing retinal ganglion cells(ip RGCs) innervate the medial amygdala(Me A). However, whether conventional RGCs(c RGCs) project to the Me A remains unknown. The goal of this study was to determine if c RGCs project to the Me A and to determine the morphological properties of Me A-projecting RGCs(Me A-RGCs). Retrogradely labeled RGCs in whole-mount retinas were intracellularly injected to reveal their dendritic morphologies. Immunohistochemical staining was performed to selectively label ip RGCs(Me A-ip RGCs) and c RGCs(Me A-c RGCs). The results showed that 95.7% of the retrogradely labeled cells were c RGCs and that the rest were ip RGCs. Specifically, Me A-c RGCs consist of two morphological types. The majority of them exhibit small but dense dendritic fields and diffuse ramification patterns as previously reported in RG_(B2)(95%), while the rest exhibit small but sparse dendritic branching patterns resembling those of RG_(B3) cells(5%). Me Aip RGCs consist of M1 and M2 subtypes. The Me A-RGCs showed an even retinal distribution patterns. The soma and dendritic field sizes of the Me A-RGCs did not vary with eccentricity. In conclusion, the present results suggest that Me A-RGCs are structurally heterogeneous. These direct RGCs that input to the Me A could be important for regulating amygdala functions.     

Strategies for Expanding the Operational Range of Channelrhodopsin in Optogenetic Vision

Some hereditary diseases, such as retinitis pigmentosa, lead to blindness due to the death of photoreceptors, though the rest of the visual system might be only slightly affected. Optogenetics is a promising tool for restoring vision after retinal degeneration. In optogenetics, light-sensitive ion channels ("channelrhodopsins") are expressed in neurons so that the neurons can be activated by light. Currently existing variants of channelrhodopsin – engineered for use in neurophysiological research – do not necessarily support the goal of vision restoration optimally, due to two factors: First, the nature of the light stimulus is fundamentally different in "optogenetic vision" compared to "optogenetic neuroscience". Second, the retinal target neurons have specific properties that need to be accounted for, e.g. most retinal neurons are non-spiking. In this study, by using a computational model, we investigate properties of channelrhodopsin that might improve successful vision restoration. We pay particular attention to the operational brightness range and suggest strategies that would allow optogenetic vision over a wider intensity range than currently possible, spanning the brightest 5 orders of naturally occurring luminance. We also discuss the biophysical limitations of channelrhodopsin, and of the expressing cells, that prevent further expansion of this operational range, and we suggest design strategies for optogenetic tools which might help overcoming these limitations. Furthermore, the computational model used for this study is provided as an interactive tool for the research community.     

AAV-Mediated Clarin-1 Expression in the Mouse Retina: Implications for USH3A Gene Therapy

Usher syndrome type III (USH3A) is an autosomal recessive disorder caused by mutations in clarin-1 (CLRN1) gene, leading to progressive retinal degeneration and sensorineural deafness. Efforts to develop therapies for preventing photoreceptor cell loss are hampered by the lack of a retinal phenotype in the existing USH3 mouse models and by conflicting reports regarding the endogenous retinal localization of clarin-1, a transmembrane protein of unknown function. In this study, we used an AAV-based approach to express CLRN1 in the mouse retina in order to determine the pattern of its subcellular localization in different cell types. We found that all major classes of retinal cells express AAV-delivered CLRN1 driven by the ubiquitous, constitutive small chicken β-actin promoter, which has important implications for the design of future USH3 gene therapy studies. Within photoreceptor cells, AAV-expressed CLRN1 is mainly localized at the inner segment region and outer plexiform layer, similar to the endogenous expression of other usher proteins. Subretinal delivery using a full strength viral titer led to significant loss of retinal function as evidenced by ERG analysis, suggesting that there is a critical limit for CLRN1 expression in photoreceptor cells. Taken together, these results suggest that CLRN1 expression is potentially supported by a variety of retinal cells, and the right combination of AAV vector dose, promoter, and delivery method needs to be selected to develop safe therapies for USH3 disorder.     

In vivo gene therapy in young and adult RPE65-/- dogs produces long-term visual improvement

Defects in the RPE65 gene, which is selectively expressed in the retinal pigment epithelium (RPE), result in blindness and gradual photoreceptor cell degeneration. Experiments were conducted to assess the efficacy of gene replacement therapy in restoring retinal function in RPE65-/- dogs. Long-term effects of RPE65 gene therapy were assessed using visual behavioral testing and electroretinographic (ERG) recordings at 10-12 weeks and 6-9 months after surgery in five affected dogs. Subretinal injections of similar dosages of two constructs were performed in affected dogs at the ages of 4-30 months: rAAV.RPE65 into one eye and, in four of five dogs, rAAV.GFP contralaterally. Before surgery all RPE65-/- dogs were behaviorally blind with either no or very low-amplitude ERG responses to light stimuli. Marked improvements in visual behavior and ERG responses were observed as early as 4 weeks after surgery in affected animals. Except for light-adapted 50 Hz ERG flicker responses, all ERG parameters tested increased significantly in the eyes treated with the rAAV.RPE65 construct at the early follow-up. Gradual progressive improvements in ERG responses were observed in the RPE65-treated eyes over time. An unexpected finding was that on long-term follow-up, marked improvement of photopic ERG responses were also observed in the contralateral control eye in both young and older dogs. These results are promising for future clinical trials of human patients with retinal degenerative diseases, such as Leber congenital amaurosis, that result from RPE65 gene defects.     

In vivo imaging reveals dendritic targeting of laminated afferents by zebrafish retinal ganglion cells

Targeting of axons and dendrites to particular synaptic laminae is an important mechanism by which precise patterns of neuronal connectivity are established. Although axons target specific laminae during development, dendritic lamination has been thought to occur largely by pruning of inappropriately placed arbors. We discovered by in vivo time-lapse imaging that retinal ganglion cell (RGC) dendrites in zebrafish show growth patterns implicating dendritic targeting as a mechanism for contacting appropriate synaptic partners. Populations of RGCs labeled in transgenic animals establish distinct dendritic strata sequentially, predominantly from the inner to outer retina. Imaging individual cells over successive days confirmed that multistratified RGCs generate strata sequentially, each arbor elaborating within a specific lamina. Simultaneous imaging of RGCs and subpopulations of presynaptic amacrine interneurons revealed that RGC dendrites appear to target amacrine plexuses that had already laminated. Dendritic targeting of prepatterned afferents may thus be a novel mechanism for establishing proper synaptic connectivity.     

Systemic Gene Delivery Protects the Photoreceptors in the Retinal Degeneration Slow Mouse

The retinal degeneration slow (rds/rds) mouse was used to test photoreceptor protection by systemic gene delivery of non-erythropoietic forms of erythropoietin (EPO). Two Epo mutants were generated and packaged into recombinant adeno-associated virus (rAAV) serotype 2/5, controls included rAAV2/5.Epo and rAAV2/5.enhanced green fluorescent protein (eGFP). Mice were injected in the quadriceps at postnatal day seven and analyses were performed at postnatal day 90. Hematocrit, serum EPO levels, and outer nuclear layer (ONL) thickness were quantified. Hematocrit and serum EPO levels in rAAV2/5.eGFP, rAAV2/5.Epo, and rAAV2/5.EpoR103E treated mice were: 46%, 8 mU/ml; 63%, 117 mU/ml; and 52%, 332 mU/ml, respectively. The ONL from rds/rds mice treated with the Epo vectors were approximately twice as thick as the negative controls. This demonstrates that the photoreceptors can be protected without performing an intraocular injection and without increasing the hematocrit to unsafe levels. Intramuscular delivery of rAAV.EpoR103E is an attractive treatment for retinal degenerative diseases.