Purpose: Particle-mediated gene transfer has been used in animal models to study the morphology and connectivity of retinal ganglion cells. The aim of the present study was to apply this method to transfect ganglion cells in postmortem human retina. Methods: Postmortem human eyes from male and female donors aged 40 to 76 years old were obtained within 15 hours after death. In addition, two marmoset retinas were obtained immediately after death. Ganglion cells were transfected with an expression plasmid for the postsynaptic density 95 protein conjugated to green or yellow fluorescent protein. Retinas were cultured for 3 days, fixed and then processed with immunohistochemical markers to reveal their stratification in the inner plexiform layer. Results: The retinas maintained their morphology and immunohistochemical properties for at least 3 days in culture. Bipolar and ganglion cell morphology was comparable to that observed in noncultured tissue. The quality of transfected cells in human retina was similar to that in freshly enucleated marmoset eyes. Based on dendritic field size and stratification, at least 11 morphological types of retinal ganglion cell were distinguished. Conclusions: Particle-mediated gene transfer allows efficient targeting of retinal ganglion cells in cultured postmortem human retina. Translational Relevance: The translational value of this methodology lies in the provision of an in vitro platform to study structural and connectivity changes in human eye diseases that affect the integrity and organization of cells in the retina.
Last. Ulrike Grünert(USYD: University of Sydney)H-Index: 49
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In primate retina, the midget, parasol, and small bistratified cell populations form the large majority of ganglion cells. In addition, there is a variety of low-density wide-field ganglion cell types, which are less well characterized. Here we studied retinal ganglion cells in the common marmoset, Callithrix jacchus, using particle-mediated gene-transfer. Ganglion cells were transfected with an expression plasmid for the postsynaptic density 95-green fluorescent protein. The retinas were proces...
#2Sammy C.S. Lee(ARC: Australian Research Council)H-Index: 14
Last. Ulrike Grünert(ARC: Australian Research Council)H-Index: 49
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Melanopsin-expressing retinal ganglion cells are intrinsically photosensitive cells that are involved in non-image forming visual processes such as the pupillary light reflex and circadian entrainment but also contribute to visual perception. Here we used immunohistochemistry to study the morphology, density, distribution, and synaptic connectivity of melanopsin-expressing ganglion cells in four post mortem human donor retinas. Two types of melanopsin-expressing ganglion cells were distinguished...
Choline acetyltransferase (ChAT) expressing retinal amacrine cells are present across vertebrates. These interneurons play important roles in the development of retinal projections to the brain and in motion detection, specifically in generating direction-selective responses to moving stimuli. ChAT amacrine cells typically comprise two spatially segregated populations that form circuits in the ‘ON’ or ‘OFF’ synaptic layers of the inner retina. This stereotypic arrangement is also found across th...
Last. Knut Stieger(University of Giessen)H-Index: 17
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The purpose of this study was characterization of adult murine neuroretina in organ culture to investigate its suitability for use in preclinical therapeutic applications. In retinal disorders, neurodegeneration of mature retinal cells takes place. Therefore, neonatal retina cultures are not adequate for therapeutic applications, such as genome editing, as the retina is still developing with cells dividing and differentiating into highly specialized cell types such as photoreceptors.Retinal expl...
#1Abhishek Sengupta(French Institute of Health and Medical Research)H-Index: 2
#1Abhishek Sengupta(French Institute of Health and Medical Research)H-Index: 1
Last. Jens Duebel(French Institute of Health and Medical Research)H-Index: 18
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Targeting the photosensitive ion channel channelrhodopsin-2 (ChR2) to the retinal circuitry downstream of photoreceptors holds promise in treating vision loss caused by retinal degeneration. However, the high intensity of blue light necessary to activate channelrhodopsin-2 exceeds the safety threshold of retinal illumination because of its strong potential to induce photochemical damage. In contrast, the damage potential of red-shifted light is vastly lower than that of blue light. Here, we show...
Last. Dennis M. Dacey(UW: University of Washington)H-Index: 6
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The long-term goal of this research is to understand how retinal ganglion cells that express the photopigment melanopsin, also known as OPN4, contribute to vision in humans and other primates. Here we report the results of anatomical studies using our polyclonal antibody specifically against human melanopsin that confirm and extend previous descriptions of melanopsin cells in primates. In macaque and human retina, two distinct populations of melanopsin cells were identified based on dendritic st...
Last. Andrew White(USYD: University of Sydney)H-Index: 23
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The study aims to validate mouse retinal explants for use as an efficient tool to investigate neuroprotective therapies for treatment in diseases such as glaucoma. Eyes from C57BL/6 mice were enucleated immediately post-mortem to make two retinal explants per eye. Explants were cultured at an air/medium interface on membrane inserts for 7 days ex-vivo. Explants were treated either with Z-VAD-FMK (a pan-caspase inhibitor; 100 μM) or vehicle. Retinal Ganglion cell (RGC) density was analysed by βII...
Macromolecular cell markers are essential for the classification and characterization of the highly complex and cellularly diverse vertebrate retina. Although a plethora of markers are described in the current literature, the immunoreactivity of these markers in normal human tissue has not been fully determined. This is problematic as they are quintessential to the characterization of morphological changes associated with human retinal disease. This review provides an overview of the macromolecu...
We used cultured adult mouse retinae as a model system to follow and quantify the retraction of dendrites using diolistic labelling of retinal ganglion cells (RGCs) following explantation. Cell death was monitored in parallel by nuclear staining as ‘labelling’ with RGC and apoptotic markers was inconsistent and exceedingly difficult to quantify reliably. Nuclear staining allowed us to delineate a lengthy time window during which dendrite retraction can be monitored in the absence of RGC death. T...
Last. Ulrike Grünert(USYD: University of Sydney)H-Index: 49
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Parallel visual pathways originate at the first synapse in the retina, where cones make connections with cone bipolar cells that in turn contact ganglion cells. There are more ganglion cell types than bipolar types, suggesting that there must be divergence from bipolar to ganglion cells. Here we analyze the contacts between an OFF bipolar type (DB3a) and six ganglion cell types in the retina of the marmoset monkey (Callithrix jacchus). Ganglion cells were transfected via particle-mediated gene t...
The prevalence of retinal disorders associated with visual impairment and blindness is increasing worldwide, while most of them remain without effective treatment. Pharmacological and molecular therapy development is hampered by the lack of effective drug delivery into the posterior segment of the eye. Among molecular approaches, RNA-interference (RNAi) features strong advantages, yet delivering it to the inner layer of the retina appears extremely challenging. To address this, we developed an o...
Summary In humans, midget and parasol ganglion cells account for most of the input from the eyes to the brain. Yet, how they encode visual information is unknown. Here, we perform large-scale multi-electrode array recordings from retinas of treatment-naive patients who underwent enucleation surgery for choroidal malignant melanomas. We identify robust differences in the function of midget and parasol ganglion cells, consistent asymmetries between their ON and OFF types (that signal light increme...