scholarly journals Optimized culture of retinal ganglion cells and amacrine cells from adult mice

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0242426
Author(s):  
Yong H. Park ◽  
Joshua D. Snook ◽  
Iris Zhuang ◽  
Guofu Shen ◽  
Benjamin J. Frankfort
Keyword(s):  
Retinal Ganglion Cells ◽  
Ganglion Cells ◽  
Culture Media ◽  
Neuronal Cell ◽  
Amacrine Cells ◽  
Mouse Retina ◽  
Neuronal Cultures ◽  
Dependent Manner ◽  
Retinal Ganglion

Cell culture is widely utilized to study the cellular and molecular biology of different neuronal cell populations. Current techniques to study enriched neurons in vitro are primarily limited to embryonic/neonatal animals and induced pluripotent stem cells (iPSCs). Although the use of these cultures is valuable, the accessibility of purified primary adult neuronal cultures would allow for improved assessment of certain neurological diseases and pathways at the cellular level. Using a modified 7-step immunopanning technique to isolate for retinal ganglion cells (RGCs) and amacrine cells (ACs) from adult mouse retinas, we have successfully developed a model of neuronal culture that maintains for at least one week. Isolations of Thy1.2+ cells are enriched for RGCs, with the isolation cell yield being congruent to the theoretical yield of RGCs in a mouse retina. ACs of two different populations (CD15+ and CD57+) can also be isolated. The populations of these three adult neurons in culture are healthy, with neurite outgrowths in some cases greater than 500μm in length. Optimization of culture conditions for RGCs and CD15+ cells revealed that neuronal survival and the likelihood of neurite outgrowth respond inversely to different culture media. Serially diluted concentrations of puromycin decreased cultured adult RGCs in a dose-dependent manner, demonstrating the potential usefulness of these adult neuronal cultures in screening assays. This novel culture system can be used to model in vivo neuronal behaviors. Studies can now be expanded in conjunction with other methodologies to study the neurobiology of function, aging, and diseases.

scholarly journals Optimized culture of retinal ganglion cells and amacrine cells from adult mice

2020 ◽  
Author(s):  
Yong H Park ◽  
Joshua D Snook ◽  
Iris Zhuang ◽  
Guofu Shen ◽  
Benjamin J Frankfort
Keyword(s):  
Retinal Ganglion Cells ◽  
Ganglion Cells ◽  
Culture Media ◽  
Neuronal Cell ◽  
Amacrine Cells ◽  
Mouse Retina ◽  
Neuronal Cultures ◽  
Dependent Manner ◽  
Retinal Ganglion

AbstractCell culture is widely utilized to study the cellular and molecular biology of different neuronal cell populations. Current techniques to study enriched neurons in vitro are primarily limited to embryonic/neonatal animals and induced pluripotent stem cells (iPSC). Although the use of these cultures is valuable, the accessibility of purified primary adult neuronal cultures would allow for improved assessment of certain neurological diseases and pathways at the cellular level. Using a modified 7-step immunopanning technique to isolate for retinal ganglion cells (RGCs) and amacrine cells (ACs) from adult mouse retinas, we have successfully developed a model of neuronal culture that maintains for at least one week. Isolations of Thy1.2+ cells are enriched for RGCs, with the isolation cell yield being congruent to the theoretical yield of RGCs in a mouse retina. ACs of two different populations (CD15+ and CD57+) can also be isolated. The populations of these three adult neurons in culture are healthy, with neurite outgrowths in some cases greater than 500µm in length. Optimization of culture conditions for RGCs and CD15+ cells revealed that neuronal survival and the likelihood of neurite outgrowth respond inversely to different culture media. Serially diluted concentrations of puromycin decreased cultured adult RGCs in a dose-dependent manner, demonstrating the potential usefulness of these adult neuronal cultures in screening assays. This novel culture system can be used to model adult neurons in vivo. Studies can now be expanded in conjunction with other methodologies to study the neurobiology of function, aging, and diseases.

scholarly journals PTEN Expression Regulates Gap Junction Connectivity in the Retina

2021 ◽  
Vol 15 ◽  
Author(s):  
Ashley M. Chen ◽  
Shaghauyegh S. Azar ◽  
Alexander Harris ◽  
Nicholas C. Brecha ◽  
Luis Pérez de Sevilla Müller
Keyword(s):  
Gap Junction ◽  
Retinal Ganglion Cells ◽  
Vision Loss ◽  
Ganglion Cells ◽  
Dendritic Structure ◽  
Amacrine Cells ◽  
Mouse Retina ◽  
Retinal Ganglion ◽  
Cell Coupling ◽  
Cell Degeneration

Manipulation of the phosphatase and tensin homolog (PTEN) pathway has been suggested as a therapeutic approach to treat or prevent vision loss due to retinal disease. In this study, we investigated the effects of deleting one copy of Pten in a well-characterized class of retinal ganglion cells called α-ganglion cells in the mouse retina. In Pten+/– retinas, α-ganglion cells did not exhibit major changes in their dendritic structure, although most cells developed a few, unusual loop-forming dendrites. By contrast, α-ganglion cells exhibited a significant decrease in heterologous and homologous gap junction mediated cell coupling with other retinal ganglion and amacrine cells. Additionally, the majority of OFF α-ganglion cells (12/18 cells) formed novel coupling to displaced amacrine cells. The number of connexin36 puncta, the predominant connexin that mediates gap junction communication at electrical synapses, was decreased by at least 50% on OFF α-ganglion cells. Reduced and incorrect gap junction connectivity of α-ganglion cells will affect their functional properties and alter visual image processing in the retina. The anomalous connectivity of retinal ganglion cells would potentially limit future therapeutic approaches involving manipulation of the Pten pathway for treating ganglion cell degeneration in diseases like glaucoma, traumatic brain injury, Parkinson’s, and Alzheimer’s diseases.

scholarly journals Defocused Images Change Multineuronal Firing Patterns in the Mouse Retina

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 530 ◽  
Author(s):  
Seema Banerjee ◽  
Qin Wang ◽  
Chung Him So ◽  
Feng Pan
Keyword(s):  
Edge Detection ◽  
Retinal Ganglion Cells ◽  
Ganglion Cells ◽  
Public Health Problem ◽  
Amacrine Cells ◽  
The United States ◽  
Mouse Retina ◽  
Major Public Health Problem ◽  
Retinal Ganglion ◽  
Firing Patterns

Myopia is a major public health problem, affecting one third of the population over 12 years old in the United States and more than 80% of people in Hong Kong. Myopia is attributable to elongation of the eyeball in response to defocused images that alter eye growth and refraction. It is known that the retina can sense the focus of an image, but the effects of defocused images on signaling of population of retinal ganglion cells (RGCs) that account either for emmetropization or refractive errors has still to be elucidated. Thorough knowledge of the underlying mechanisms could provide insight to understanding myopia. In this study, we found that focused and defocused images can change both excitatory and inhibitory conductance of ON alpha, OFF alpha and ON–OFF retinal ganglion cells in the mouse retina. The firing patterns of population of RGCs vary under the different powers of defocused images and can be affected by dopamine receptor agonists/antagonists’ application. OFF-delayed RGCs or displaced amacrine cells (dACs) with time latency of more than 0.3 s had synchrony firing with other RGCs and/or dACs. These spatial synchrony firing patterns between OFF-delayed cell and other RGCs/dACs were significantly changed by defocused image, which may relate to edge detection. The results suggested that defocused images induced changes in the multineuronal firing patterns and whole cell conductance in the mouse retina. The multineuronal firing patterns can be affected by dopamine receptors’ agonists and antagonists. Synchronous firing of OFF-delayed cells is possibly related to edge detection, and understanding of this process may reveal a potential therapeutic target for myopia patients.

scholarly journals Tracer coupling of intrinsically photosensitive retinal ganglion cells to amacrine cells in the mouse retina

2010 ◽  
Vol 518 (23) ◽  
pp. 4813-4824 ◽  
Author(s):  
Luis Pérez de Sevilla Müller ◽  
Michael Tri H. Do ◽  
King-Wai Yau ◽  
Shigang He ◽  
William H. Baldridge
Keyword(s):  
Retinal Ganglion Cells ◽  
Ganglion Cells ◽  
Amacrine Cells ◽  
Mouse Retina ◽  
Retinal Ganglion

scholarly journals POU6F2 Positive Retinal Ganglion Cells a Novel Group of ON-OFF Directionally Selective Subtypes in the Mouse Retina

2020 ◽  
Author(s):  
Ying Li ◽  
Jiaxing Wang ◽  
Rebecca King ◽  
Eldon E. Geisert
Keyword(s):  
Retinal Ganglion Cells ◽  
Ganglion Cells ◽  
Yellow Fluorescent Protein ◽  
Corneal Thickness ◽  
Amacrine Cells ◽  
Molecular Signature ◽  
Retinal Cell ◽  
Mouse Retina ◽  
Inner Plexiform Layer ◽  
Retinal Ganglion

AbstractPurposePreviously we identified POU6F2 as a genetic link between central corneal thickness (CCT) and risk of open-angle glaucoma. The present study is designed to characterize the POU6F2-positive retinal ganglion cells (RGCs).MethodsThe Thy1-YFP-H mouse was used to identify the structure of POU6F2-positive RGCs in the retina. In the retina of the Thy1-YFP-H mouse approximately 3% of the RGCs were labeled with yellow fluorescent protein. These retinas were stained for POU6F2 to identify the morphology of the POU6F2 subtypes in 3D reconstructions of the labeled RGCs. Multiple retinal cell markers were also co-stained with POU6F2 to characterize the molecular signature of the POU6F2-positive RGCs. DBA/2J glaucoma models were used to test the role of POU6F2 in injury.ResultsIn the retina POU6F2 labels 32.9% of the RGCs in the DBA/2J retina (16.1% heavily and 16.8% lightly labeled). In 3D constructions of Thy1-YFP-H positive RGCs, the heavily labeled POU6F2-positive cells had dendrites in the inner plexiform layer that were bistratified and appeared to be ON-OFF directionally selective cells. The lightly labeled POU6F2 RGCs displayed 3 different dendritic distributions, with dendrites in the ON sublaminae only, OFF sublaminae only, or bistratified. The POU6F2-positive cells partially co-stained with Cdh6. The POU6F2-positive cells do not co-stain with CART and SATB2 (markers for ON-OFF directionally selective RGC), SMI32 (a marker for alpha RGCs), or ChAT and GAD67(markers for amacrine cells). The POU6F2-positive cells were sensitive to injury. In DBA/2J glaucoma model, at 8 months of age there was a 22% loss of RGCs (labeled with RBPMS) while there was 73% loss of the heavily labeled POU6F2 RGCs.ConclusionsPOU6F2 is a marker for a novel group of RGC subtypes that are ON-OFF directionally selective RGCs that are sensitive to glaucomatous injury.

Photovoltaic stimulation efficiently evokes network-mediated activity of retinal ganglion cells

2019 ◽  
Author(s):  
Naïg A. L. Chenais ◽  
Marta J. I. Airaghi Leccardi ◽  
Diego Ghezzi
Keyword(s):  
Retinal Ganglion Cells ◽  
Ganglion Cells ◽  
Amacrine Cells ◽  
Biophysical Model ◽  
High Frequency Stimulation ◽  
Retinal Ganglion ◽  
Cell Coupling ◽  
Inner Retina ◽  
Stimulation Of

AbstractObjectivePhotovoltaic retinal prostheses theoretically offer the possibility of standalone high-resolution electrical stimulation of the retina. However, in artificial vision, achieving locally selective epiretinal stimulation is particularly challenging, on the grounds of axonal activation and electrical cell coupling.ApproachHere we show that electrical and photovoltaic stimulation of dystrophic retinal circuits with capacitive-like pulses leads to a greater efficiency for indirect network-mediated activation of retinal ganglion cells. In addition, a biophysical model of the inner retina stimulation is proposed to investigate the waveform and duration commitments in the genesis of indirect activity of retinal ganglion cells.Main resultsBoth in-vitro and in-silico approaches suggest that the application of long voltage pulses or gradual voltage changes are more effective to sustainably activate the inner excitatory and inhibitory layers of the retina, thus leading to a reproducible indirect response. The involvement of the inhibitory feedback from amacrine cells in the forming of indirect patterns represents a novel biological tool to locally cluster the response of the retinal ganglion cells.SignificanceThese results demonstrate that recruiting inner retina cells with epiretinal stimulation enables not only to bypass axonal stimulation but also to obtain a more focal activation thanks to the natural lateral inhibition. In this perspective, the use of capacitive-like waveforms generated by photovoltaic prostheses may allow improving the neural response resolution while standing high-frequency stimulation.

scholarly journals Parallel Inhibition of Dopamine Amacrine Cells and Intrinsically Photosensitive Retinal Ganglion Cells in a Non-Image-Forming Visual Circuit of the Mouse Retina

2015 ◽  
Vol 35 (48) ◽  
pp. 15955-15970 ◽  
Author(s):  
H. E. Vuong ◽  
C. N. Hardi ◽  
S. Barnes ◽  
N. C. Brecha
Keyword(s):  
Retinal Ganglion Cells ◽  
Ganglion Cells ◽  
Amacrine Cells ◽  
Mouse Retina ◽  
Retinal Ganglion

scholarly journals Nel positively regulates the genesis of retinal ganglion cells by promoting their differentiation and survival during development

2014 ◽  
Vol 25 (2) ◽  
pp. 234-244 ◽  
Author(s):  
Chizu Nakamoto ◽  
Soh-Leh Kuan ◽  
Amy S. Findlay ◽  
Elaine Durward ◽  
Zhufeng Ouyang ◽  
...  
Keyword(s):  
Retinal Ganglion Cells ◽  
Molecular Mechanisms ◽  
Ganglion Cells ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Neuronal Cell ◽  
Amacrine Cells ◽  
Progression Rate ◽  
Retinal Ganglion ◽  
Displaced Amacrine Cells

For correct functioning of the nervous system, the appropriate number and complement of neuronal cell types must be produced during development. However, the molecular mechanisms that regulate the production of individual classes of neurons are poorly understood. In this study, we investigate the function of the thrombospondin-1–like glycoprotein, Nel (neural epidermal growth factor [EGF]-like), in the generation of retinal ganglion cells (RGCs) in chicks. During eye development, Nel is strongly expressed in the presumptive retinal pigment epithelium and RGCs. Nel overexpression in the developing retina by in ovo electroporation increases the number of RGCs, whereas the number of displaced amacrine cells decreases. Conversely, knockdown of Nel expression by transposon-mediated introduction of RNA interference constructs results in decrease in RGC number and increase in the number of displaced amacrine cells. Modifications of Nel expression levels do not appear to affect proliferation of retinal progenitor cells, but they significantly alter the progression rate of RGC differentiation from the central retina to the periphery. Furthermore, Nel protects RGCs from apoptosis during retinal development. These results indicate that Nel positively regulates RGC production by promoting their differentiation and survival during development.

scholarly journals Rod bipolar cells dysfunction occurs before ganglion cells loss in excitotoxin-damaged mouse retina

2019 ◽  
Vol 10 (12) ◽  
Author(s):  
Yumeng Shen ◽  
Xue Luo ◽  
Shiliang Liu ◽  
Ying Shen ◽  
Scott Nawy ◽  
...  
Keyword(s):  
Retinal Ganglion Cells ◽  
Ganglion Cells ◽  
Experimental Models ◽  
Bipolar Cells ◽  
Diagnostic Tools ◽  
Mouse Retina ◽  
Receptor Subtype ◽  
Dependent Manner ◽  
Retinal Ganglion ◽  
Rod Bipolar Cells

AbstractProgressive degeneration of retinal ganglion cells (RGCs) will cause a blinding disease. Most of the study is focusing on the RGCs itself. In this study, we demonstrate a decline of the presynaptic rod bipolar cells (RBCs) response precedes RGCs loss and a decrease of protein kinase Cα (PKCα) protein expression in RBCs dendrites, using whole-cell voltage-clamp, electroretinography (ERG) measurements, immunostaining and co-immunoprecipitation. We present evidence showing that N-methyl D-aspartate receptor subtype 2B (NR2B)/protein interacting with C kinase 1 (PICK1)-dependent degradation of PKCα protein in RBCs contributes to RBCs functional loss. Mechanistically, NR2B forms a complex with PKCα and PICK1 to promote the degradation of PKCα in a phosphorylation- and proteasome-dependent manner. Similar deficits in PKCα expression and response sensitivity were observed in acute ocular hypertension and optic never crush models. In conclusion, we find that three separate experimental models of neurodegeneration, often used to specifically target RGCs, disrupt RBCs function prior to the loss of RGCs. Our findings provide useful information for developing new diagnostic tools and treatments for retinal ganglion cells degeneration disease.

Spatial resolution, contrast sensitivity, and sensitivity to defocus of chicken retinal ganglion cells in vitro

2009 ◽  
Vol 26 (5-6) ◽  
pp. 467-476 ◽  
Author(s):  
ERICH DIEDRICH ◽  
FRANK SCHAEFFEL
Keyword(s):  
Contrast Sensitivity ◽  
Retinal Ganglion Cells ◽  
Spatial Resolution ◽  
Microelectrode Array ◽  
Ganglion Cells ◽  
Amacrine Cells ◽  
Retinal Ganglion ◽  
Modulation Transfer ◽  
Eye Growth

AbstractThe chicken has been extensively studied as an animal model for myopia because its eye growth is tightly controlled by visual experience. It has been found that the retina controls the axial eye growth rates depending on the amount and the sign of defocus imposed in the projected image. Glucagonergic amacrine cells were discovered that appear to encode for the sign of imposed defocus. It is not clear whether the downstream neurons, the retinal ganglion cells, still have access to this information—and whether it ultimately reaches the brain. We have analyzed the spike rates of chicken retinal ganglion cells in vitro using a microelectrode array. For this purpose, we initially defined spatial resolution and contrast sensitivity in vitro. Two classes of chicken retinal ganglions were found, depending on the linearity of their responses with increasing contrast. Responses generally declined with increasing defocus of the visual stimulus. These responses were well predicted by the modulation transfer function for a diffraction-limited defocused optical system, the first Bessel function. Thus, the studied retinal ganglion cells did not distinguish between a loss of contrast at a given spatial frequency due to reduced contrast of the stimulus pattern or because the pattern was presented out of focus. Furthermore, there was no indication that the retinal ganglion cells responded differently to defocus of either sign, at least for the cells that were recorded in this study.

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