Fluorescent identification of axons, dendrites and soma of neuronal retinal ganglion cells with a genetic marker as a tool for facilitating the study of neurodegeneration
Abstract This study characterizes a fluorescent Slc17a6‐tdTomato neuronal reporter mouse line with strong labeling of axons throughout the optic nerve, of retinal ganglion cell (RGC) soma in the ganglion cell layer (GCL), and of RGC dendrites in the inner plexiform layer (IPL). The model facilitated...
Saved in:
| Main Authors: | , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Wiley
2025-01-01
|
| Series: | FASEB BioAdvances |
| Subjects: | |
| Online Access: | https://doi.org/10.1096/fba.2024-00095 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1841560710275923968 |
|---|---|
| author | Puttipong Sripinun Wennan Lu Sergei Nikonov Suhani Patel Sarah Hennessy Tianyuan Yao Qi N. Cui Brent A. Bell Claire H. Mitchell |
| author_facet | Puttipong Sripinun Wennan Lu Sergei Nikonov Suhani Patel Sarah Hennessy Tianyuan Yao Qi N. Cui Brent A. Bell Claire H. Mitchell |
| author_sort | Puttipong Sripinun |
| collection | DOAJ |
| description | Abstract This study characterizes a fluorescent Slc17a6‐tdTomato neuronal reporter mouse line with strong labeling of axons throughout the optic nerve, of retinal ganglion cell (RGC) soma in the ganglion cell layer (GCL), and of RGC dendrites in the inner plexiform layer (IPL). The model facilitated assessment of RGC loss in models of degeneration and of RGC detection in mixed neural/glial cultures. The tdTomato signal showed strong overlap with >98% cells immunolabeled with RGC markers RBPMS or BRN3A, consistent with the ubiquitous presence of the vesicular glutamate transporter 2 (VGUT2, SLC17A6) in all RGC subtypes. There was no cross‐labeling of ChAT‐positive displaced amacrine cells in the GCL, although some signal emanated from the outer plexiform layer, consistent with horizontal cells. The fluorescence allowed rapid screening of RGC loss following optic nerve crush and intraocular pressure (IOP) elevation. The bright fluorescence also enabled non‐invasive monitoring of extensive neurite networks and neuron/astrocyte interactions in culture. Robust Ca2+ responses to P2X7R agonist BzATP were detected from fluorescent RGCs using Ca2+‐indicator Fura‐2. Fluorescence from axons and soma was detected in vivo with a confocal scanning laser ophthalmoscope (cSLO); automatic RGC soma counts enhanced through machine learning approached the numbers found in retinal wholemounts. Controls indicated no impact of Slc17a6‐tdTomato expression on light‐dependent neuronal function as measured with a microelectrode array (MEA), or on retinal structure as measured with optical coherence tomography (OCT). In summary, the bright fluorescence in axons, dendrites and soma of ~all RGCs in the Slc17a6‐tdTomato reporter mouse may facilitate the study of RGCs. |
| format | Article |
| id | doaj-art-7a6f478177604677a70210351f1db397 |
| institution | Kabale University |
| issn | 2573-9832 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | FASEB BioAdvances |
| spelling | doaj-art-7a6f478177604677a70210351f1db3972025-01-03T15:59:11ZengWileyFASEB BioAdvances2573-98322025-01-0171n/an/a10.1096/fba.2024-00095Fluorescent identification of axons, dendrites and soma of neuronal retinal ganglion cells with a genetic marker as a tool for facilitating the study of neurodegenerationPuttipong Sripinun0Wennan Lu1Sergei Nikonov2Suhani Patel3Sarah Hennessy4Tianyuan Yao5Qi N. Cui6Brent A. Bell7Claire H. Mitchell8Department of Basic and Translational Science University of Pennsylvania Philadelphia Pennsylvania USADepartment of Basic and Translational Science University of Pennsylvania Philadelphia Pennsylvania USADepartment of Neuroscience University of Pennsylvania Philadelphia Pennsylvania USADepartment of Basic and Translational Science University of Pennsylvania Philadelphia Pennsylvania USADepartment of Basic and Translational Science University of Pennsylvania Philadelphia Pennsylvania USADepartment of Ophthalmology University of Pennsylvania Philadelphia Pennsylvania USADepartment of Ophthalmology University of Pennsylvania Philadelphia Pennsylvania USADepartment of Ophthalmology University of Pennsylvania Philadelphia Pennsylvania USADepartment of Basic and Translational Science University of Pennsylvania Philadelphia Pennsylvania USAAbstract This study characterizes a fluorescent Slc17a6‐tdTomato neuronal reporter mouse line with strong labeling of axons throughout the optic nerve, of retinal ganglion cell (RGC) soma in the ganglion cell layer (GCL), and of RGC dendrites in the inner plexiform layer (IPL). The model facilitated assessment of RGC loss in models of degeneration and of RGC detection in mixed neural/glial cultures. The tdTomato signal showed strong overlap with >98% cells immunolabeled with RGC markers RBPMS or BRN3A, consistent with the ubiquitous presence of the vesicular glutamate transporter 2 (VGUT2, SLC17A6) in all RGC subtypes. There was no cross‐labeling of ChAT‐positive displaced amacrine cells in the GCL, although some signal emanated from the outer plexiform layer, consistent with horizontal cells. The fluorescence allowed rapid screening of RGC loss following optic nerve crush and intraocular pressure (IOP) elevation. The bright fluorescence also enabled non‐invasive monitoring of extensive neurite networks and neuron/astrocyte interactions in culture. Robust Ca2+ responses to P2X7R agonist BzATP were detected from fluorescent RGCs using Ca2+‐indicator Fura‐2. Fluorescence from axons and soma was detected in vivo with a confocal scanning laser ophthalmoscope (cSLO); automatic RGC soma counts enhanced through machine learning approached the numbers found in retinal wholemounts. Controls indicated no impact of Slc17a6‐tdTomato expression on light‐dependent neuronal function as measured with a microelectrode array (MEA), or on retinal structure as measured with optical coherence tomography (OCT). In summary, the bright fluorescence in axons, dendrites and soma of ~all RGCs in the Slc17a6‐tdTomato reporter mouse may facilitate the study of RGCs.https://doi.org/10.1096/fba.2024-00095axonsdendritesglial/neuron cultureIOPmachine learningMEA |
| spellingShingle | Puttipong Sripinun Wennan Lu Sergei Nikonov Suhani Patel Sarah Hennessy Tianyuan Yao Qi N. Cui Brent A. Bell Claire H. Mitchell Fluorescent identification of axons, dendrites and soma of neuronal retinal ganglion cells with a genetic marker as a tool for facilitating the study of neurodegeneration FASEB BioAdvances axons dendrites glial/neuron culture IOP machine learning MEA |
| title | Fluorescent identification of axons, dendrites and soma of neuronal retinal ganglion cells with a genetic marker as a tool for facilitating the study of neurodegeneration |
| title_full | Fluorescent identification of axons, dendrites and soma of neuronal retinal ganglion cells with a genetic marker as a tool for facilitating the study of neurodegeneration |
| title_fullStr | Fluorescent identification of axons, dendrites and soma of neuronal retinal ganglion cells with a genetic marker as a tool for facilitating the study of neurodegeneration |
| title_full_unstemmed | Fluorescent identification of axons, dendrites and soma of neuronal retinal ganglion cells with a genetic marker as a tool for facilitating the study of neurodegeneration |
| title_short | Fluorescent identification of axons, dendrites and soma of neuronal retinal ganglion cells with a genetic marker as a tool for facilitating the study of neurodegeneration |
| title_sort | fluorescent identification of axons dendrites and soma of neuronal retinal ganglion cells with a genetic marker as a tool for facilitating the study of neurodegeneration |
| topic | axons dendrites glial/neuron culture IOP machine learning MEA |
| url | https://doi.org/10.1096/fba.2024-00095 |
| work_keys_str_mv | AT puttipongsripinun fluorescentidentificationofaxonsdendritesandsomaofneuronalretinalganglioncellswithageneticmarkerasatoolforfacilitatingthestudyofneurodegeneration AT wennanlu fluorescentidentificationofaxonsdendritesandsomaofneuronalretinalganglioncellswithageneticmarkerasatoolforfacilitatingthestudyofneurodegeneration AT sergeinikonov fluorescentidentificationofaxonsdendritesandsomaofneuronalretinalganglioncellswithageneticmarkerasatoolforfacilitatingthestudyofneurodegeneration AT suhanipatel fluorescentidentificationofaxonsdendritesandsomaofneuronalretinalganglioncellswithageneticmarkerasatoolforfacilitatingthestudyofneurodegeneration AT sarahhennessy fluorescentidentificationofaxonsdendritesandsomaofneuronalretinalganglioncellswithageneticmarkerasatoolforfacilitatingthestudyofneurodegeneration AT tianyuanyao fluorescentidentificationofaxonsdendritesandsomaofneuronalretinalganglioncellswithageneticmarkerasatoolforfacilitatingthestudyofneurodegeneration AT qincui fluorescentidentificationofaxonsdendritesandsomaofneuronalretinalganglioncellswithageneticmarkerasatoolforfacilitatingthestudyofneurodegeneration AT brentabell fluorescentidentificationofaxonsdendritesandsomaofneuronalretinalganglioncellswithageneticmarkerasatoolforfacilitatingthestudyofneurodegeneration AT clairehmitchell fluorescentidentificationofaxonsdendritesandsomaofneuronalretinalganglioncellswithageneticmarkerasatoolforfacilitatingthestudyofneurodegeneration |